CN120603606A

CN120603606A - Viral protein blockers/inhibitors as anti-influenza agents

Info

Publication number: CN120603606A
Application number: CN202380092555.0A
Authority: CN
Inventors: I·阿尔金; H·拉希里
Original assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2022-12-14
Filing date: 2023-12-14
Publication date: 2025-09-05
Also published as: AU2023393490A1; KR20250148563A; WO2024127407A1; EP4633677A1; IL321421A

Abstract

Provided are pharmaceutical compositions comprising an influenza A M2 protein channel blocker for use in treating or preventing the virulence of influenza A (e.g., H1N1 subtype) that is resistant to aminoadamantine in a subject. Further provided are pharmaceutical compositions comprising an influenza A M2 protein channel blocker for use in preventing influenza A cell entry, uncoating, and/or release from cells.

Description

Viral protein blockers/inhibitors as anti-influenza agents

Reference to electronic sequence Listing

The entire contents of the electronic sequence list (VRB-P-004-PCT ST26.Xml; size: 2035 bytes; and date of creation: 2023, 12, 6) are incorporated herein by reference.

Cross Reference to Related Applications

The present application claims the benefit of priority from U.S. provisional patent application No. 63/432,446, entitled "VIROPORINS BLOCKERS/INHIBITORS AS ANTI-INFLUENZAAGENTS (as a blocker/inhibitor of viral proteins against influenza agents)" filed on 12 months 14 of 2022, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention belongs to the field of antiviral treatment.

Background

Influenza is a segmented single stranded negative sense RNA virus, which belongs to the orthomyxoviridae family. It is one of the leading causes of morbidity and mortality from infectious diseases. In particular, it was the leading cause of infectious death in the western world, before COVID-19. Influenza is effectively combated by vaccine and antiviral therapies. However, continued gene transfer and drift continuously impair vaccine effectiveness and antiviral drug benefits. For example, the annual vaccine protection rate is about 40%, and resistance to each anti-influenza drug has been reported. In particular, 98% of the currently popular varieties are p-aminoadamantanes @And) Aminoadamantanes are the first anti-influenza antiviral agent developed in the 60 s of the 20 th century (Davies, W.L. et al, "ANTIVIRAL ACTIVITY of 1-ADAMANTANAMINE (AMANTADINE)." Science1964;144:862-863, the entire contents of which are incorporated herein by reference).

There is an unmet need for methods and compounds for improving or treating influenza viruses, particularly influenza variants that are resistant to aminoadamantanes.

Disclosure of Invention

According to a first aspect there is provided a method of 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 fludarabine, asuprevir, raffmonazole (Ravuconzole), amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glaconvir, isaconazol, voriconazole, palipervir, vidarabine or a metabolite thereof, and any combination thereof, thereby treating or preventing influenza a virulence in the subject.

According to another aspect, there is provided a pharmaceutical composition comprising a molecule for use in the treatment or prevention of influenza a virulence in a subject in need thereof, wherein the molecule is selected from the group consisting of fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatiravir, isaconazol, voriconazole, paliprevir, vidarabine or a metabolite thereof, and any combination thereof.

According to another aspect, a kit is provided comprising at least two molecules selected from the group consisting of fludarabine or a derivative thereof, asuprevir, amikacin, xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, glatirevir, voriconazole, paliprovir, and any combination thereof.

In some embodiments, the molecule is selected from the group consisting of fludarabine, asuprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, glatirivir, voriconazole, peramivir, vidarabine, or a metabolite thereof, and any combination thereof.

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, asuprevir, amikacin, xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, glatiramivir, voriconazole, paliprovir, and any combination thereof.

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

In some embodiments, the metabolite of arabinoside is arabininosine (Arainosine).

In some embodiments, the xanthine derivative is theobromine.

In some embodiments, the kit is used to prepare a medicament for treating a subject suffering from influenza or infected with influenza virus.

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

In some embodiments, the influenza a virus is an H1N1 subtype.

In some embodiments, the influenza a virus is resistant to aminoadamantane.

In some embodiments, the molecule is used at a daily dose of 0.01 to 500mg/kg of body weight of the subject.

In some embodiments, the administration is a therapeutically effective amount of 2 molecules selected from the group consisting of fludarabine, asuprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, glatiramivir, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof.

In some embodiments, the 2 molecules are arabinoside or a metabolite thereof, and a molecule selected from the group consisting of theobromine and glatiramir.

According to another aspect, there is provided a combination for use in the treatment or prevention of influenza a virulence in a subject in need thereof, wherein the combination comprises at least two molecules selected from the group consisting of fludarabine, asuprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, glatiramivir, voriconazole, paliprovir, vidarabine or a metabolite thereof, and any combination thereof.

In some embodiments, the at least two molecules are (i) vidarabine or a metabolite thereof, and (ii) a molecule selected from theobromine and glatirivir.

In some embodiments, the metabolite of arabinoside is or includes arabininosine.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those 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 case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Further embodiments and full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

FIG. 1 includes a graph demonstrating negative detection of influenza M2 virus porins, where protein expression is detrimental to bacterial growth. Growth of DH10B bacteria varied with varying concentrations of the protein inducer isopropyl-beta-D-thiogalactoside (IPTG).

Figure 2 includes a vertical bar graph demonstrating positive detection of influenza M2 virus porin. In the presence (10. Mu.M) or absence of the inducer IPTG, the maximum growth rate of K ⁺ uptake-deficient bacteria expressing influenza M2 virus porins was monitored as a function of different K ⁺ concentrations.

Figure 3 includes a graph demonstrating the acidity detection of influenza M2 virus porins. The change in the concentration of cytoplasmic H ⁺ was monitored over time, and at time 0, an acidic solution was injected into the medium. The results shown are from bacteria, where the viral porin induction varies with IPTG concentration as shown.

Fig. 4 includes graphs showing the results of negative assays for influenza M2 virus porin blocker screening. Bacteria that did not induce channel expression ("no channel") or that did not add drug ("no drug") were used as controls.

Fig. 5 includes graphs showing the results of a positive assay for influenza M2 virus porin blocker screening. Bacteria that did not induce channel expression ("no channel") or that did not add drug ("no drug") were used as controls.

Figure 6 includes a graph demonstrating the acidity detection of influenza M2 virus porins performed in the presence of a test drug.

Fig. 7 includes vertical bar graphs showing cell viability in the presence of various drugs at a concentration of 10 μm (unless otherwise indicated). The dashed line indicates the viability of cells in the "vehicle" treatment.

Fig. 8 includes vertical bar graphs showing cell viability in the presence of various drugs at a concentration of 3 μm (unless otherwise indicated). The dashed line indicates the viability of the cells in the "vehicle" treatment.

Fig. 9 includes vertical bar graphs showing that the effect of test drug on cell viability is dose dependent. The dashed line indicates the viability of the cells in the "vehicle" treatment.

Fig. 10 includes vertical bar graphs showing that the effect of test drug on cell viability is dose dependent. The dashed line indicates the viability of the cells in the "vehicle" treatment.

Fig. 11 includes vertical bar graphs showing that the effect of test drug on cell viability is dose dependent. The dashed line indicates the viability of the cells in the "vehicle" treatment.

Fig. 12 includes vertical bar graphs showing that the effect of test drug on cell viability is dose dependent. The dashed line indicates the viability of the cells in the "vehicle" treatment.

Fig. 13 includes graphs showing half maximal effective concentration (EC 50) values of test compounds.

Figure 14 includes a graph showing half maximal effective concentration (EC 50) values of test compounds.

Fig. 15 includes graphs showing drug synergy. All drugs were administered at a concentration of 0.01 μm. The dashed line indicates the viability of the cells in the "vehicle" treatment.

Fig. 16 includes vertical bar graphs showing relative cell viability after 48 hours of infection in the presence of various drugs or combinations thereof. The combination of theobromine and ara-adenosine was observed to synergistically increase cell survival. Furthermore, the effect of the combination of theobromine and ara-adenosin is even more pronounced than the drug oseltamivir and fapirat Wei Geng obtained in the batch.

Fig. 17 includes vertical bar graphs showing pulmonary viral RNA (%) measured after in vivo studies in mice treated with the indicated drugs. The combination of theobromine and inosine was observed to be more effective in vivo than the leading drug oseltamivir on the market, even though it was administered at significantly lower doses.

Fig. 18 includes chemical structures of xanthines and other closely related compounds having a xanthine backbone and further modifications. The modified position is indicated by a dashed line.

Fig. 19 includes graphs showing structure-activity relationship (SAR) analysis of xanthine derivatives. Cell viability was determined as described. Higher values indicate better protection against virus-induced cell death. As shown in the top panel, different xanthine derivatives include modifications at the position of xanthine R ₁-R₃.

Fig. 20 includes chemical structures of xanthines and other closely related compounds with xanthine backbones and further modifications (top row). The modified position is indicated by a dashed line. Further presented is the antiviral drug arabinoside and the natural metabolite arabininosine (bottom row).

Figure 21 includes a table summarizing combination therapies with the indicated drugs. Higher values indicate better protection against virus-induced cell death. As presented, the compounds are used in a variety of concentrations.

Fig. 22 includes graphs 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 panel, different fludarabine derivatives include modifications at the R ₁-R₅ position of fludarabine.

Detailed Description

In some embodiments, the invention provides compositions comprising a matrix protein 2 or M2 protein channel blocker for treating or preventing influenza virulence in a subject. In some embodiments, the invention provides compositions comprising influenza M2 protein channel blockers for preventing entry, uncoating, and/or release of influenza cells from the cells. As used herein, the terms "matrix protein 2" and "M2 protein" may be used interchangeably.

In some embodiments, the invention is based on the discovery that at least one molecule selected from the group consisting of fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatirevir, isaconazole, voriconazole, palependvir, vidarabine, or a metabolite thereof, and any combination thereof, inhibits the M2 channel of influenza a and is therefore useful in the treatment and/or prevention of 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 aminoadamantane.

In some embodiments, at least one molecule selected from the group consisting of fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatiramivir, isaconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, may be used to treat and/or prevent influenza a virulence.

M2 protein of influenza virus

The M2 protein of influenza virus is a transmembrane protein, known to be present in all influenza a types. 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 equilibrate the pH across the membrane of influenza virus during cell entry and across the golgi membrane of the infected cell during viral maturation. In some embodiments, M2 is critical for viral replication. In some embodiments, M2 is a target for anti-influenza drugs (e.g., amantadine and rimantadine). In some embodiments, the M2 protein belongs to the class of viral porins (e.g., ion channel forming small proteins that increase membrane permeability of virus infected cells). In some embodiments, M2 of influenza a (also referred to as AM 2) and M2 of influenza b (also referred to as BM 2) are predominantly proton channels. As used herein, the terms "M2 of influenza a" and "AM2" may be used interchangeably. The terms "M2 of influenza b" and "BM2" are used interchangeably. In some embodiments, the M2 protein (CM 2) in influenza c and the M2 protein (DM 2) in influenza delta have a particular selectivity for chloride ions, possibly with some permeability for protons.

In some embodiments, the influenza viruses disclosed herein comprise influenza a viruses. In some embodiments, M2 disclosed herein comprises AM2. In some embodiments, the influenza virus comprises influenza a, and M2 disclosed herein comprises AM2.

Influenza a viruses are generally divided into subtypes based on the relationship between the antigens hemagglutinin and neuraminidase in the surface glycoproteins. In some embodiments, the influenza A virus comprises 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 a hemagglutinin and a neuraminidase antigen. In some embodiments, the influenza a virus comprises an antigen selected from H1-H16 and an antigen selected from N1-N9 in any combination. In some embodiments, the subtype may contain differences in the rest of the genome in addition to the antigenic similarities on the surface glycoproteins. In some embodiments, not all H1N1 (i.e., having the same hemagglutinin 1 and neuraminidase 1 antigens) have the same characteristics.

In some embodiments, the influenza a virus comprises an H1N1 subtype. In some embodiments, the H1N1 influenza virus comprises an M2 protein.

In some embodiments, the influenza virus is resistant to aminoadamantane. Amantadine (brand names Gocovri, symadine and symmetry) includes the organic compounds 1-amantadine or 1-aminoadamantane, which consists of an adamantane backbone and an amino group substituted at one of four methine positions. Amantadine (also known as meglumine (flumadine)) includes adamantane derivatives having similar biological properties. In some embodiments, amantadine and rimantadine target the M2 proton channel of influenza a virus. In some embodiments, the aminoadamantane comprises amantadine (adamantan-1-amine hydrochloride) and amantadine (1- (1-adamantyl) ethylamine hydrochloride).

As used herein, the term "resistance" or "resistance" refers to a decrease in the ability of an antiviral drug to prevent or reduce viral infection. In some embodiments, resistance to aminoadamantane may 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 blocks or reduces the ability of aminoadamantane to inhibit the M2 protein. In some embodiments, the mutation of a nucleic acid encoding an M2 protein results in at least one non-synonymous amino acid substitution. In some embodiments, the virus is or is characterized as resistant or resistant to a drug.

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

In some embodiments, the amino acid substitution of the M2 protein is caused by a missense mutation. In some embodiments, the amino acid substitutions in the M2 protein comprise at least one amino acid substitution. Examples of mutations that result in aminoadamantane resistance are known in the art, such as amino acid substitutions at amino acid residues selected from 27, 30, 31, and 34, as described by Hay AJ et al, "The molecular basis of THE SPECIFIC ANTI-Influenza Action of amantadine," EMBO J1985;4:3021-3024, the entire contents of which are incorporated herein by reference. In some embodiments, the amino acid substitutions in the M2 protein comprise at least one amino acid substitution selected from the group consisting of S31N, V27A, V27G, V27D, I S, I27T, I27A, A30T, A P and G34E.

Examples of M2 mutations that provide resistance to aminoadamantane are known in the art and 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/german/34 or H7N1 Rostock or a/chicken/german/27 or H7N7 Weybridge are known to be resistant to amantadine. In some embodiments, human strain a/singapore/1/57 (H2N 2) is resistant to amantadine when amantadine is associated with a previous infection. In some embodiments, several H3N2 and H1N1 strains are resistant to amantadine.

In some embodiments, at least one mutation selected from S31N (serine 31 to asparagine) and V27A (valine 27 to alanine) results in aminoadamantane resistance (e.g., in the case of WSN/33H1N1 strain). In some embodiments, influenza a disclosed herein comprises an aminoadamantane-resistant variant of HIN 1. In some embodiments, the amino adamantane resistant variant of HIN1 comprises an S31N amino acid substitution.

Method of

In some embodiments, the invention provides a method of treating or preventing influenza disease or at least one symptom associated therewith. In some embodiments, the invention provides a method of 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 fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatiramivir, isaconazol, voriconazole, peramivir, vidarabine, or a metabolite thereof, or any combination thereof.

In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a molecule selected from fludarabine, a derivative thereof or a metabolite thereof, asuprevir, raffmonazole, amikacin, xanthine or a derivative thereof, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatiramivir, isaconazole, voriconazole, pariprevir or a metabolite thereof, and any combination thereof.

In some embodiments, the fludarabine derivative is selected from the group consisting of vidarabine, nelarabine (Nelarabine), cordycepin, clofarabine, cladribine, or any metabolite thereof.

In some embodiments, the fludarabine derivative is selected from the group consisting of vidarabine, nelarabine, cordycepin, or any metabolite thereof.

In some embodiments, the fludarabine derivative is selected from the group consisting of vidarabine, nelarabine, or any metabolite thereof.

In some embodiments, the fludarabine derivative is selected from the group consisting of vidarabine or any metabolite thereof.

In some embodiments, the xanthine derivative is selected from the group consisting of theobromine, 1-methylxanthine, 3-methylxanthine, caffeine, theophylline, enpropyltheophylline, parathyroxanthine (paraxanthine), 7-methylxanthine, or any combination thereof.

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.

In some embodiments, the xanthine derivative is or includes theobromine. In some embodiments, the 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.

In some embodiments, the arabinoside metabolite comprises or is arabininosine.

In some embodiments, the invention provides compositions comprising an influenza a M2 channel blocker for treating or preventing influenza a virulence in a subject. In some embodiments, the invention provides compositions comprising M2 protein channel blockers for preventing entry, uncoating, and/or release of influenza a virus cells from the cells.

In some embodiments, the present invention is based on the discovery that at least one molecule selected from the group consisting of fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatiramivir, isaconazol, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, inhibits the M2 channel of influenza a and is therefore useful in the treatment and/or prevention of influenza a virulence.

In some embodiments, at least one molecule selected from fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, cm4620, levamlodipine, emamectin, glatiravir, isaconazole, voriconazole, paliprevir, vidarabine, or a metabolite thereof, and any combination thereof, may be used to treat and/or prevent 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 aminoadamantane.

In some embodiments, at least one molecule selected from the group consisting of fludarabine, asuprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, glatiramir, voriconazole, paliprovir, vidarabine, or a metabolite thereof, and any combination thereof inhibits the M2 channel of influenza a.

In some embodiments, the matrix protein 2 or M2 protein of influenza a virus (a/Bellamy/1942H 1N1 strain) is disclosed under GenBank accession No. ABW 75846.1.

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

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 and range therebetween. Each possibility represents a separate embodiment of the 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 invention.

The term "analog" as used herein refers to a polypeptide similar to, but not identical to, a polypeptide of the present invention, which still belongs to influenza a virus. In some embodiments, the analog is resistant to aminoadamantane. Analogs may have deletions or mutations which result in amino acid sequences that differ from the amino acid sequences of the polypeptides of the invention. It will be appreciated that all analogues of the polypeptides of the invention are still capable of creating ion channels. Furthermore, an analog may be similar to a fragment of a polypeptide of the invention, however, in this case the fragment must comprise at least 50 consecutive amino acids in the polypeptide of the invention.

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.

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

According to some embodiments, the present invention provides a method of preventing cellular entry of influenza virus, the method comprising contacting the cells with an influenza virus M2 protein channel blocker, thereby preventing cellular entry of influenza virus.

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

According to some embodiments, the cell is a cell of a subject. According to some embodiments, the contacting is to a subject. According to some embodiments, the subject is a subject infected or suspected of being infected with 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.

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, asuprevir or a salt thereof, raffmonazole or a salt thereof, amikacin or a salt thereof, theobromine or a salt thereof, flunisolide or a salt thereof, alvimopan or a salt thereof, irinotecan or a salt thereof, CM4620 or a salt thereof, levamlodipine or a salt thereof, emamectin or a salt thereof, glaconvir or a salt thereof, isaconazol 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.

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, asuprolide 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, glatiravir or a salt thereof, voriconazole or a salt thereof, peramivir or a salt thereof, vidarabine or any metabolite thereof or a salt thereof, and any combination thereof.

According to some embodiments, treating a subject in need of treatment with an influenza virus M2 channel blocker comprises treating with two molecules selected from the group consisting of theobromine + ara-adenosine or a metabolite thereof, and ara-adenosine or a metabolite thereof + glatiramir. In some embodiments, the influenza virus M2 channel blocker comprises treatment with vidarabine or a metabolite thereof and at least one molecule selected from the group consisting of theobromine and glatiramir.

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

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

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

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

According to some embodiments, the M2 channel blocker is within a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

According to some embodiments, the present invention provides a pharmaceutical composition comprising fludarabine, an analog or salt thereof for use in the treatment of a viral infection. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

Fludarabine as 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 comprises a chemotherapeutic agent for treating leukemia and/or lymphoma.

According to some embodiments, the present invention provides a pharmaceutical composition comprising asuprolide, an analogue or salt thereof for use in the treatment of viral infections. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

As used herein, asunaprevir includes asunaprevir (original name BMS-650032, cas:630420-165-5, iupac: 3-methyl-N- { [ (2-methyl-2-propyl) oxy ] carbonyl } -L-isovaleryl- (4R) -4- [ (7-chloro-4-methoxy-1-isoquinolinyl) oxy ] -N- { (1R, 2 s) -1- [ (cyclopropylsulfonyl) carbamoyl ] -2-vinylcyclopropyl } -L-prolinamide), as well as pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

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

According to some embodiments, the influenza virus M2 channel blocker comprises raffmonazole, an analog or salt thereof.

As used herein, raffinazole includes raffinazole (also known as BMS-207147 or ER-30346, cas:18276-06-1, iupac:4- [2- [ (2 r,3 r) -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.

According to some embodiments, the present invention provides a pharmaceutical composition comprising amikacin, an analog or salt thereof for use in the treatment of viral infections. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

Amikacin as 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) oxalan-2-yl ] oxy-4- [ (2R, 3R,4S,5R, 6R) -6- (aminomethyl) -3,4, 5-trihydroxy-oxalan-2-yl ] oxy-3-hydroxy-cyclohexyl ] -2-hydroxybutyramide), as well as pharmaceutically acceptable salts, solvates, hydrates thereof, and mixtures thereof.

According to some embodiments, the present invention provides a pharmaceutical composition comprising theobromine, an analogue or salt thereof for use in the treatment of viral infections. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

Theobromine as 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.

According to some embodiments, the present invention provides a pharmaceutical composition comprising flunisolide, an analogue or salt thereof for use in the treatment of a viral infection. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

The flunisolide as 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-dioxapentacyclo [10.8.0.02,9.04,8.013,18] twenty-14, 17-dien-16-one), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

According to some embodiments, the present invention provides a pharmaceutical composition comprising alvimopan, an analogue or salt thereof for use in the treatment of viral infections. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

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

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

According to some embodiments, the influenza virus M2 channel blocker comprises irinotecan, an analog or salt thereof.

As used herein, irinotecan includes irinotecan (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.

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 viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

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.

According to some embodiments, the present invention provides a pharmaceutical composition comprising levamlodipine, an analogue or salt thereof for the treatment of viral infections. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

As used herein, levamlodipine includes levamlodipine (also known as levamlodipine (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.

According to some embodiments, the present invention provides a pharmaceutical composition comprising emamectin, an analogue or salt thereof, for use in the treatment of a viral infection. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

According to some embodiments, the influenza virus M2 channel blocker comprises emamectin, an analogue or salt thereof.

Emamectins as used herein include Emamectins (CAS: 119791-41-2 or 155569-91-8, also known as4 "-deoxy-4" -epi-methylamino-avermectin B1, epi-methylamino-4 "-deoxy-avermectin, MK 243, EMA, or GWN 1972), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

According to some embodiments, the present invention provides a pharmaceutical composition comprising glatiramir, an analogue or salt thereof for use in the treatment of a viral infection. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

According to some embodiments, the influenza virus M2 channel blocker comprises glatiramir, an analog or salt thereof.

As used herein, glatiramer Wei Baokuo glatiramer (also designated MK-5172, cas:135014-68-9, iupac:1r,18r,20r,24s,27 s) -N- { (1 r,2 s) -1- [ (cyclopropylsulfonyl) carbamoyl ] -2-vinylcyclopropyl } -7-methoxy-24- (2-methyl-2-propyl) -22, 25-dioxo-2, 21-dioxa-4,11,23,26-tetraazapentacyclo [24.2.1.0 ^3,12.0^5,10. 018,20] icosacarbon-3,5,7,9,11-pentene-27-carboxamide), and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

According to some embodiments, the present invention provides a pharmaceutical composition comprising isaconazole, an analogue or salt thereof for use in the treatment of viral infections. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

According to some embodiments, the influenza virus M2 channel blocker comprises isaconazole, an analogue or salt thereof.

As used herein, isaconazole includes isaconazole or isaconazole-onium 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, the isaconazole is hydrolyzed by esterases in the blood or gastrointestinal tract to an active form comprising isaconazole.

According to some embodiments, the present invention provides a pharmaceutical composition comprising voriconazole, an analogue or salt thereof for use in the treatment of a viral infection. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

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

Voriconazole as used herein includes voriconazole (CAS: 137234-62-9, iupac) (2 r,3 s) -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.

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

According to some embodiments, the influenza virus M2 channel blocker comprises paliprevir, an analog or salt thereof.

As used herein, pariprine Wei Baokuo pariprevir (also known as ABT-450, cas:1216941-48-8, iupac, (2 r,6s,12z,13as,14ar,16 as) -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 a-decatetrahydrocyclopropa [ e ] pyrrolo [1,2-a ] [1,4] diazacyclopentadecine-14 a (5H) -carboxamide) and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof.

According to some embodiments, the present invention provides a pharmaceutical composition comprising arabinoside, a metabolite thereof, an analogue or salt thereof for use in the treatment of a viral infection. In some embodiments, the viral infection comprises an influenza viral 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 a viral infection that is resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection caused by a virus comprising influenza M2 protein.

According to some embodiments, the influenza virus M2 channel blocker comprises arabinoside, a metabolite thereof, an analogue or a salt thereof.

The arabinosides as used herein include arabinosides (also known as 9-beta-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.

Pharmaceutical composition

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

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

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

In some embodiments, a therapeutically effective dose of a composition of the 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 to achieve the desired therapeutic or prophylactic effect at the dosages and for periods of time necessary. The exact dosage form and regimen will be determined by the physician in light of the patient's condition.

The dosage administered will depend on the age, health and weight of the recipient, the type of concurrent therapy (if any), the frequency of the therapy, 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, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other injection means known in the art. While the bioavailability of peptides administered by other routes may be lower than that administered by parenteral injection, it is contemplated that the compositions of the invention will be administered by transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular therapy by use of appropriate compositions. Furthermore, it may be desirable to introduce the pharmaceutical composition of the present invention by any suitable route, including intraventricular and intrathecal injection, which may be facilitated by an intraventricular catheter attached to a reservoir, for example.

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

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

According to some embodiments, the influenza virus M2 channel blocker comprises fludarabine or a salt thereof in a daily dose of about 0.1mg/M ²/day to about 50mg/M ²/day, 1mg/M ²/day to 30 mg/kg/day, and 0.1mg/M ²/day to 10mg/M ²/day.

According to some embodiments, the influenza virus M2 channel blocker comprises asunaprevir or a salt thereof in 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.

According to some embodiments, the influenza virus M2 channel blocker comprises raffmonazole or a salt thereof in 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.

According to some embodiments, the influenza virus M2 channel blocker comprises amikacin or a salt thereof in 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.

According to some embodiments, the influenza virus M2 channel blocker comprises theobromine or a salt thereof in 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.

According to some embodiments, the influenza virus M2 channel blocker comprises flunisolide or a salt thereof in 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.

According to some embodiments, the influenza virus M2 channel blocker comprises alvimopan or a salt thereof in a daily dose of about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1 mg/kg/day to10 mg/kg/day, and 0.5 mg/kg/day to 5 mg/kg/day.

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.

According to some embodiments, the influenza virus M2 channel blocker comprises glatiramir 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 to10 mg/kg/day, and 0.5 mg/kg/day to 5 mg/kg/day.

According to some embodiments, the influenza virus M2 channel blocker comprises voriconazole or a salt thereof in 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.

According to some embodiments, the influenza virus M2 channel blocker comprises paliperidone 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.

According to some embodiments, the influenza virus M2 channel blocker comprises ara-adenosine or a metabolite thereof, or a salt thereof, in 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.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, or excipient.

As used herein, the term "carrier," "adjuvant," or "excipient" refers to any ingredient in a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, dextrose, and sucrose, starches such as corn starch and potato starch, celluloses and derivatives thereof such as sodium carboxymethyl cellulose, ethyl cellulose, 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 non-toxic compatible materials for pharmaceutical formulations. Some non-limiting examples of materials that may be used as carriers herein include sugar, starch, cellulose and derivatives thereof, tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifying agents and other non-toxic pharmaceutically compatible materials for use in other pharmaceutical preparations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, 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 for this purpose are well known to those of ordinary skill in The art, such as those described in The Merck Index, thirteenth edition, budavari et al, eds, merck & co.inc., rahway, n.j. (2001), CTFA (cosmetic, toiletry and perfume association) international cosmetic ingredient dictionary and handbook (International Cosmetic Ingredient Dictionary and Handbook), tenth edition (2004), and The american Food and Drug Administration (FDA) drug evaluation and research Center (CDER), the "inactive ingredient Guide (INACTIVE INGREDIENT Guide)" of The regulatory office, the contents of 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. Such additional inactive ingredients, as well as effective formulations and administration procedures, are known in the art and described in standard textbooks, such as Goodman and Gillman's pharmacological basis for treatment (The Pharmacological Bases of Therapeutics), 8 th edition, gilman et al, editors, pergamon Press (1990), remington's Pharmaceutical Sciences, 18 th edition, mack publishing company, easton, pa., pa., 1990, and Remington: THE SCIENCE AND PRACTICE of Pharmmaccy, 21 st edition, lippincott Williams & Wilkins, philadelphia, pa., pa., 2005, each of which is incorporated herein by reference in its entirety. The presently described compositions may also be included in artificially constructed structures such as liposomes, ISCOMS, slow 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. Liposomes for use 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 is generally dependent on factors such as liposome size and stability in blood. A variety of methods are available for preparing liposomes, for example, reviewed by Coligan, J.E. et al, current Protocols in Protein Science,1999,John Wiley&Sons company (New York), and also see U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

The carrier may comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical composition described herein in total.

Kit for detecting a substance in a sample

According to another aspect, there is provided a kit comprising at least two molecules selected from the group consisting of fludarabine or a derivative thereof, asuprevir, amikacin, xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, glatiramivir, voriconazole, paliprovir, and any combination thereof.

In some embodiments, the molecule is selected from the group consisting of fludarabine, asuprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, glatirivir, voriconazole, paliprovir, arabinoside, or a metabolite thereof, and any combination thereof.

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, asuprolide, amikacin, xanthine or a derivative thereof, flunisolide, alvimopan, levamlodipine, glatiramivir, voriconazole, paliprovir, and any combination thereof.

In some embodiments, the metabolite of ara-adenosine is ara-inosine.

In some embodiments, the xanthine derivative is theobromine.

In some embodiments of the subject kits, at least two molecules are packaged in a container.

In some embodiments, the container is made of a material selected from thin-walled films or plastics (transparent or opaque), cardboard, foil, rigid plastics, metals (e.g., aluminum), glass, and the like.

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

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

In some embodiments of the subject kits, at least two molecules are stored in separate containers, e.g., cassettes or similar structures, within the main kit containment element, which may or may not be airtight containers, e.g., to further maintain sterility of some or all of the components of the kit.

In some embodiments, the dosage of at least two molecules provided in the kit may be sufficient for a single application or multiple applications.

In these embodiments, the kit may have multiple doses of at least two molecules packaged in a single container, such as a single tube, bottle, vial, 1.5-2ml tube, e.g., eppendorf, etc.

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

In some embodiments, multiple doses of at least two molecules may be packaged in a single, separate container.

In some embodiments, the kit comprises instructions for preparing the compositions used therein and how to perform the methods of the invention.

In some embodiments, the kit further comprises a measuring instrument, such as a syringe, a measuring spoon, or a measuring cup.

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

In some embodiments, the instructions may be present in the kit as a package insert, container label of the kit or component thereof (i.e., associated with a package or sub-package), or the like. In other embodiments, the instructions are in the form of electronically stored data files on a suitable computer-readable storage medium, such as a CD-ROM, floppy disk, or the like. In other embodiments, no physical instructions are present in the kit, but methods of obtaining instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit comprising a website where the instructions can be reviewed and/or downloaded. As with the instructions, this method of obtaining the instructions is recorded on a suitable substrate.

Screening test

By another aspect, a method of screening for the effectiveness of an agent in treating or preventing influenza virus infection is provided. According to some embodiments, the method comprises providing a cell comprising a membrane permeated by an influenza 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 is indicative of the agent being effective to treat or prevent an influenza infection, thereby screening the agent for its effectiveness in treating or preventing an influenza infection. In some embodiments, the influenza virus comprises influenza a virus. In some embodiments, the influenza a virus comprises an H1N1 subtype. In some embodiments, the influenza virus is resistant to aminoadamantane.

In some embodiments, the method comprises a negative assay. In some embodiments, the cells are characterized by retarded growth due to membrane penetration by influenza M2 channels. In some embodiments, the agent that alleviates growth retardation is indicated to be effective in treating or preventing influenza virus infection.

In some embodiments, the method comprises a positive assay. In some embodiments, the cells are K ⁺ uptake defective cells that cannot grow in low [ K ⁺ ] medium, but undergo growth due to the channels formed by the influenza M2 channels. In some embodiments, the agent that induces growth retardation is indicated to be effective in treating or preventing influenza virus infection.

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

In some embodiments, the method further comprises acidity determination. In some embodiments, the cell is a cell that includes a pH reporter gene or protein product thereof, such as, but not limited to, a pH sensitive Green Fluorescent Protein (GFP). In some embodiments, H ⁺ is induced to flow into cells grown in a medium supplemented with an acidic solution. In some embodiments, agents that block changes in intracellular pH are useful for treating or preventing influenza virus infection.

In some embodiments, the method further comprises a validation step comprising checking for molecules of all three bacterial assays that pass positive in a mammalian cell-based assay. In some embodiments, the validating step entails infecting mammalian cells with influenza a virus and examining the effect of the molecule on cell viability and/or growth.

Non-limiting examples of bacterial cell growth suitable for use in 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, I.T.PLoS One 9, e105387 (2014).

In some embodiments, the assay comprises determining a susceptibility of the virus to developing resistance to the agent.

As used herein, the term "about" when combined 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.

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 (apolynucleotide)" includes a plurality of such polynucleotides, reference to "the polypeptide" includes reference to one or more polypeptides known to those of ordinary skill in the art and equivalents thereof, and so forth. It should also be noted that drafting of the claims may exclude any optional elements. Accordingly, such recitations are intended to serve as a basis for reference to such exclusive terminology as "only (solely)", "only (only)", and the like, as used in recitation of claim elements or in the use of a "negative" limitation.

Where a convention analogous to "at least one of A, B and C, etc." is used, in general such a grammar construction has the meaning that one having ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include but not be limited to a system 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.). It will be further understood by those within the art that virtually any conjunctive and/or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" will be understood to include the possibilities of "a" or "B" or "a and B".

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of embodiments related to the invention are specifically contemplated by the present invention and disclosed herein as if each and every combination were individually and specifically disclosed. Moreover, the invention specifically encompasses all subcombinations of the various embodiments and elements thereof and is disclosed herein as if each and every such subcombination were individually and specifically disclosed herein.

Additional objects, advantages and novel features of the present invention will become apparent to those of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. Furthermore, various embodiments and aspects of the invention described above and claimed in the claims section are experimentally supported in the following examples.

Various embodiments and aspects of the invention described above and claimed in the claims section are experimentally supported in the following examples.

Examples

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbial, and recombinant DNA techniques. These techniques are well explained in the literature. See, for example, "Molecular Cloning: alaboratory Manual (molecular cloning: laboratory Manual)", sambrook et al (1989); "Current Protocols in Molecular Biology (Current protocols in molecular biology)", ausubel, volumes I-III, R.M. editions (1994) ", ausubel et al," Current Protocols in Molecular Biology ", john Wiley andSons, maryland, barkat (1989); perbal", "APRACTICAL GUIDE TO MOLECULAR CLONING (molecular cloning practice guide)", john Wiley & Sons, new York (1988) ", watson et al," Recombant DNA (Recombinant DNA) ", SCIENTIFIC AMERICAN Books, new York", "Birren et al (editions)" Genome Analysis: ALaboratory Manual Series (Genome Analysis: laboratory Manual series) ", volumes 1-4, cold spring laboratory Press, new York (1998)", e.g., U.S. patent No.4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057, "Cell Biology: ALaboratory Handbook (cytobiology: laboratory Manual)", cellis, volumes I-III, J.E. editions (1994), "Culture of ANIMAL CELLS-AManual of Basic Technique (animal Cell Culture-Basic technical handbook)", wiley-List, new York (1994), third edition, "Current Protocols in Immunology (immunoCurrent protocol)", volumes I-III, coligan J.E. editions (1994), "Stites et al (editions)," Basic AND CLINICAL Immunology) "(8 th edition), appleton & Lange, norwalk, CT (1994)," Mishell and Shiigi (editions), "STRATEGIES FOR PROTEIN PURIFICATION AND CHARACTERIZATION-A Laboratory Course Manual (protein purification and characterization strategy-laboratory curriculum)", CSHL publication (1996), all of which are incorporated herein by reference. other general references are provided in this document.

Materials and methods

Bacterial-based channel detection

Three bacterial-based channel assays were used to study influenza channel activity and blockers thereof. In all assays, MBP (maltose binding protein) fusion purification system (New England Biolabs; ipswich, mass.) was used, in which the M2 channel from an aminoadamantane resistant H1N1 strain was expressed as a chimera by fusion to the carboxy terminus of the maltose binding protein. The system ensures that proteins target the bacterial endomembrane and are successfully used to express and verify many other viral ion channels. The strains used by the inventors are H1N1 strains known to be resistant to aminoadamantane, due to the mutation of Ser31 to Asn in their M2 protein (Hay AJ et al, "The molecular basis of THE SPECIFIC ANTI-InfluenzaAction of amantadine", EMBO J1985; 4:3021-3024, cited herein in their entirety), and H1N1 strains sensitive to aminoadamantane.

Negative detection

Bacterial cultures (DH 10B) were grown overnight, eventually diluted 500-fold, and grown again until their O.D.600 reached 0.2. Mu.l of the culture was added to a 96-well flat bottom plate, and the plate was pretreated with 50. Mu.l of a specific chemical and isopropyl-. Beta. -d-1-thiogalactopyranoside (IPTG) inducer. Induction was achieved by using different concentrations of IPTG. Using a multi-plate incubator (INFINITE M pro of Tecan Group; switzerland)Or LogPhase to BioTek; SANTA CLARA, california, U.S.A.) plates were incubated at 37℃for 16 hours with a constant shaking rate (700 rpm). Bacterial growth was monitored by measuring o.d.600 every 15 min. Each measurement was repeated or performed three times.

Positive detection

The positive assay uses the same protocol as the negative assay, but in this case a K ⁺ -uptake defective bacterial strain is used. In addition, overnight growth was performed in LB medium, with KCl of the indicated different concentrations replacing NaCl.

Acidity detection

Acidity detection is based on bacteria expressing chromosomal copies of pH-sensitive GFP. The overnight bacterial culture was diluted to 1:500 in LB medium and then grown to O.D.600 of 0.6-0.8. As previously described, IPTG induced protein expression at different concentrations. After induction for one hour, the cells were diluted to 0.2 o.d.600 and precipitated at 3500g for 10min. Subsequently, the cells were resuspended in MCILVAINE buffer containing 200mM Na ₂HPO₄ and 0.9% NaCl, adjusted to pH 7.6 with 0.1M citric acid. 200 μl of the cell suspension was added to a 96-well plate (Nunclonf microwell black polystyrene, simer Feishmania technologies; waltham, mass.) containing 30 μ L MCILVAINE buffer per well. Plates included three wells with MCILVAINE buffer and three wells without induced culture as controls. Fluorescence measurements were performed at ambient temperature in microplate readers (INFINITE F Pro, tecan Group; switzerland)) The emission is fixed at 520nm, alternating between 390 and 466nm excitation. At the start point, 70. Mu.l of 300mM citric acid was added to the bacterial culture and a fluorescence reading was performed for 30s for each wavelength. Finally, the proton concentration is calculated from the ratio of the two excitation wavelengths.

Chemical screening

The chemical library was purchased from MedChem Express (HY-L035, monmouth Junction, NJ, USA). At that time, the library contained 2839 re-used drugs and indicated that the amount 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 operations and growths were performed in a robotic system (EVO 75Tecan,Switzerland) or LogPhase 600 microorganism reader (Agilent, SANTACLARA, CA, USA).

For each growth test, two indicators were measured, maximum growth rate and final bacterial density. However, in practice, visual inspection is far superior in identifying individual hits due to spurious factors affecting the above-mentioned indicators, such as compound absorbance, solubility, and the like.

Chemical product

Isopropyl-. Beta. -d-1-thiogalactopyranoside (IPTG) is obtained from biochem-Fluka (Buchs, switzerland). All other chemicals were purchased from Sigma-Aldrich laboratory (israel rehovit).

Bacterial growth medium

In most cases lysogenic medium (LB) was used, only LBK was used for positive detection, with NaCl replaced by 10mg/lt KCL. All media contained 100g/ml ampicillin.

Antiviral activity of the compounds was evaluated in BSL-3 facility at Hiberlaia university (the Hebrew University) by maintaining motor-Dishi canine kidney (MDCK) [ ATCC MDCK NBL-2] cells in Dulbecco's Modified Eagle's Medium (DMEM); (Biological Industries; israel Beit Haemek), supplemented with 10% fetal bovine serum, 2mM L-glutamine, 10IU/mL penicillin and 10. Mu.g/mL streptomycin, aureomycin-3, (Biological Industries; israel Beit Haemek). Influenza a virus a/Wisconsin/629-D02152/2009 (H1N 1) pdm09 was saved by the U.S. disease control and prevention center and obtained by BEI Resources, NIAID and NIH. The same viral stock aliquots were prepared from the mother liquor supplied by BEI Resources. Sub-stock solutions were prepared for infection at 1:1000 fold dilutions from each aliquot. Subsequent infection of MDCK cells was performed in MEM (Biological Industries; beit Haemek, israel) containing 0.3% bovine serum albumin (Sigma: A4503, lot number: SLCK 2178) and 3 μg/ml TPCK-treated trypsin (Sigma, T8802), and further incubated at 35℃for 48h in a 5% CO ₂ atmosphere. All infection experiments were performed in a BSL-3 facility. Stock solutions of the listed compounds were prepared at 10mM in DMSO and stored in aliquots at-80 ℃ until further use. MDCK cells were seeded at a density of 15000 cells per well in 200 μl of medium on 96-well flat bottom plates and grown overnight. Dilutions of test compounds were prepared in MEM with 0.3% BSA, 3. Mu.g/ml TPCK treated trypsin and 50. Mu.L was added to the cells. The effect of the drug on the metabolic activity of MDCK cells was assessed 48h after treatment using CellTiter 96 aqueous non-radioactive cell proliferation reagent (Promega; madison, wis., USA). To examine the effect of the different drugs, cells were infected with 400tcid 50/well influenza a virus for two hours and then treated with the listed drugs. Three tests were performed for each compound concentration and each assay plate contained controls of no cells (background control), cells treated with medium (mock infection normalized), infected/untreated cells and infected/solvent treated cells (infection control). Two days after infection, the efficacy of drug control toxicity was evaluated for 3h in a 5% CO ₂ atmosphere at 37℃using CellTiter 96 aqueous non-radioactive cell proliferation reagent (Promega; madison, wis.). the reaction was stopped and 30. Mu.l of 4% formaldehyde inactivated virus was added. Using Tecan plate readerSwiss) absorbance was measured at 492 nm. Finally, the data were normalized to a simulated infection control, and then EC ₅₀ values were calculated by fitting the data to the Monod equation.

Animal study

Mice were infected with H1N1 virus and given oral treatment (BID) for 5 days. After 9 days, the number of pulmonary viruses was quantified by q-RT PCR. BALB/c mice were selected for animal experiments. The inventors first performed tolerability and pharmacokinetic studies on the corresponding drugs, 3 mice per group, 5 groups total. The pharmaceutical combinations of the groups (1:1 molar ratio) were (i) 1.5mg/kg of inosine and 1mg/kg of theobromine, (ii) 4.5mg/kg of inosine and 3mg/kg of theobromine, (iii) 15mg/kg of inosine and 10mg/kg of theobromine, (iv) 45mg/kg of inosine and 30mg/kg of theobromine, and (v) 150mg/kg of inosine and 100mg/kg of theobromine. For efficacy studies, 56 BALB/c mice were divided into 7 groups, so each group contained 8 mice for this group of experiments. The 7 groups were treated by (i) vehicle (control group), (ii) 20mg/kg oseltamivir, (iii) 1.5mg/kg of inosine and 1mg/kg of theobromine, (iv) 4.5mg/kg of inosine and 3mg/kg of theobromine, (v) 15mg/kg of inosine and 10mg/kg of theobromine, (vi) 45mg/kg of inosine and 30mg/kg of theobromine, and (vii) 150mg/kg of inosine and 100mg/kg of theobromine. Each group was first infected with influenza by intranasal spraying 50 μl of approximately 1000 viruses per animal. All the combination at the desired concentration was dissolved in 1% Emulfor-EL-620 in water and oseltamivir was dissolved in water. Thus, the vehicle is an aqueous solution of only 1% Emulfor-EL-620. Animals were orally fed 10ml/kg for 5 days twice daily. 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-RT PCR.

Example 1

Bacterial-based detection for ion channel activity assessment

To assess the activity of the amantadine resistant H1N1 strain, the inventors used a bacterial-based assay in which the function of the channel altered the phenotype of the bacteria. The advantage of such assays is that they are easy to screen for high throughput and the convenience of genetic manipulation in bacteria enables rapid transition from one sequence/variant to another. Finally, these assays have been performed on a variety of viral porins from a variety of 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,P.P.S, et al, viruses 2019,11, tomar, P.P.S. et al, viruses 2021,13, tomar, P.P.S. et al, pharmaceuticals (Basel) 2021,14, tomar, P.P.S. et al, krugliak, M.; singh, A.; arkin, I.T. biomedicines 2022).

Negative detection

The primary assay employed involves elevated levels of expression of viral channels in "normal" E.coli. At a certain viral porin concentration, growth retardation was observed due to excessive membrane permeability blocking the bioenergy of the bacteria. Thus, this test is called negative detection as the title, due to the detrimental effect of the protein on the bacteria.

Positive detection

The second assay requires expression of viral channels at lower levels in K ⁺ -uptake deficient bacteria. These bacteria cannot grow in conventional media unless potassium is supplemented to the bacteria or when the bacteria express channels capable of transporting K ⁺. In this case, the viral channel has a favorable effect on the bacteria, and thus the detection is referred to as positive detection. It should be noted that at high induction levels, the channels negatively affect the bacteria due to excessive membrane penetration similar to the negative assay described above while alleviating the K ⁺ shortage in bacteria.

Acidity detection

The final detection of the activity of the channels was verified based on the effect of the channels on the cytoplasmic pH of the bacteria. When concentrated acid is injected into the culture medium, the cytoplasmic pH decreases if the bacteria express channels capable of transporting H ⁺. Subsequently, by monitoring the fluorescence of the pH-sensitive GFP expressed by the chromosome, changes in cytoplasmic pH can be detected.

The negative, positive and acidity test results are shown in figures 1, 2 and 3, respectively, confirming the activity of the M2 channel in all validated tests. In all figures, the protein expression level, i.e. the level of the M2 channel, is controlled by the level of isopropyl β -D-1-thiogalactopyranoside (IPTG).

Blocking agent screening

After confirming the channel activity of the M2 channel, the inventors sought to identify drugs that could block its function. Screening is performed in three stages. First, all compounds were screened in a negative assay. There were two controls per plate, positive controls were bacteria without IPTG, i.e. without channel induction. Blank DMSO was added as a negative control. Subsequently, bacteria with growth enhancement exceeding the empirical threshold were re-examined in triplicate. Then, each compound passing the test was checked in triplicate in a positive test. Finally, two replicates of dose response assays and four replicates of pH assays were performed on compounds detected by positive and negative. Finally, examples of compound screening assays are shown in FIGS. 4-6. "channel-free" refers to the absence of IPTG to induce the M2 channel, while "drug-free" refers to the maximum induction of the M2 channel protein by IPTG in the absence of drug. IPTG concentrations in negative, positive and acidity assays were 100. Mu.M, 20. Mu.M and 50. Mu.M, respectively (FIGS. 4-6).

As shown in fig. 4-6, compounds exhibiting activity in one or more assays are listed as amantadine, fludarabine, asuprevir, raffmonazole, amikacin, theobromine, flunisolide, alvimopan, irinotecan, CM4620, levamlodipine, emamectin, glaconvir, isaconazol, voriconazole, paliprevir, vidarabine, and inosine.

* Amantadine (Amantan)Are known channel blockers of the M2 channel and are therefore identified as contemplated in the screening (Pinto, L.H. et al, "Influenza virus M2 protein has ion channel activity." Cell 1992;69:517-528, hereby incorporated by reference in its entirety).

Example 2

In vitro and in vivo analysis of mammalian systems

Tissue culture studies

The inventors next sought to characterize the antiviral activity of channel blockers identified in tissue culture cells. To this end, the present inventors cultured motor-canine kidney (MDCK) cells and examined their viability after infection with an amantadine resistant H1N1 virus and the ability of the drug to affect cell viability thereon. The virus strain used was H1N1, which is known to be resistant to aminoadamantane due to mutation of Ser31Asn in the M2 protein.

Compounds active in tissue culture

First, two concentrations of drug were used, 10. Mu.M as shown in FIG. 7, and the latter 3. Mu.M as shown in FIG. 8. In both experiments, two available anti-influenza drugs were used as positive controls, oseltamivirHefupirrevir

The results show that the following drugs show a considerable anti-influenza effect in tissue culture at 10. Mu.M, fludarabine, asuprevir, amikacin, theobromine, flunisolide, alvimopan, levamlodipine, glatirivir, voriconazole, paliprovir and vidarabine.

The following compounds are active at 3. Mu.M, fludarabine, asuprevir, theobromine, flunisolide, glatiramivir, voriconazole and vidarabine.

Finally, as expected,AndExhibits potent anti-influenza activity (thus acting as a positive control), and is also desirable because the infection is with an H1N1 virus strain resistant to aminoadamantane, amantadineCompletely ineffective (thus acting as a negative control).

The inventors subsequently performed dose-response assays to determine the ability of each active compound to inhibit virus at different concentrations. The results of these analyses are shown in figures 9-12. The shaded area in the graph represents the vehicle level. In fig. 13 and 14, a fit of EC ₅₀ values for each compound can be found.

After affinity analysis of each drug, the inventors sought potential additive and synergistic effects between positive results. The results of the combination experiment of each drug in combination with the other drug at a concentration of 0.01 μm are shown in fig. 15. It can be seen that theobromine + ara-adenosine and ara-adenosine + glatirivir show a clear synergy. Other combinations exhibit additive effects or nullifying effects as shown in table 1 below. The shaded area represents a single compound.

The combination experiments were performed at lower drug concentrations. It can be seen in fig. 16 that the combination of theobromine and arathyronine is particularly effective at 30nm, providing complete protection against virus-induced cell death.

TABLE 1 summary of combined effects between positive results

	Glatirevir	Theobromine (Theobroma cacao alkali)	Vidarabine	Flunisolide	Fludarabine	Ashorevir
							Glatirevir	40%	56%	106%	57%	37%	32%
Theobromine (Theobroma cacao alkali)	56%	-6%	101%	22%	43%	46%
							Vidarabine	106%	101%	-3%	37%	41%	21%
Flunisolide	57%	22%	37%	31%	15%	-3%
							Fludarabine	37%	43%	41%	15%	34%	-3%
Ashorevir	32%	46%	21%	-3%	-3%	33%

Additional mice were infected with H1N1 virus and given oral treatment (BID) for 5 days as described above. After 9 days, the number of pulmonary viruses was quantified by q-RT PCR. The results indicate that although the pharmaceutical combination disclosed herein comprising theobromine + vidarabine is administered at significantly lower doses, it is more effective than the leading drug oseltamivir on the market (figure 17).

Furthermore, the inventors performed structure-activity relationship (SAR) analysis, examining the effect of xanthine and its derivatives on cell viability (fig. 18). The results indicate that several closely related xanthine derivatives improved cell viability (fig. 19). In this regard, theobromine can greatly improve cell viability at concentrations of 0.3 μm to 10 μm compared to the control group. Surprisingly, caffeine, which is very similar to theobromine, did not increase cell viability compared to the control without the drug. The tendency is similar for enpropyltheophylline and parathyroid xanthine. Theophylline and 3-methylxanthine were found to greatly improve cell viability at any concentration tested (0.01. Mu.M-1. Mu.M) compared to the control group. Compared with the control group, 7-methylxanthine can be found to greatly improve the cell survival rate at the concentration of 1 mu M-10 mu M.

Furthermore, the inventors performed SAR analysis and examined the effect of fludarabine and its derivatives on cell viability (fig. 22). The results indicate that fludarabine and several closely related derivatives thereof increased cell viability compared to the control group (fig. 22). In this regard, it was found that vidarabine can greatly improve cell viability at a concentration of 0.3. Mu.M to 10. Mu.M compared to the control group. Nelarabine and cordycepin increased cell viability at concentrations of 10. Mu.M and 1. Mu.M-10. Mu.M, respectively.

In addition, the inventors performed a combination experiment with xanthine or other closely related compounds thereof (having a xanthine skeleton) and the antiviral drug arabinoside or the natural metabolite arabininosine thereof (fig. 20). The results are shown in FIG. 21.

Briefly, a combination comprising vidarabine at a concentration of 300nM and 1-methylxanthine at a concentration of 10nM-300nM improved cell viability by about 33-37% compared to the negative control (no drug).

The combination of vidarabine at a concentration of 300nM and xanthine at a concentration of 10nM-300nM improved cell viability by about 27-48% compared to the control group.

The combination comprising 10nM or 100mM of ara-adenosine and 30nM of 3-methylxanthine increased cell viability by about 63% and 28%, respectively, compared to the control group. The combination of vidarabine at a concentration of 30nM or 100mM and 3-methylxanthine at a concentration of 100nM improved cell viability by about 63% and 46%, respectively, compared to the control group.

The combination of vidarabine and theobromine has a great positive impact on cell viability. The combination of vidarabine and theobromine, including any tested concentration (e.g., 10nM to 300mM of each compound), improved cell viability by about 37% to 98% compared to the control group. In particular, the combination comprising ara-adenosine at a concentration of 10nM to 300mM and theobromine at a concentration of 30nM to 300nM increased cell viability by about 64% to 98% compared to the control group.

In addition, the inventors examined the effect of theobromine and inosine (a natural metabolite of arabinoside) on cell viability. The results show that the combination of inosine including 100nM or 300mM concentration and theobromine at 10nM concentration increased cell viability by about 10% to 75% compared to the control group. In addition, the combination comprising inosine at a concentration of 10nM to 300mM and theobromine at a concentration of 30nM to 300nM increased cell viability by about 61% to 100% compared to the control group.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, 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.