Virus protein blockers / inhibitors as anti-influenza agents

Abstract

A pharmaceutical composition comprising an influenza A M2 protein channel blocker for use in the treatment or prevention of the virulence of an aminoadamantane resistant influenza A, such as the H1N1 subtype, in a subject is provided. Further provided is a pharmaceutical composition comprising the influenza A M2 protein channel blocker for preventing entry, shelling and / or release from influenza A cells.

Description

[0001] References to electronic sequence listings

[0002] The entire contents of the electronic sequence listing (VRB-P-004-PCT ST26.xml; size: 2035 bytes; and creation date: December 6, 2023) are incorporated herein by reference.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 432,446, filed on December 14, 2022, entitled “VIROPORINS BLOCKERS / INHIBITORSAS ANTI-INFLUENZAAGENTS,” the entire contents of which are incorporated herein by reference. Technical Field

[0005] The present invention belongs to the field of antiviral therapy. Background Art

[0006] Influenza is a segmented, single-stranded, negative-sense RNA virus belonging to the family Orthomyxoviridae. It is one of the leading causes of morbidity and mortality from infectious diseases. Especially before COVID-19, it was the leading infectious cause of death in the Western world. Influenza is effectively combated by vaccines and antiviral treatments. However, ongoing gene transfer and drift continuously undermine the effectiveness of vaccines and the benefits of antiviral drugs. For example, the annual vaccine protection rate is approximately 40%, and resistance to every anti-influenza drug has been reported. In particular, 98% of the currently circulating variants are resistant to aminoadamantines ( and ), which is resistant to influenza. Aminoadamantanes are the first class of antiviral agents developed in the 1960s to fight influenza (Davies, WL et al., "Antiviral Activity of 1-Adamantanamine (Amantadine)." Science 1964; 144: 862-863, the entire contents of which are incorporated herein by reference).

[0007] There is an unmet need for methods and compounds to ameliorate or treat influenza viruses, particularly influenza variants that are resistant to aminoadamantanes. Summary of the Invention

[0008] According to a first aspect, a method for treating or preventing influenza A virus in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a molecule selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine or its metabolites and any combination thereof, thereby treating or preventing influenza A virulence in the subject.

[0009] According to another aspect, a pharmaceutical composition is provided comprising a molecule for treating or preventing influenza A virulence in a subject in need thereof, wherein the molecule is selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof.

[0010] According to another aspect, a kit is provided comprising at least two molecules selected from the group consisting of fludarabine or a derivative thereof, asunaprevir, amikacin, a xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, and any combination thereof.

[0011] In some embodiments, the molecule is selected from the group consisting of fludarabine, asunaprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof.

[0012] In some embodiments, the kit further comprises instructions for mixing at least two molecules selected from the group consisting of fludarabine or a derivative thereof, asunaprevir, amikacin, a xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, and any combination thereof.

[0013] In some embodiments, the fludarabine derivative comprises vidarabine or a metabolite thereof.

[0014] In some embodiments, the metabolite of vidarabine is inosine.

[0015] In some embodiments, the xanthine derivative is theobromine.

[0016] In some embodiments, the kit is used to prepare a medicament for treating a subject having influenza or infected with an influenza virus.

[0017] In some embodiments, the molecule is an M2 protein blocker.

[0018] In some embodiments, the influenza A virus is of the H1N1 subtype.

[0019] In some embodiments, the influenza A virus is resistant to aminoadamantanes.

[0020] In some embodiments, the molecule is administered at a daily dosage of 0.01 to 500 mg / kg body weight of the subject.

[0021] In some embodiments, administration is of therapeutically effective amounts of two molecules selected from the group consisting of fludarabine, asunaprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof.

[0022] In some embodiments, the two molecules are vidarabine or a metabolite thereof, and a molecule selected from the group consisting of theobromine and grazoprevir.

[0023] According to another aspect, a combination for treating or preventing influenza A virulence in a subject in need thereof is provided, wherein the combination comprises at least two molecules selected from the group consisting of fludarabine, asunaprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, vidarabine or its metabolites and any combination thereof.

[0024] In some embodiments, the at least two molecules are (i) vidarabine or a metabolite thereof, and (ii) a molecule selected from the group consisting of theobromine and grazoprevir.

[0025] In some embodiments, the metabolite of vidarabine is or includes inosine arabinoside.

[0026] Unless otherwise defined, all technical terms and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although similar or equivalent methods and materials as described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In the event of conflict, the patent specification (including definitions) shall prevail. In addition, materials, methods and examples are merely illustrative and are not intended to be limiting.

[0027] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Included is a graph showing negative detection of influenza M2 virorin, where protein expression is detrimental to bacterial growth. Growth of DH10B bacteria was altered by varying concentrations of the protein inducer isopropyl-β-D-thiogalactopyranoside (IPTG).

[0029] Figure 2 Included are vertical bar graphs demonstrating positive detection of influenza M2 viroporins. K values ​​for expression of influenza M2 viroporins were monitored in the presence (10 μM) or absence of the inducer IPTG. + The maximum growth rate of uptake-deficient bacteria varies with K + The concentration changes.

[0030] Figure 3 Includes a figure showing the acidity detection of influenza M2 viroporin. + Concentration changes over time, acidic solution was injected into the culture medium at time 0. Results shown are from bacteria where viroporin induction was varied with IPTG concentration as indicated.

[0031] Figure 4 Included are figures showing the results of a negative assay for screening of influenza M2 viroporin blockers. Bacteria in which channel expression was not induced ("No Channel") or drug was not added ("No Drug") were used as controls.

[0032] Figure 5 Included are figures showing the results of positive assays for screening of influenza M2 viroporin blockers. Bacteria in which channel expression was not induced ("No Channel") or drug was not added ("No Drug") were used as controls.

[0033] Figure 6 Included is a graph showing acidity assays of influenza M2 viroporins in the presence of test drugs.

[0034] Figure 7 Included are vertical bar graphs showing cell viability in the presence of various drugs at a concentration of 10 μM (unless otherwise indicated). The dashed line represents the viability of cells in "vehicle" treatment.

[0035] Figure 8Included are vertical bar graphs showing cell viability in the presence of various drugs at a concentration of 3 μM (unless otherwise indicated). The dashed line represents the viability of cells in the "vehicle" treatment.

[0036] Figure 9 Included are vertical bar graphs demonstrating the dose-dependent effect of the test drug on cell viability. The dashed line represents the viability of cells in the "vehicle" treatment.

[0037] Figure 10 Included are vertical bar graphs demonstrating the dose-dependent effect of the test drug on cell viability. The dashed line represents the viability of cells in the "vehicle" treatment.

[0038] Figure 11 Included are vertical bar graphs demonstrating the dose-dependent effect of the test drug on cell viability. The dashed line represents the viability of cells in the "vehicle" treatment.

[0039] Figure 12 Included are vertical bar graphs demonstrating the dose-dependent effect of the test drug on cell viability. The dashed line represents the viability of cells in the "vehicle" treatment.

[0040] Figure 13 Included are graphs showing the half maximal effective concentration (EC50) values ​​of the test compounds.

[0041] Figure 14 Included are graphs showing the half maximal effective concentration (EC50) values ​​of the test compounds.

[0042] Figure 15 Included are graphs demonstrating drug synergy. All drugs were administered at a concentration of 0.01 μM. The dashed line represents the viability of cells in the "vehicle" treatment.

[0043] Figure 16 Included are vertical bar graphs showing relative cell survival 48 hours after infection in the presence of various drugs or combinations thereof. It was observed that the combination of theobromine and vidarabine synergistically improved cell survival. Furthermore, the effect of theobromine and vidarabine combination was even more pronounced than that of the approved drugs oseltamivir and favipiravir.

[0044] Figure 17 Included are vertical bar graphs showing the viral RNA (%) in the lungs as measured following in vivo studies in mice where the animals were treated with the indicated drugs. The combination of theobromine and inosine arabinoside was observed to be more effective in vivo than the market leading drug, oseltamivir, even though it was administered at a significantly lower dose.

[0045] Figure 18 Included are the chemical structures of xanthine and other closely related compounds that share the xanthine skeleton and further modifications. The locations of the modifications are indicated by dashed lines.

[0046] Figure 19 Included is a figure showing structure-activity relationship (SAR) analysis of xanthine derivatives. Cell viability was measured as described. Higher values ​​indicate better protection against virus-induced cell death. As shown in the top figure, different xanthine derivatives include modifications at the xanthine R1-R3 positions.

[0047] Figure 20 Included are the chemical structures of xanthine and other closely related compounds with the xanthine skeleton and further modifications (top row). The locations of the modifications are indicated by dashed lines. Also presented are the antiviral drug adenosine and its natural metabolite inosine arabinoside (bottom row).

[0048] Figure 21 Included is a table summarizing combination therapies with the indicated drugs. Higher values ​​indicate better protection against virus-induced cell death. As presented, compounds were used at various concentrations.

[0049] Figure 22 Included are figures showing SAR analysis of fludarabine derivatives. Cell viability was determined as described. Higher values ​​indicate better protection against virus-induced cell death. As shown in the top figure, different fludarabine derivatives include modifications at the R1-R5 positions of fludarabine. DETAILED DESCRIPTION

[0050] In some embodiments, the present invention provides a composition comprising a matrix protein 2 or M2 protein channel blocker for treating or preventing influenza virulence in a subject. In some embodiments, the present invention provides a composition comprising an influenza virus M2 protein channel blocker for preventing influenza cell entry, uncoating, and / or release from cells. As used herein, the terms "matrix protein 2" and "M2 protein" can be used interchangeably.

[0051] In some embodiments, the present invention is based on the discovery that at least one molecule selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, inhibits the M2 channel of influenza A and is therefore useful for treating and / or preventing influenza A virulence. In some embodiments, the influenza A strain that can be treated or prevented by at least one of the above molecules is resistant to aminoadamantanes.

[0052] In some embodiments, at least one molecule selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, or its metabolites, and any combination thereof, can be used to treat and / or prevent influenza A virulence.

[0053] Influenza virus M2 protein

[0054] The M2 protein of influenza virus is a transmembrane protein known to be present in all influenza A strains. In some embodiments, the M2 protein forms a proton channel in the viral envelope. In some embodiments, the function of the M2 channel is to balance the pH of the influenza virus membrane during cell entry and to balance the pH of the trans-Golgi membrane of the infected cell during viral maturation. In some embodiments, M2 is essential for viral replication. In some embodiments, M2 is the target of anti-influenza drugs (e.g., amantadine and rimantadine). In some embodiments, the M2 protein belongs to the class of viroporins (e.g., small proteins that form ion channels that increase the membrane permeability of virus-infected cells). In some embodiments, M2 of influenza A (also known as AM2) and M2 of influenza B (also known as BM2) are primarily proton channels. As used herein, the terms "M2 of influenza A" and "AM2" are used interchangeably. The terms "M2 of influenza B" and "BM2" are used interchangeably. In some embodiments, the influenza C M2 protein (CM2) and the influenza D M2 protein (DM2) are particularly selective for chloride ions and may have some permeability to protons.

[0055] In some embodiments, the influenza virus disclosed herein comprises influenza A. In some embodiments, the M2 disclosed herein comprises AM2. In some embodiments, the influenza virus comprises influenza A, and the M2 disclosed herein comprises AM2.

[0056] Influenza A viruses are typically divided into subtypes based on the relationship between the antigens hemagglutinin and neuraminidase in their surface glycoproteins. In some embodiments, influenza A viruses include at least 16 hemagglutinin subtypes (designated H1-H16) and at least 9 neuraminidase subtypes (designated N1-N9). In some embodiments, each influenza A virus has one hemagglutinin and one neuraminidase antigen. In some embodiments, influenza A viruses include one antigen selected from H1-H16 and one antigen selected from N1-N9 in any combination. In some embodiments, in addition to the antigenic similarity on the surface glycoproteins, subtypes may include differences in the rest of the genome. In some embodiments, not all H1N1 viruses (i.e., those with the same hemagglutinin 1 and neuraminidase 1 antigens) have the same characteristics.

[0057] In some embodiments, the influenza A virus comprises an H1N1 subtype. In some embodiments, the H1N1 influenza virus comprises an M2 protein.

[0058] In some embodiments, influenza viruses are resistant to aminoadamantanes. Amantadine (brand names Gocovri, Symadine, and Symmetrel) includes the organic compound 1-adamantanamine or 1-aminoadamantane, which consists of an adamantane backbone with an amino group substituted at one of the four methine positions. Rimantadine (also known as flumadine) includes adamantane derivatives with similar biological properties. In some embodiments, amantadine and rimantadine target the M2 proton channel of influenza A viruses. In some embodiments, aminoadamantanes include amantadine (adamantan-1-amine hydrochloride) and rimantadine (1-(1-adamantyl)ethylamine hydrochloride).

[0059] As used herein, the term "resistance" or "resistant" refers to a reduced ability of an antiviral drug to prevent or reduce viral infection. In some embodiments, resistance to aminoadamantane can be observed when aminoadamantane is administered before or after infection. In some embodiments, the nucleic acid encoding the M2 protein of the influenza virus has a mutation that hinders or reduces the ability of aminoadamantane to inhibit the M2 protein. In some embodiments, the mutation in the nucleic acid encoding the M2 protein results in at least one non-synonymous amino acid substitution. In some embodiments, the virus has or is characterized as being drug-resistant or resistant to the drug.

[0060] As used herein, the term "non-synonymous amino acid substitution" refers to any nucleotide mutation that changes the amino acid sequence of a protein. In some embodiments, the amino acid substitution is caused by at least one of the following: (i) a missense mutation, i.e., a non-synonymous substitution caused by a point mutation in a single nucleotide, (ii) a nonsense mutation (a non-synonymous substitution that occurs when a mutation causes a protein to terminate prematurely by changing an amino acid to a stop codon); and (iii) a non-stop mutation or read-through mutation that occurs when a stop codon is replaced with an amino acid codon, resulting in a protein that is longer than specified.

[0061] In some embodiments, the amino acid substitution in the M2 protein is caused by a missense mutation. In some embodiments, the amino acid substitution in the M2 protein includes at least one amino acid substitution. Examples of mutations that confer resistance to aminoadamantanes are known in the art, such as amino acid substitutions at amino acid residues selected from 27, 30, 31, and 34, as described in Hay AJ et al., "The molecular basis of the specific anti-Influenza Action of amantadine." EMBO J 1985; 4: 3021-3024, the entire contents of which are incorporated herein by reference. In some embodiments, the amino acid substitution in the M2 protein includes at least one amino acid substitution selected from S31N, V27A, V27G, V27D, I27S, I27T, I27A, A30T, A30P, and G34E.

[0062] Examples of M2 mutations that confer resistance to aminoadamantane are known in the art and are described in Astrahan P and Arkin IT, Resistance characteristics of influenza to amino-adamantyls. Biochim Biophys Acta. 2011; 1808: 547-553, the entire contents of which are incorporated herein by reference. In some embodiments, avian strains including A / chicken / Germany / 34 or H7N1 Rostock or A / chicken / Germany / 27 or H7N7 Weybridge are known to be resistant to amantadine. In some embodiments, human strain A / Singapore / 1 / 57 (H2N2) is resistant to amantadine when amantadine is associated with a prior infection. In some embodiments, several H3N2 and H1N1 strains are resistant to amantadine.

[0063] In some embodiments, at least one mutation selected from S31N (serine 31 to asparagine) and V27A (valine 27 to alanine) confers aminoadamantane resistance (e.g., in the case of the WSN / 33 H1N1 strain). In some embodiments, the influenza A disclosed herein includes aminoadamantane-resistant variants of HIN1. In some embodiments, the aminoadamantane-resistant variants of HIN1 include the S31N amino acid substitution.

[0064] method

[0065] In some embodiments, the present invention provides a method for treating or preventing influenza disease or at least one symptom associated therewith. In some embodiments, the present invention provides a method for treating or preventing a disease induced by influenza A virus in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a molecule selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, or any combination thereof.

[0066] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a molecule selected from the group consisting of fludarabine, a derivative or metabolite thereof, asunaprevir, ravuconazole, amikacin, a xanthine or a derivative thereof, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir or a metabolite thereof, and any combination thereof.

[0067] In some embodiments, the fludarabine derivative is selected from the group consisting of: vidarabine, nelarabine, cordycepin, clofarabine, cladribine, or any metabolites thereof.

[0068] In some embodiments, the fludarabine derivative is selected from the group consisting of: vidarabine, nelarabine, cordycepin, or any metabolites thereof.

[0069] In some embodiments, the fludarabine derivative is selected from: vidarabine, nelarabine, or any metabolite thereof.

[0070] In some embodiments, the fludarabine derivative is selected from: vidarabine or any metabolite thereof.

[0071] In some embodiments, the metabolite of vidarabine is or includes inosine arabinoside.

[0072] In some embodiments, the xanthine derivative is selected from the group consisting of theobromine, 1-methylxanthine, 3-methylxanthine, caffeine, theophylline, enprophylline, paraxanthine, 7-methylxanthine, or any combination thereof.

[0073] In some embodiments, the xanthine derivative is selected from the group consisting of theobromine, 1-methylxanthine, 3-methylxanthine, theophylline, 7-methylxanthine, or any combination thereof.

[0074] In some embodiments, the xanthine derivative is or includes theobromine. In some embodiments, theobromine is a theobromine precursor. In some embodiments, the theobromine precursor is or includes caffeine, so long as it is metabolized to a therapeutically effective amount of theobromine.

[0075] In some embodiments, the vidarabine metabolite includes or is inosine arabinoside.

[0076] In some embodiments, the present invention provides a composition comprising an influenza A virus M2 channel blocker for treating or preventing influenza A virulence in a subject. In some embodiments, the present invention provides a composition comprising an M2 protein channel blocker for preventing influenza A virus cell entry, uncoating, and / or release from a cell.

[0077] In some embodiments, the present invention is based on the discovery that at least one molecule selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, inhibits the M2 channel of influenza A and is therefore useful in treating and / or preventing influenza A virulence.

[0078] In some embodiments, at least one molecule selected from the group consisting of fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, can be used to treat and / or prevent influenza A virulence.

[0079] In some embodiments, the influenza A strain treatable or preventable by at least one of the molecules described above is resistant to aminoadamantanes.

[0080] In some embodiments, at least one molecule selected from the group consisting of fludarabine, asunaprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, inhibits the M2 channel of influenza A.

[0081] In some embodiments, the matrix protein 2 or M2 protein of influenza A virus (strain A / Bellamy / 1942 H1N1) is disclosed under GenBank accession number ABW75846.1.

[0082] According to some embodiments, the M2 protein of influenza A virus comprises the amino acid sequence: MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIVGILHLILWILDRLFFKCIYRLFKHGLKR GPSTEGVPESMREEYRKEQQSAVDADDSHFVNIEL (SEQ ID NO: 1).

[0083] According to some embodiments, the M2 protein of the influenza virus comprises an analog of SEQ ID NO: 1 having at least 70%, at least 75%, at least 85%, at least 90%, at least 95% sequence identity or homology thereto, or any value or range therebetween. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the M2 protein comprises an analog of SEQ ID NO: 1 having homology thereto in the range of 85-100%, 91-100%, and 96-100%. Each possibility represents a separate embodiment of the present invention.

[0084] As used herein, the term "analog" refers to a polypeptide that is similar, but not identical, to a polypeptide of the present invention, yet still belongs to influenza A virus. In some embodiments, the analog is resistant to aminoadamantane. Analogs may have deletions or mutations that result in an amino acid sequence that differs from the amino acid sequence of the polypeptide of the present invention. It should be understood that all analogs of the polypeptide of the present invention are still capable of producing ion channels. In addition, analogs may resemble fragments of the polypeptide of the present invention; however, in such cases, the fragment must include at least 50 contiguous amino acids of the polypeptide of the present invention.

[0085] According to some embodiments, the present invention provides a method of treating or preventing influenza virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an influenza virus M2 protein channel blocker, thereby treating or preventing influenza virulence in the subject.

[0086] According to some embodiments, the present invention provides a method for preventing influenza virus release from a cell, the method comprising contacting the cell with an influenza virus M2 protein channel blocker, thereby preventing influenza virus release from the cell.

[0087] According to some embodiments, the present invention provides a method for preventing influenza virus entry into a cell, the method comprising contacting a cell with an influenza virus M2 protein channel blocker, thereby preventing influenza virus entry into the cell.

[0088] According to some embodiments, the present invention provides a method for preventing influenza virus uncoating, the method comprising contacting a cell with an influenza virus M2 channel blocker, thereby preventing influenza virus uncoating.

[0089] According to some embodiments, the cell is a cell of a subject. According to some embodiments, the contacting is administration to a subject. According to some embodiments, the subject is a subject infected or suspected of being infected with an influenza virus. In some embodiments, the influenza virus comprises influenza A virus. In some embodiments, the influenza virus comprises an H1N1 subtype. In some embodiments, the influenza virus is resistant to aminoadamantane.

[0090] According to some embodiments, the influenza virus M2 channel blocker is at least one molecule selected from the following: fludarabine or a salt thereof, asunaprevir or a salt thereof, ravuconazole or a salt thereof, amikacin or a salt thereof, theobromine or a salt thereof, flunisolide or a salt thereof, alvimopan or a salt thereof, eliglukast or a salt thereof, CM4620 or a salt thereof, levamlodipine or a salt thereof, emamectin or a salt thereof, grazoprevir or a salt thereof, isavuconazole or a salt thereof, voriconazole or a salt thereof, paliprevir or a salt thereof, vidarabine or any metabolite or salt thereof, and any combination thereof.

[0091] According to some embodiments, the influenza virus M2 channel blocker is at least one molecule selected from the group consisting of fludarabine or a salt thereof, asunaprevir or a salt thereof, amikacin or a salt thereof, theobromine or a salt thereof, flunisolide or a salt thereof, alvimopan or a salt thereof, levamlodipine or a salt thereof, grazoprevir or a salt thereof, voriconazole or a salt thereof, paliprevir or a salt thereof, vidarabine or any metabolite thereof or a salt thereof, and any combination thereof.

[0092] According to some embodiments, treating a subject in need thereof with an influenza virus M2 channel blocker comprises treating with two molecules selected from the group consisting of: theobromine + vidarabine or a metabolite thereof, and vidarabine or a metabolite thereof + grazoprevir. In some embodiments, the influenza virus M2 channel blocker comprises treating with vidarabine or a metabolite thereof and at least one molecule selected from theobromine and grazoprevir.

[0093] According to some embodiments, treating a subject in need thereof with an influenza virus M2 channel blocker comprises treating with two molecules selected from the group consisting of: theobromine + inosine arabinoside and inosine arabinoside + grazoprevir. In some embodiments, the influenza virus M2 channel blocker comprises treating with inosine arabinoside and at least one molecule selected from theobromine and grazoprevir.

[0094] According to some embodiments, treating a subject in need thereof with an influenza virus M2 channel blocker comprises treating with theobromine and vidarabine or a metabolite thereof.

[0095] According to some embodiments, the present invention provides an M2 channel blocker for use in treating or preventing influenza virulence in a subject in need thereof.

[0096] According to some embodiments, the present invention provides an M2 channel blocker for preventing the release of influenza virus from cells.

[0097] According to some embodiments, the M2 channel blocker is in a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

[0098] According to some embodiments, the present invention provides a pharmaceutical composition comprising fludarabine, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0099] According to some embodiments, the influenza virus M2 channel blocker comprises fludarabine, an analog or salt thereof.

[0100] Fludarabine used herein includes fludarabine (CAS: 21679-14-1, IUPAC: (2R, 3S, 4S, 5R) -2- (6-amino-2-fluoropurin-9-yl) -5- (hydroxymethyl) tetrahydrofuran-3, 4-diol), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof. In some embodiments, fludarabine includes chemotherapy drugs used to treat leukemia and / or lymphoma.

[0101] According to some embodiments, the present invention provides a pharmaceutical composition comprising asunaprevir, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0102] According to some embodiments, the influenza virus M2 channel blocker comprises asunaprevir, an analog or salt thereof.

[0103] As used herein, asunaprevir includes asunaprevir (formerly known as BMS-650032, CAS: 630420-165-5, IUPAC: 3-methyl-N-{[(2-methyl-2-propyl)oxy]carbonyl}-L-isovalinoyl-(4R)-4-[(7-chloro-4-methoxy-1-isoquinolinyl)oxy]-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-vinylcyclopropyl}-L-prolinamide), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0104] According to some embodiments, the present invention provides a pharmaceutical composition comprising ravuconazole, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0105] According to some embodiments, the influenza virus M2 channel blocker comprises ravuconazole, an analog or a salt thereof.

[0106] As used herein, ravuconazole includes ravuconazole (also known as BMS-207147 or ER-30346, CAS: 18276-06-1, IUPAC: 4-[2-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1,2,4-triazol-1-yl)butan-2-yl]-1,3-thiazol-4-yl]benzonitrile), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0107] According to some embodiments, the present invention provides a pharmaceutical composition comprising amikacin, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0108] According to some embodiments, the influenza virus M2 channel blocker comprises amikacin, an analog or a salt thereof.

[0109] Amikacin used herein includes amikacin (CAS: 37517-28-5, IUPAC: (2S)-4-amino-N-[(2S,3S,4R,5S)-5-amino-2-[(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-[(2R,3R,4S,5R,6R)-6-(aminomethyl)-3,4,5-trihydroxy-oxan-2-yl]oxy-3-hydroxy-cyclohexyl]-2-hydroxybutanamide), and pharmaceutically acceptable salts, solvates, hydrates and mixtures thereof.

[0110] According to some embodiments, the present invention provides a pharmaceutical composition comprising theobromine, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0111] According to some embodiments, the influenza virus M2 channel blocker comprises theobromine, an analog or a salt thereof.

[0112] Theobromine used herein includes theobromine (CAS: 83-67-0, IUPAC: 3,7-dimethyl-1H-purine-2,6-dione), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0113] According to some embodiments, the present invention provides a pharmaceutical composition comprising flunisolide, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0114] According to some embodiments, the influenza virus M2 channel blocker comprises flunisolide, an analog or salt thereof.

[0115] Flunisolide used herein includes flunisolide (CAS: 3385-03-3, IUPAC: (1S,2S,4R,8S,9S,11S,12S,13R,19S)-19-fluoro-11-hydroxy-8-(2-hydroxyacetyl)-6,6,9,13-tetramethyl-5,7-dioxopentacyclo[10.8.0.02,9.04,8.013,18]eicos-14,17-dien-16-one), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0116] According to some embodiments, the present invention provides a pharmaceutical composition comprising alvimopan, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0117] According to some embodiments, the influenza virus M2 channel blocker comprises alvimopan, an analog or a salt thereof.

[0118] Alvimopan as used herein includes alvimopan (CAS: 156053-89-3, IUPAC: 2-([(2S)-2-([(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl)phenylpropionyl]amino)acetic acid), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0119] According to some embodiments, the present invention provides a pharmaceutical composition comprising eliglukast, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0120] According to some embodiments, the influenza virus M2 channel blocker comprises eliglukast, an analog or salt thereof.

[0121] The iliglikast used herein includes iliglikast (CAS: 491833-29-5, IUPAC: N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-(1-pyrrolidinyl)-2-propyl]octanamide), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0122] According to some embodiments, the present invention provides a pharmaceutical composition comprising CM4620, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0123] According to some embodiments, the influenza virus M2 channel blocker comprises CM4620, an analog or salt thereof.

[0124] CM4620 as used herein includes CM4620 (CAS: 1712240-67-5, IUPAC: N-[5-(6-chloro-2,2-difluoro-1,3-benzodioxol-5-yl)pyrazin-2-yl]-2-fluoro-6-methylbenzamide), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0125] According to some embodiments, the present invention provides a pharmaceutical composition comprising levamlodipine, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0126] According to some embodiments, the influenza virus M2 channel blocker comprises levamlodipine, an analog or a salt thereof.

[0127] The levamlodipine used herein includes levamlodipine (also known as levoamlodipine or S-amlodipine, CAS: 103129-82-4, IUPAC: 5-methyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid (S)-3-ethyl ester), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0128] According to some embodiments, the present invention provides a pharmaceutical composition comprising emamectin, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an infection with a virus resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0129] According to some embodiments, the influenza virus M2 channel blocker comprises emamectin, an analog or salt thereof.

[0130] Emamectin as used herein includes emamectin (CAS: 119791-41-2 or 155569-91-8, also known as 4"-deoxy-4"-epi-methylamino-avermectin B1, epi-methylamino-4"-deoxy-avermectin, MK 243, EMA or GWN1972), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0131] According to some embodiments, the present invention provides a pharmaceutical composition comprising grazoprevir, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0132] According to some embodiments, the influenza virus M2 channel blocker comprises grazoprevir, an analog or salt thereof.

[0133] As used herein, grazoprevir includes grazoprevir (also designated as MK-5172, CAS: 135014-68-9, IUPAC: 1R,18R,20R,24S,27S)-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-vinylcyclopropyl}-7-methoxy-24-(2-methyl-2-propyl)-22,25-dioxy-2,21-dioxa-4,11,23,26-tetraazapentacyclo[24.2.1.0 3,12 .0 5,10 .018,20] nonacosa-3,5,7,9,11-pentene-27-carboxamide), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0134] According to some embodiments, the present invention provides a pharmaceutical composition comprising isavuconazole, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0135] According to some embodiments, the influenza virus M2 channel blocker comprises isavuconazole, an analog or salt thereof.

[0136] Isavuconazole as used herein includes isavuconazole or isavuconazolium sulfate (CAS: 742049-41-8, 946075-13-4 or 241479-67-4, IUPAC: 4-{2-[(1R,2R)-(2,5-difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-1,3-thiazol-4-yl}benzonitrile), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. In some embodiments, after oral or intravenous (IV) administration, isavuconazolium is hydrolyzed by esterases in the blood or gastrointestinal tract to an active form including isavuconazole.

[0137] According to some embodiments, the present invention provides a pharmaceutical composition comprising voriconazole, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza A virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0138] According to some embodiments, the influenza virus M2 channel blocker comprises voriconazole, an analog or a salt thereof.

[0139] The voriconazole used herein includes voriconazole (CAS: 137234-62-9, IUPAC: (2R, 3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0140] According to some embodiments, the present invention provides a pharmaceutical composition comprising pariprevir, an analog or salt thereof, for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza A virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0141] According to some embodiments, the influenza virus M2 channel blocker comprises pariprevir, an analog or a salt thereof.

[0142] As used herein, pariprevir includes pariprevir (also known as ABT-450, CAS: 1216941-48-8, IUPAC: (2R,6S,12Z,13aS,14aR,16aS)-N-(cyclopropylsulfonyl)-6-{[(5-methyl-2-pyrazinyl)carbonyl]amino}-5,16-dioxo-2-(6-phenanthridinyloxy)-1,2,3,6,7,8,9,10,11,13a,14,15,16,16a-tetradecahydrocyclopropyl[e]pyrrolo[1,2-a][1,4]diazacyclopentadecane-14a(5H)-carboxamide), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[0143] According to some embodiments, the present invention provides a pharmaceutical composition comprising vidarabine, a metabolite, an analog or a salt thereof for use in treating a viral infection. In some embodiments, the viral infection comprises an influenza virus infection. In some embodiments, the viral infection comprises an influenza A virus infection. In some embodiments, the influenza A virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises an aminoadamantane-resistant viral infection. In some embodiments, the viral infection comprises an infection caused by a virus that comprises the influenza virus M2 protein.

[0144] According to some embodiments, the influenza virus M2 channel blocker comprises vidarabine, a metabolite, an analog or a salt thereof.

[0145] The vidarabine used herein includes vidarabine (also known as 9-β-D-arabinofuranosyl adenine or ara-A, CAS: 24356-66-9, IUPAC: (2R, 3S, 4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- (hydroxymethyl) tetrahydrofuran-3, 4-diol hydrate), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

[0146] In some embodiments, the metabolite of vidarabine is or includes inosine arabinoside.

[0147] Pharmaceutical composition

[0148] As used herein, the terms "treatment" or "treating" a disease, disorder, or condition encompasses alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, the useful compositions herein simply reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or improve the quality of life of the patient or subject.

[0149] As used herein, the terms "administering," "administration," and like terms refer to any method, within sound medical practice, of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect.

[0150] As used herein, the term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for whom treatment is desired, particularly a mammalian subject, eg, a human.

[0151] In some embodiments, a therapeutically effective dose of the composition of the present invention is administered. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. The term "therapeutically effective amount" refers to an amount effective at the dosage and for the necessary time period to achieve the desired therapeutic or preventive effect. The exact dosage form and regimen will be determined by the physician based on the patient's condition.

[0152] The dosage will depend on the age, health and weight of the recipient, the type of simultaneous treatment (if any), the frequency of treatment, and the nature of the desired effect. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injection, for example, intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other injection known in the art. Although the bioavailability of peptides administered by other routes may be lower than that administered by parenteral injection, by using appropriate compositions, it is conceivable that the compositions of the present invention will be administered by transdermal, oral, rectal, vaginal, topical, nasal, inhalation, and ophthalmic treatments. In addition, it may be desirable to introduce the pharmaceutical composition of the present invention by any suitable route, including intraventricular and intrathecal injections; intraventricular injections can be facilitated by, for example, an intraventricular catheter attached to a reservoir.

[0153] In some embodiments, the compositions of the present invention include oral delivery. In some embodiments, the compositions of the present invention include oral compositions. In some embodiments, the compositions of the present invention further comprise an orally acceptable carrier, excipient, or diluent.

[0154] According to some embodiments, the daily dose of the influenza virus M2 channel blocker is 0.01 to 500 mg / kg.

[0155] According to some embodiments, the influenza virus M2 channel blocker comprises fludarabine or a salt thereof, and the daily dose thereof is about 0.1 mg / m 2 / day to approximately 50 mg / m 2 / day, 1mg / m 2 / day to 30 mg / kg / day and 0.1 mg / m 2 / day to 10 mg / m2 / sky.

[0156] According to some embodiments, the influenza virus M2 channel blocker comprises asunaprevir or a salt thereof at a daily dose of about 1 mg / kg / day to about 100 mg / day, about 5 mg / kg / day to 60 mg / kg / day, and 20 mg / kg / day to 50 mg / kg / day.

[0157] According to some embodiments, the influenza virus M2 channel blocker comprises ravuconazole or a salt thereof at a daily dose of about 1 mg / kg / day to about 100 mg / kg / day, about 1 mg / kg / day to 50 mg / kg / day, and 5 mg / kg / day to 30 mg / kg / day.

[0158] According to some embodiments, the influenza virus M2 channel blocker comprises amikacin or a salt thereof at a daily dose of about 1 mg / kg / day to about 100 mg / kg / day, about 5 mg / kg / day to 50 mg / kg / day, and 10 mg / kg / day to 20 mg / kg / day.

[0159] According to some embodiments, the influenza virus M2 channel blocker comprises theobromine or a salt thereof at a daily dose of about 1 mg / kg / day to about 200 mg / kg / day, about 5 mg / kg / day to 100 mg / kg / day, and 10 mg / kg / day to 50 mg / kg / day.

[0160] According to some embodiments, the influenza virus M2 channel blocker comprises flunisolide or a salt thereof at a daily dose of about 0.1 mg / kg / day to about 50 mg / kg / day, about 0.1 mg / kg / day to 20 mg / kg / day, and 0.2 mg / kg / day to 5 mg / kg / day.

[0161] According to some embodiments, the influenza virus M2 channel blocker comprises alvimopan or a salt thereof at a daily dose of about 0.1 mg / kg / day to about 50 mg / kg / day, about 0.1 mg / kg / day to 10 mg / kg / day, and 0.5 mg / kg / day to 5 mg / kg / day.

[0162] According to some embodiments, the influenza virus M2 channel blocker comprises levamlodipine or a salt thereof at a daily dose of about 0.01 mg / kg / day to about 10 mg / kg / day, 0.01 mg / kg / day to 5 mg / kg / day, and 0.01 mg / kg / day to 1 mg / kg / day.

[0163] According to some embodiments, the influenza virus M2 channel blocker comprises grazoprevir or a salt thereof at a daily dose of about 0.1 mg / kg / day to about 50 mg / kg / day, about 0.1 mg / kg / day to 10 mg / kg / day, and 0.5 mg / kg / day to 5 mg / kg / day.

[0164] According to some embodiments, the influenza virus M2 channel blocker comprises voriconazole or a salt thereof at a daily dose of about 1 mg / kg / day to about 50 mg / kg / day, about 2 mg / kg / day to 20 mg / kg / day, and 2 mg / kg / day to 10 mg / kg / day.

[0165] According to some embodiments, the influenza virus M2 channel blocker comprises pariprevir or a salt thereof at a daily dose of about 0.5 mg / kg / day to about 500 mg / day, about 1 mg / kg / day to 300 mg / day, and 0.5 mg / day to 10 mg / day.

[0166] According to some embodiments, the influenza virus M2 channel blocker comprises vidarabine or a metabolite thereof, or a salt thereof, at a daily dose of about 0.5 mg / kg / day to about 100 mg / day, about 1 mg / kg / day to 50 mg / day, and 5 mg / day to 40 mg / day.

[0167] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0168] As used herein, the terms "carrier," "adjuvant," or "excipient" refer to any ingredient in a pharmaceutical composition that is not the active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a nontoxic, inert solid, semisolid liquid filler, diluent, encapsulating material, formulation aid of any type, or a simple sterile aqueous medium such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can be used as carriers herein include sugars, starches, cellulose and its derivatives, tragacanth powder, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions described herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those skilled in the art, such as those described in The Merck Index, 13th edition, Budavari et al., Eds., Merck & Co. Inc., Rahway, NJ (2001); CTFA (Cosmetic, Toiletries, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004); and the U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management's "Inactive Ingredient Guide," all of which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents that can be used in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO.These additional inactive ingredients, as well as effective formulations and administration procedures, are known in the art and are described in standard textbooks such as Goodman and Gillman, The Pharmacological Bases of Therapeutics, 8th ed.; Gilman et al., eds., Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005), each of which is incorporated herein by reference in its entirety. The presently described compositions may also be contained in artificially constructed structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles that increase the half-life of the peptide or polypeptide in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. The liposomes used with the presently described peptides are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid generally depends on factors such as liposome size and stability in blood. A variety of methods can be used to prepare liposomes, for example, as reviewed by Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc. (New York), and also see U.S. Patents Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0169] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions described herein.

[0170] Reagent test kit

[0171] According to another aspect, a kit comprising at least two molecules is provided, the molecules being selected from the group consisting of fludarabine or a derivative thereof, asunaprevir, amikacin, a xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, and any combination thereof.

[0172] In some embodiments, the molecule is selected from the group consisting of fludarabine, asunaprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof.

[0173] In some embodiments, the kit further comprises instructions for mixing at least two molecules selected from the group consisting of fludarabine or a derivative thereof, asunaprevir, amikacin, a xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, and any combination thereof.

[0174] In some embodiments, the fludarabine derivative comprises vidarabine or a metabolite thereof.

[0175] In some embodiments, the metabolite of vidarabine is inosine arabinoside.

[0176] In some embodiments, the xanthine derivative is theobromine.

[0177] In some embodiments of the subject kits, at least two molecules are packaged within a container.

[0178] In some embodiments, the container is made of a material selected from thin-walled films or plastics (clear or opaque), cardboard, foil, rigid plastic, metal (eg, aluminum), glass, and the like.

[0179] In some embodiments, the contents of the kit are packaged, as described below, to allow storage of the components until they are needed.

[0180] In some embodiments, some or all of the components of the kit may be packaged in suitable packaging to maintain sterility.

[0181] In some embodiments of the subject kits, at least two molecules are stored in separate containers within a main kit containing member, e.g., a box or similar structure, which may or may not be an airtight container, e.g., to further maintain the sterility of some or all components of the kit.

[0182] In some embodiments, the dosages of at least two molecules provided in the kit can be sufficient for a single application or for multiple applications.

[0183] In these embodiments, the kit can have multiple doses of at least two molecules packaged in a single container, such as a single tube, bottle, vial, 1.5-2 ml tube, e.g., Eppendorf, or the like.

[0184] In some embodiments, the kits can have multiple doses of at least two molecules packaged separately, such that certain kits can have more than one container of at least two molecules.

[0185] In some embodiments, multiple doses of at least two molecules can be packaged in a single individual container.

[0186] In some embodiments, the kits contain instructions for preparing the compositions for use therein and how to practice the methods of the invention.

[0187] In some embodiments, the kit further comprises a measuring device, such as a syringe, a measuring spoon, or a measuring cup.

[0188] In some embodiments, the instructions can be recorded on a suitable recording medium or substrate. For example, the instructions can be printed on a substrate such as paper or plastic.

[0189] In some embodiments, the instructions may be present in the kit as a package insert, a container label for the kit or its components (i.e., associated with a package or subpackage), or the like. In other embodiments, the instructions are present in the form of an electronically stored data file on a suitable computer-readable storage medium, such as a CD-ROM, floppy disk, or the like. In other embodiments, the actual instructions are not present in the kit, but a method for obtaining the instructions from a remote source (e.g., via the Internet) is provided. An example of this embodiment is a kit that includes a website address where the instructions can be viewed and / or downloaded. Like the instructions, this method for obtaining the instructions is recorded on a suitable substrate.

[0190] Screening test

[0191] According to another aspect, a method for screening an agent for effectiveness in treating or preventing influenza virus infection is provided. According to some embodiments, the method comprises providing a cell comprising a membrane permeable to the influenza virus M2 channel, contacting the cell with an agent, and determining the effect of the agent on cell growth, wherein a substantial effect of the agent on cell growth indicates that the agent is effective in treating or preventing influenza virus infection, thereby screening the agent for effectiveness in treating or preventing influenza virus infection. In some embodiments, the influenza virus comprises influenza A virus. In some embodiments, the influenza A virus comprises the H1N1 subtype. In some embodiments, the influenza virus is resistant to aminoadamantane.

[0192] In some embodiments, the method comprises a negative assay. In some embodiments, the cell is characterized by growth retardation caused by membrane permeation through the influenza virus M2 channel. In some embodiments, the agent that alleviates growth retardation is shown to be effective in treating or preventing influenza virus infection.

[0193] In some embodiments, the method comprises a positive assay. In some embodiments, the cell is a K + Uptake-deficient cells, which cannot + ] medium, but undergo growth due to the channels formed by the influenza virus M2 channel. In some embodiments, the agent that induces growth retardation is shown to be effective in treating or preventing influenza virus infection.

[0194] In some embodiments, the method comprises performing a negative assay and a positive assay.

[0195] In some embodiments, the method further comprises an acidity measurement. In some embodiments, the cell is a cell comprising a pH reporter gene or its protein product, such as but not limited to pH-sensitive green fluorescent protein (GFP). In some embodiments, the induction of H + into cells grown in a culture medium supplemented with an acidic solution. In some embodiments, agents that block changes in intracellular pH are useful in treating or preventing influenza virus infection.

[0196] In some embodiments, the method further comprises a validation step comprising testing the molecule for positive passage of all three bacterial assays in a mammalian cell-based assay. In some embodiments, the validation step entails infecting mammalian cells with influenza A virus and testing the effect of the molecule on cell viability and / or growth.

[0197] Non-limiting examples of bacterial cell growth suitable for the screening methods provided herein include: Astrahan, P. et al., Acta 1808, 394-8 (2011); Santner, P. et al., Biochemistry 57, 5949-5956 (2018), and Taube, R., Alhadeff, R., Assa, D., Krugliak, M. and Arkin, ITPLoS One 9, e105387 (2014).

[0198] In some embodiments, the assay comprises determining the susceptibility of the virus to developing resistance to an agent.

[0199] As used herein, the term "about" in conjunction with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0200] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this recitation is intended to serve as a basis for incorporating such exclusive terminology as "solely," "only," and the like when reciting claim elements or using a "negative" limitation.

[0201] Where a convention similar to "at least one of A, B, and C, etc." is used, generally, the meaning of such grammatical construction is what one of ordinary skill in the art understands the meaning of the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One of ordinary skill in the art would further understand that virtually any conjunction and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one, either, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0202] It should be understood that certain features of the invention that, for the sake of clarity, are described in the context of different embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that, for the sake of brevity, are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the invention are specifically contemplated by the invention and are disclosed herein, just as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of various embodiments and elements thereof are specifically contemplated by the invention and are disclosed herein, just as if each and every such subcombination were individually and expressly disclosed herein.

[0203] By examining the following examples, those skilled in the art will understand additional objects, advantages and novel features of the present invention, which are not intended to be limiting. In addition, various embodiments and aspects of the present invention as described above and as claimed in the claims are experimentally supported in the following examples.

[0204] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section herein find experimental support in the following examples.

[0205] Example

[0206] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are fully explained in the literature. For example, see "Molecular Cloning: A Laboratory Manual", Sambrook et al. (1989); "Current Protocols in Molecular Biology", Ausubel Vols. I-III, RM ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (ed.) "Genome Analysis: A Laboratory Manual" Series (Genome Analysis: A Laboratory Handbook Series), Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methods as described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; Cell Biology: A Laboratory Handbook, Vols. I-III, Cellis, JE ed. (1994); Freshney, Culture of Animal Cells—A Manual of Basic Technique, Wiley-Liss, New York (1994), 3rd ed.; Coligan, JE ed., Current Protocols in Immunology, Vols. I-III, ed.Editors (1994); Stites et al. (eds.), "Basic and Clinical Immunology" (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Strategies for Protein Purification and Characterization—A Laboratory Course Manual," CSHL Press (1996); all of which are incorporated herein by reference. Other general references are provided throughout this document.

[0207] Materials and methods

[0208] Bacteria-based channel detection

[0209] Three bacterial-based channel assays were used to study influenza channel activity and its blockers. In all assays, an MBP (maltose binding protein) fusion purification system (New England Biolabs; Ipswich, Massachusetts, USA) was used, in which the M2 channel from an aminoadamantane-resistant H1N1 strain was expressed as a chimera by being fused to the carboxyl terminus of maltose binding protein. This system ensures that the protein targets the bacterial inner membrane and has been successfully used to express and verify many other viral ion channels. The viral strains used by the inventors were H1N1 strains known to be resistant to aminoadamantane, due to a mutation of Ser31 to Asn in their M2 protein (Hay AJ et al., "The molecular basis of the specific anti-Influenza Action of amantadine", EMBO J 1985; 4: 3021-3024, hereby cited in full), as well as H1N1 strains sensitive to aminoadamantane.

[0210] Negative test

[0211] Bacterial cultures (DH10B) were grown overnight, diluted 500-fold, and subcultured until their OD600 reached 0.2. 50 μl of culture was added to 96-well flat-bottom plates, which were pretreated with 50 μl of the specific chemical and isopropyl-β-d-1-thiogalactopyranoside (IPTG) inducer. Induction was achieved by using different concentrations of IPTG. The plates were incubated in a multiplate incubator (infinite M200 pro, Tecan Group; Switzerland). Plates were incubated at 37°C for 16 hours with a constant shaking rate (700 rpm) using a 1% OD600 microplate reader (or BioTek LogPhase 600; Santa Clara, CA, USA). Bacterial growth was monitored by measuring OD600 every 15 minutes. Each measurement was performed in duplicate or three times.

[0212] Positive test

[0213] A positive test follows the same protocol as a negative test, but in this case, K + - Uptake-deficient bacterial strains. In addition, overnight growth was performed in LB medium in which KCl replaced NaCl at various concentrations as indicated.

[0214] Acidity test

[0215] The acidity assay is based on bacteria expressing a chromosomal copy of a pH-sensitive GFP. Overnight bacterial cultures were diluted 1:500 in LB medium and subsequently grown to an OD600 of 0.6–0.8. Protein expression was induced with IPTG at various concentrations as described above. After one hour of induction, cells were diluted to an OD600 of 0.2 and pelleted at 3500 g for 10 min. Cells were then resuspended in McILvaine buffer containing 200 mM Na2HPO4 and 0.9% NaCl, adjusted to pH 7.6 with 0.1 M citric acid. 200 μl of the cell suspension was added to a 96-well plate (Nunclon f96 Microwell Black Polystyrene, Thermo Fisher Scientific; Waltham, MA, USA), containing 30 μl of McILvaine buffer per well. The plate contained three wells containing McILvaine buffer and three wells of uninduced cultures as controls. Fluorescence measurements were performed at ambient temperature in a microplate reader (Infinite F200 Pro, Tecan Group; Switzerland ) with emission fixed at 520 nm and excitation alternating between 390 and 466 nm. At the start, 70 μl of 300 mM citric acid was added to the bacterial culture, and fluorescence readings were taken for 30 seconds at each wavelength. Finally, proton concentration was calculated based on the ratio of the two excitation wavelengths.

[0216] Chemical screening

[0217] The chemical library was purchased from MedChem Express (HY-L035, Monmouth Junction, NJ, USA). At the time, the library contained 2839 repurposed drugs, with the notation that the number of chemicals would change over time. Each chemical was tested at a final concentration of 100 μM. The final concentration of dimethyl sulfoxide was 2%. All manipulations and growth were performed on a robotic system (EVO 75 Tecan, The results were performed on a LogPhase 600 microbial reader (Agilent, Santa Clara, CA, USA).

[0218] For each growth test, two metrics were measured: maximum growth rate and final bacterial density. However, in practice, due to spurious factors that can affect the above metrics, such as compound absorbance, solubility, etc., visual inspection is far superior in identifying individual hits.

[0219] Chemicals

[0220] Isopropyl-β-d-1-thiogalactopyranoside (IPTG) was purchased from Biochemika-Fluka (Buchs, Switzerland). All other chemicals were purchased from Sigma-Aldrich Laboratories (Rehovot, Israel).

[0221] Bacterial growth medium

[0222] Lysogenic medium (LB) was used in most cases, with the exception of LBK for positive detection, where NaCl was replaced with 10 mg / l KCl. All media contained 100 g / ml ampicillin.

[0223] The antiviral activity of the compounds was evaluated in a BSL-3 facility at the Hebrew University as follows: Madin-Darwin canine kidney (MDCK) [ATCC MDCK NBL-2] cells were maintained in Dulbecco's modified Eagle's medium (DMEM); (Biological Industries; Beit Haemek, Israel), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 IU / mL penicillin, and 10 μg / mL streptomycin and chlortetracycline-3, (Biological Industries; Beit Haemek, Israel). Influenza A virus A / Wisconsin / 629-D02452 / 2009 (H1N1) pdm09 was stored by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, and NIH. Identical virus stock aliquots were prepared from mother solutions provided by BEI Resources. A 1:1000 dilution of a substock was prepared from each aliquot for infection. Subsequent infection of MDCK cells was performed in MEM (Biological Industries; Beit Haemek, Israel) containing 0.3% bovine serum albumin (Sigma: A4503, batch number: SLCK2178) and 3 μg / ml TPCK-treated trypsin (Sigma, T8802) and further incubated at 35°C for 48h in a 5% CO2 atmosphere. All infection experiments were performed in a BSL-3 facility. 10mM stocks of the listed compounds were prepared in DMSO and stored in aliquots at -80°C until further use. MDCK cells were seeded in 200 μL of culture medium on a 96-well flat-bottom plate at a density of 15,000 cells per well and grown overnight. Dilutions of the test compounds were prepared in MEM with 0.3% BSA, 3 μg / ml TPCK-treated trypsin, and 50 μL were added to the cells. The effects of drugs on the metabolic activity of MDCK cells were assessed 48 h after treatment using CellTiter 96 aqueous non-radioactive cell proliferation reagent (Promega; Madison, WI, USA). To examine the effects of different drugs, cells were infected with influenza A virus at 400 TCID50 / well for two hours and then treated with the listed drugs. Each compound concentration was tested in triplicate, and each assay plate contained the following controls: no cells (background control), cells treated with culture medium (mock infection for normalization), infected / untreated cells, and infected / solvent-treated cells (infection control).Two days after infection, the efficacy of the drugs in controlling toxicity was assessed using CellTiter 96 aqueous non-radioactive cell proliferation reagent (Promega; Madison, WI, USA) at 37°C in a 5% CO2 atmosphere for 3 h. The reaction was stopped and 30 μl of 4% formaldehyde was added to inactivate the virus. The cells were analyzed using a Tecan plate reader (. The absorbance was measured at 492 nm by using the Monod (Switzerland) method. Finally, the data were normalized to the mock-infected control, and the EC was calculated by fitting the data to the Monod equation. 50 value.

[0224] Animal studies

[0225] Mice were infected with the H1N1 virus and given oral treatment (BID) for 5 days. After 9 days, the amount of virus in the lungs was quantified by q-RT PCR. BALB / c mice were selected for animal experiments. The inventors first conducted tolerability and pharmacokinetic studies on the corresponding drugs, with 3 mice in each group, for a total of 5 groups. The drug combinations (1:1 molar ratio) in each group were as follows: (i) 1.5 mg / kg inosine and 1 mg / kg theobromine; (ii) 4.5 mg / kg inosine and 3 mg / kg theobromine; (iii) 15 mg / kg inosine and 10 mg / kg theobromine; (iv) 45 mg / kg inosine and 30 mg / kg theobromine, and (v) 150 mg / kg inosine and 100 mg / kg theobromine. For the efficacy study, a total of 56 BALB / c mice were divided into 7 groups, so each group contained 8 mice for this group of experiments. The seven groups were treated as follows: (i) vehicle (control); (ii) 20 mg / kg oseltamivir; (iii) 1.5 mg / kg inosine and 1 mg / kg theobromine; (iv) 4.5 mg / kg inosine and 3 mg / kg theobromine; (v) 15 mg / kg inosine and 10 mg / kg theobromine; (vi) 45 mg / kg inosine and 30 mg / kg theobromine; and (vii) 150 mg / kg inosine and 100 mg / kg theobromine. Each group was first infected with influenza virus via intranasal spray (50 μl) at approximately 1,000 viruses per animal. All combination drugs were dissolved in a 1% aqueous solution of Emulfor-EL-620 at the desired concentrations, and oseltamivir was dissolved in water. Thus, the vehicle consisted solely of a 1% aqueous solution of Emulfor-EL-620. Animals were treated by oral gavage at 10 ml / kg twice daily for five days. The body weight of each group was monitored daily, and animals were sacrificed after 9 days and lungs were collected for further RNA quantification by q-RTPCR.

[0226] Example 1

[0227] Bacteria-based assays for the assessment of ion channel activity

[0228] To assess the activity of aminoadamantane against H1N1 strains, the inventors used a bacteria-based assay in which the function of the channel alters the bacterial phenotype. The advantages of such assays are that they are amenable to high-throughput screening, and the ease of genetic manipulation in bacteria enables rapid transition from one sequence / variant to another. Finally, these assays have been tested on a variety of viroporins from various viruses (Assa, D. et al. J Mol Biol 2016, 428, 4209-4217, Astrahan, P. et al. 2011, 1808, 394-398, Taube, R, PLoS One 2014, 9, e105387, Tomar, PPS et al. Viruses 2019, 11, Tomar, PPS et al. Viruses 2021, 13, Tomar, PPS et al. Pharmaceuticals (Basel) 2021, 14, Tomar, PPS et al. Krugliak, M.; Singh, A.; Arkin, ITBiomedicines 2022).

[0229] Negative test

[0230] The primary assay employed involves elevated expression levels of viroporins in "normal" E. coli. At certain viroporin concentrations, growth arrest is observed due to excessive membrane permeability that hinders bacterial bioenergetics. Therefore, the test is, as the title suggests, a negative test due to the protein's deleterious effects on the bacteria.

[0231] Positive test

[0232] The second test requires + - The viral channel is expressed at low levels in bacteria with potassium uptake defects. + In this case, the viral passage has a favorable effect on the bacteria and the test is therefore called a positive test. It should be noted that at high induction levels, the K + While short, the channel negatively impacts the bacteria due to excessive membrane permeation similar to the negative assay described above.

[0233] Acidity test

[0234] The final test to verify channel activity is based on the effect of the channel on the bacterial cytoplasmic pH. When concentrated acid is injected into the culture medium, if the bacteria express a protein that can transport H +The cytoplasmic pH decreases when the cell is cleaved. This change in cytoplasmic pH can then be detected by monitoring the fluorescence of a pH-sensitive GFP expressed on the chromosome.

[0235] Negative, positive and acidity test results are as follows Figure 1 、 2 M2 channel activity was confirmed in all validated assays as shown in Figures 3 and 4. In all figures, protein expression levels, i.e., M2 channel levels, were controlled by the levels of isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0236] Blocker screening

[0237] After confirming the channel activity of the M2 channel, the inventors sought to identify drugs that could block its function. The screening was performed in three stages. First, all compounds were screened in a negative assay. Each plate had two controls: the positive control was bacteria without IPTG, i.e., bacteria without channel induction. Blank DMSO was added as a negative control. Subsequently, bacteria with growth enhancement exceeding an empirical threshold were rechecked in triplicate. Each compound that passed this assay was then checked in triplicate in a positive test. Finally, compounds that passed both the positive and negative assays were subjected to a dose-response analysis with two replicates and a pH assay with four replicates. Finally, an example of a compound screening assay is shown in FIG. Figure 4-6 “No channel” refers to the absence of IPTG induction of M2 channels, while “No drug” refers to the maximum induction of M2 channel protein by IPTG in the absence of drugs. The IPTG concentrations in the negative, positive, and acidity assays were 100 μM, 20 μM, and 50 μM, respectively. Figure 4-6 ).

[0238] like Figure 4-6 As indicated, compounds that showed activity in one or more of the assays are listed below: amantadine*, fludarabine, asunaprevir, ravuconazole, amikacin, theobromine, flunisolide, alvimopan, eliglukast, CM4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, vidarabine, and inosine.

[0239] *Amantadine is a known channel blocker of the M2 channel, so its identification in the screen was expected (Pinto, LH et al., "Influenza virus M2 protein has ion channel activity." Cell 1992; 69: 517-528, incorporated herein by reference in its entirety).

[0240] Example 2

[0241] In vitro and in vivo analysis of mammalian systems

[0242] Tissue culture research

[0243] The inventors next sought to characterize the antiviral activity of the channel blockers identified in tissue culture cells. To this end, the inventors cultured Madin-Darwin canine kidney (MDCK) cells and examined their survival after infection with an aminoadamantane-resistant H1N1 virus, as well as the ability of the drugs to affect cell survival. The viral strain used was H1N1, which is known to be resistant to aminoadamantane due to a mutation at Ser31Asn in its M2 protein.

[0244] Compounds active in tissue culture

[0245] First, two concentrations of drugs were used: 10 μM, e.g. Figure 7 as shown, and the latter at 3 μM, as Figure 8 In both experiments, two available anti-influenza drugs were used as positive controls: oseltamivir and favipiravir

[0246] The results showed that the following drugs exhibited considerable anti-influenza activity at 10 μM in tissue culture: fludarabine, asunaprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, and vidarabine.

[0247] The following compounds were active at 3 μM: fludarabine, asunaprevir, theobromine, flunisolide, grazoprevir, voriconazole, and vidarabine.

[0248] Finally, as expected, and showed strong anti-influenza activity (and therefore served as a positive control), which was also satisfactory because the infection was performed with an H1N1 virus strain resistant to aminoadamantane. Completely ineffective (thus serving as a negative control).

[0249] The inventors then performed dose-response analyses to determine the ability of each active compound to inhibit the virus at different concentrations. The results of these analyses are shown in Table 1. Figure 9-12 The shaded area in the graph represents the solvent level. Figure 13 and 14 The EC of each compound can be found in 50 The fitting value.

[0250] After affinity analysis for each drug, the inventors looked for potential additive and synergistic effects between the positive results. The results of the combination experiments of each drug in combination with another drug at a concentration of 0.01 μM are shown in Figure 2. Figure 15 As shown in Table 1, theobromine + vidarabine and vidarabine + grazoprevir exhibited significant synergistic effects. Other combinations exhibited additive or no effects, as shown in Table 1. The shaded areas represent individual compounds.

[0251] Combination experiments were performed at lower drug concentrations. Figure 16 As can be seen in Figure 3, the combination of theobromine and vidarabine was particularly effective at 30 nm, providing complete protection against virus-induced cell death.

[0252] Table 1. Summary of the combination effects between positive results

[0253] Grazoprevir Theobromine Adenosine Flunisolide Fludarabine Asunaprevir Grazoprevir 40％ 56％ 106％ 57％ 37％ 32％ Theobromine 56％ -6％ 101％ 22％ 43％ 46％ Adenosine 106％ 101％ -3％ 37％ 41％ 21％ Flunisolide 57％ 22％ 37％ 31％ 15％ -3％ Fludarabine 37％ 43％ 41％ 15％ 34％ -3％ Asunaprevir 32％ 46％ 21％ -3％ -3％ 33％

[0254] As described above, additional mice were infected with H1N1 virus and given oral treatment (BID) for 5 days. After 9 days, the amount of virus in the lungs was quantified by q-RT PCR. The results showed that the drug combination disclosed herein, including theobromine + vidarabine, was more effective than the leading drug on the market, oseltamivir, despite being administered at a significantly lower dose. Figure 17 ).

[0255] In addition, the inventors conducted structure-activity relationship (SAR) analysis to examine the effects of xanthine and its derivatives on cell viability ( Figure 18 ). The results showed that several closely related xanthine derivatives increased cell survival ( Figure 19 ). In this regard, theobromine was found to increase cell survival to a great extent at concentrations of 0.3μM-10μM compared to the control group. Surprisingly, caffeine, which is very similar to theobromine, did not increase cell survival compared to the no-drug control. Similar trends were observed for enprophylline and paraxanthine. Theophylline and 3-methylxanthine were found to increase cell survival to a great extent at any tested concentration (0.01μM-1μM) compared to the control group. 7-methylxanthine was found to increase cell survival to a great extent at concentrations of 1μM-10μM compared to the control group.

[0256] In addition, the inventors performed SAR analysis to examine the effects of fludarabine and its derivatives on cell viability ( Figure 22 The results showed that fludarabine and several of its closely related derivatives increased cell survival compared with the control group ( Figure 22In this regard, it was found that vidarabine at a concentration of 0.3 μM-10 μM could largely improve cell survival compared to the control group. Nelarabine and cordycepin increased cell survival at concentrations of 10 μM and 1 μM-10 μM, respectively.

[0257] In addition, the inventors conducted a combination experiment using xanthine or its other closely related compounds (having a xanthine skeleton) with the antiviral drug adenosine or its natural metabolite inosine arabinoside ( Figure 20 ). The result is as follows Figure 21 shown.

[0258] Briefly, a combination comprising vidarabine at a concentration of 300 nM and 1-methylxanthine at concentrations ranging from 10 nM to 300 nM increased cell viability by approximately 33-37% compared to a negative control (no drug).

[0259] The combination including vidarabine at a concentration of 300 nM and xanthine at a concentration of 10 nM to 300 nM increased the cell survival rate by approximately 27-48% compared to the control group.

[0260] The combination comprising vidarabine at a concentration of 10 nM or 100 mM and 3-methylxanthine at a concentration of 30 nM increased cell survival by approximately 63% and 28%, respectively, compared to the control group. The combination comprising vidarabine at a concentration of 30 nM or 100 mM and 3-methylxanthine at a concentration of 100 nM increased cell survival by approximately 63% and 46%, respectively, compared to the control group.

[0261] The combination of vidarabine and theobromine had a significant positive effect on cell survival. Combinations of vidarabine and theobromine at any tested concentration (e.g., 10 nM to 300 mM of each compound) increased cell survival by approximately 37% to 98% compared to the control group. In particular, combinations of vidarabine at concentrations of 10 nM to 300 mM and theobromine at concentrations of 30 nM to 300 nM increased cell survival by approximately 64% to 98% compared to the control group.

[0262] In addition, the inventors examined the effects of theobromine and inosine (a natural metabolite of adenosine) on cell viability. The results showed that, compared to the control group, a combination of inosine at a concentration of 100 nM or 300 mM and theobromine at a concentration of 10 nM increased cell viability by approximately 10% to 75%. In addition, a combination of inosine at a concentration of 10 nM to 300 mM and theobromine at a concentration of 30 nM to 300 nM increased cell viability by approximately 61% to 100% compared to the control group.

[0263] Although the present invention has been described in conjunction with the specific embodiments of the present invention, it will be apparent to those skilled in the art that many substitutions, modifications and variations are apparent. Therefore, the present invention is intended to cover all such substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A method for treating or preventing influenza A virus in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule selected from the group consisting of theobromine, vidarabine, inosine arabinoside, fludarabine, asunaprevir, ravuconazole, amikacin, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, and any combination thereof, thereby treating or preventing influenza A virulence in the subject.

2. The method of claim 1, wherein the molecule is selected from the group consisting of theobromine, vidarabine, inosine arabinoside, fludarabine, asunaprevir, amikacin, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, pariprevir, and any combination thereof.

3. The method of claim 1 or 2, wherein the molecule is an M2 protein blocker.

4. The method according to any one of claims 1 to 3, wherein the influenza A virus is of the H1N1 subtype.

5. The method according to any one of claims 1 to 4, wherein the influenza A virus is resistant to aminoadamantane.

6. The method of any one of claims 1 to 5, wherein the molecule is administered at a daily dose of 0.01 to 500 mg / kg body weight of the subject.

7. The method according to any one of claims 1 to 6, wherein the administration is a therapeutically effective amount of two molecules selected from the group consisting of theobromine, vidarabine, inosine arabinoside, fludarabine, asunaprevir, amikacin, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, paliprevir, and any combination thereof.

8. The method of claim 7, wherein the two molecules are vidarabine or inosine arabinoside and a molecule selected from the group consisting of theobromine and grazoprevir.

9. The method of claim 7, wherein the two molecules are vidarabine or inosine arabinoside and theobromine.

10. A pharmaceutical composition comprising a molecule for use in treating or preventing influenza A virulence in a subject in need thereof, wherein the molecule is selected from the group consisting of theobromine, vidarabine, inosine arabinoside, fludarabine, asunaprevir, ravuconazole, amikacin, flunisolide, alvimopan, eliglukast, Cm4620, levamlodipine, emamectin, grazoprevir, isavuconazole, voriconazole, paliprevir, and any combination thereof.

11. The pharmaceutical composition for use according to claim 10, wherein the molecule is selected from the group consisting of theobromine, vidarabine, inosine arabinoside, fludarabine, asunaprevir, amikacin, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, pariprevir, and any combination thereof.

12. The pharmaceutical composition for use according to claim 10 or 11, wherein the molecule is an M2 protein blocker.

13. The pharmaceutical composition for use according to any one of claims 10 to 12, wherein the influenza A virus is of the H1N1 subtype.

14. The pharmaceutical composition for use according to any one of claims 10 to 13, wherein the influenza A virus is resistant to aminoadamantane.

15. The pharmaceutical composition for use according to any one of claims 10 to 14, wherein the molecule comprises vidarabine or inosine arabinoside and theobromine or grazoprevir.

16. A pharmaceutical composition for use according to any one of claims 10 to 15, wherein the molecule comprises vidarabine or inosine arabinoside and theobromine.

17. A combination for use in treating or preventing influenza A virulence in a subject in need thereof, wherein the combination comprises at least two molecules selected from the group consisting of theobromine, vidarabine, inosine arabinoside, fludarabine, asunaprevir, amikacin, flunisolide, alvimopan, levamlodipine, grazoprevir, voriconazole, and paliprevir.

18. The combination for use according to claim 17, wherein the at least two molecules are (i) vidarabine and (ii) a molecule selected from the group consisting of theobromine and grazoprevir.

19. The combination for use according to claim 17 or 18, wherein the at least two molecules are (i) vidarabine or inosine arabinoside and (ii) theobromine.

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