EP4633677A1 - Viroporins blockers/inhibitors as anti-influenza agents - Google Patents

Viroporins blockers/inhibitors as anti-influenza agents

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Publication number
EP4633677A1
EP4633677A1 EP23902961.4A EP23902961A EP4633677A1 EP 4633677 A1 EP4633677 A1 EP 4633677A1 EP 23902961 A EP23902961 A EP 23902961A EP 4633677 A1 EP4633677 A1 EP 4633677A1
Authority
EP
European Patent Office
Prior art keywords
influenza
theobromine
virus
vidarabine
arainosine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23902961.4A
Other languages
German (de)
French (fr)
Inventor
Isaiah Arkin
Hiya Lahiri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yissum Research Development Co of Hebrew University of Jerusalem
Original Assignee
Yissum Research Development Co of Hebrew University of Jerusalem
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yissum Research Development Co of Hebrew University of Jerusalem filed Critical Yissum Research Development Co of Hebrew University of Jerusalem
Publication of EP4633677A1 publication Critical patent/EP4633677A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention is in the field of anti-viral therapy.
  • Influenza is a segmented, single-stranded negative-sense RNA virus that belongs to the Orthomyxoviridae. It represents one of the leading causes of morbidity and mortality due to an infectious disease. In particular, before COVID- 19, it was the primary communicable cause of death in the western world.
  • Combating influenza is achieved by vaccines and antiviral therapy. However, constant genomic shifts and drifts continuously vitiate vaccine effectiveness and antiviral drug benefits. For example, the annual vaccine protection rate revolves around 40%, and resistance to every anti-flu agent has already been reported.
  • a method of treating or preventing an Influenza A virus in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a molecule selected from the group consisting of: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, thereby treating or preventing an Influenza A virulence in the subject.
  • a molecule selected from the group consisting of: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Is
  • 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 a group consisting of: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
  • kits comprising at least two molecules selected from the group consisting of: Fludarabine or a derivative thereof, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
  • the molecule is selected from a group consisting of: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
  • the kit further comprises instructions for mixing the at least two molecules selected from the group consisting of: Fludarabine or a derivative thereof, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
  • the Fludarabine derivative comprises Vidarabine or a metabolite thereof.
  • the metabolite of Vidarabine is Arainosine.
  • the xanthine derivative is Theobromine.
  • the kit is for preparing a medicament for treating a subject afflicted with influenza or infected with an Influenza virus.
  • the molecule is a M2 protein blocker.
  • the Influenza A virus is a H1N1 subtype.
  • the Influenza A virus is resistant to aminoadamantanes.
  • the molecule is for use at a daily dose of 0.01 to 500 mg/kg per body weight of the subject.
  • administering is a therapeutically effective amount of 2 molecules selected from: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
  • the 2 molecules are Vidarabine or a metabolite thereof, and a molecule selected from: theobromine and grazoprevir.
  • a combination for use in treating or preventing Influenza A virulence in a subject in need thereof comprising at least two molecules selected from a group consisting of: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and Vidarabine or a metabolite thereof.
  • the at least two molecules are (i) Vidarabine or a metabolite thereof, and (ii) a molecule selected from: Theobromine and Grazoprevir.
  • a metabolite of Vidarabine is or comprises arainosine.
  • Fig. 1 includes a graph showing a negative assay of influenza M2 viroporin, in which protein expression is detrimental to bacterial growth. Growth of DH10B bacteria as a function of different concentrations of the protein inducer Isopropyl-P-D-thiogalactoside (IPTG).
  • IPTG Isopropyl-P-D-thiogalactoside
  • Fig. 2 includes a vertical bar graph showing a positive assay of influenza M2 viroporin. Maximal growth rates of K + -uptake deficient bacteria expressing the influenza M2 viroporin is monitored as a function of different K + concentrations in the presence (10 pM) or absence of the inducer IPTG.
  • Fig. 3 includes a graph showing an acidity assay of the influenza M2 viroporin. Cytoplasmic H + concentration is monitored as a function of time whereby at time 0, an acidic solution is injected into the media. Results are shown from bacteria in which viroporin induction is changed a as function of IPTG concentration as noted.
  • Fig. 4 include a graph showing results of a negative assay used in a blocker screening against influenza M2 viroporin. Bacteria in which the channel expression was not induced (“no channel”), or the drug was not added (“no drug”) were used as controls.
  • Fig. 5 includes a graph showing results of a positive assay used in a blocker screening against influenza M2 viroporin. Bacteria in which the channel expression was not induced (“no channel”), or the drug was not added (“no drug”) were used as controls.
  • Fig. 6 includes a graph showing an acidity assay of the influenza M2 viroporin conducted in the presence of the tested drugs.
  • Fig. 7 includes a vertical bar graph showing cell survival in the presence of various drugs at a concentration of 10 pM (unless indicated otherwise). Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 8 includes a vertical bar graph showing cell survival in the presence of various drugs at a concentration of 3 pM (unless indicated otherwise). Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 9 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 10 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 11 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 12 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 13 includes graphs showing the half maximal effective concentration (EC50) values of the tested compounds.
  • Fig. 14 includes graphs showing the half maximal effective concentration (EC50) values of the tested compounds.
  • Fig. 15 includes a graph showing drug synergism. All drugs were applied at a concentration of 0.01 pM. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
  • Fig. 16 includes a vertical bar graph showing relative cell viability after 48 hours of infection, in the presence of various drugs or a combination thereof. It is observed that a combination of Theobromine and Vidarabine synergistically improves cell viability. Further, the effect attributed to the combination of Theobromine and Vidarabine was even more pronounced than that of the approved drugs Oseltamivir, and favipiravir.
  • Fig. 17 includes a vertical bar graph showing viral RNA per (%) as determined following an in vivo study in mice wherein animals were treated with the indicated drugs. It is observed that a combination Theobromine and arainosine is more effective in vivo than Oseltamivir, the leading drug on the market, even though it is given at a significantly lower dosage.
  • Fig. 18 includes chemical structures of xanthine and other closely related compounds having a xanthine backbone and further modifications. Positions of modifications are indicated by dashed lines.
  • Fig. 19 includes a graph showing structure-activity relationship (SAR) analysis of xanthine derivatives. Cell viability was determined as described. Higher values indicate better protection against viral-induced cell death. The different xanthine derivatives including modifications in positions R1-R3 of xanthine, as shown on top.
  • SAR structure-activity relationship
  • Fig. 20 includes chemical structures of xanthine and other closely related compounds having a xanthine backbone and further modifications (top row). Positions of modifications are indicated by dashed lines. Further presented are the antiviral drug vidarabine and its natural metabolite arainosine (bottom row). [046] Fig. 21 includes tables summarizing combination therapies with the indicated drug. Higher values indicate better protection against viral-induced cell death. Compounds were used at several concentrations, as presented.
  • Fig. 22 includes a graph showing SAR analysis of Fludarabine derivatives. Cell viability was determined as described. Higher values indicate better protection against viral-induced cell death. The different Fludarabine derivatives including modifications in positions R1-R5 of Fludarabine, as shown on top.
  • the present invention in some embodiments, provides compositions comprising a matrix protein 2 or M2 protein channel blocker for treating or preventing an influenza virulence in a subject.
  • the present invention in some embodiments, provides compositions comprising an influenza M2 protein channel blocker for preventing influenza cell entry, uncoating and/or release from a cell.
  • matrix protein 2 and “M2 protein” are used interchangeably.
  • the present invention in some embodiments, is based, on the finding that at least one molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, inhibits M2 channel from an Influenza A, and therefore, can be used to treat and/or prevent Influenza A virulence.
  • the Influenza A strain that can be treated or prevented by the at least one of the abovementioned molecules is resistant to an aminoadamantane .
  • the M2 protein of the Influenza virus is a transmembrane protein known to be present in all Influenza A types.
  • the M2 protein forms a proton channel in the viral envelope.
  • the M2 channel function is to equilibrate pH across the influenza membrane during cell entry and across the trans-Golgi membrane of an infected cell during viral maturation.
  • M2 is vital for viral replication.
  • M2 is a target for the anti-influenza drugs (e.g., amantadine and rimantadine).
  • M2 protein belongs to the class of viroporins (e.g., small proteins that form ion channels that increase membrane permeability in virus-infected cells).
  • M2 of Influenza A also named AM2
  • B also named BM2
  • M2 proteins in influenza C CM2) and D (DM2), are especially selective for chloride ions, with possibly some permeability to protons.
  • the Influenza virus disclosed herein comprises an Influenza A virus.
  • the M2 disclosed herein comprises AM2.
  • the Influenza virus comprises Influenza A and the M2 disclosed herein comprises AM2.
  • Type A Influenza viruses are commonly divided into subtypes based on the relationships between the antigens hemagglutinin and neuraminidase in the surface glycoproteins.
  • Influenza A virus comprises at least 16 hemagglutinin subtypes (named H1-H16) and at least nine neuraminidase subtypes (named N1-N9).
  • each Influenza A virus has one hemagglutinin and one neuraminidase antigen.
  • Influenza A virus comprises one antigen selected from H1-H16, and one antigen selected from N1-N9, in any combination thereof.
  • the subtypes apart from the antigenic similarity on the surface glycoproteins, may contain differences in the rest of the genome. In some embodiments, not all the H1N1 (i.e. having the same hemagglutinin 1 and neuraminidase 1 antigens), have the same characteristics.
  • Influenza A virus comprises a H1N1 subtype.
  • the H1N1 Influenza virus comprises M2 protein.
  • Influenza virus is resistant to aminoadamantanes.
  • Amantadine brand names Gocovri, Symadine, and Symmetrel
  • Amantadine comprises the organic compound 1- adamantylamine or 1 -aminoadamantane, which consists of an adamantane backbone with an amino group substituted at one of the four methyne positions.
  • Rimantadine also named flumadine
  • amantadine and rimantadine target the M2 proton channel of Influenza A virus.
  • an aminoadamantane comprises amantadine (adamantan-1 -amine hydrochloride) and rimantadine (l-(l-Adamantyl)ethanamine hydrochloride).
  • resistance or “resistant” refers to reduced ability of the antiviral drug to prevent or to reduce the viral infection.
  • resistance to aminoadamantane can be observed when aminoadamantanes are applicated prior or post infection.
  • the nucleic acid encoding M2 protein of the Influenza virus has a mutation that hinders or reduces the ability of an aminoadamantane to inhibit the M2 protein.
  • the mutation of the nucleic acid encoding M2 protein results in at least one nonsynonymous amino acid substitution.
  • the virus is or characterized by resistance or being resistant to the drug.
  • nonsynonymous amino acid substitution refers to any nucleotide mutation that alters the amino acid sequence of a protein.
  • the amino acid substitution results from at least one of: (i) a missense mutation, i.e., a nonsynonymous substitution that arises from a point mutation in a single nucleotide, (ii) a nonsense mutation (nonsynonymous substitution that arises when a mutation causes a protein to prematurely terminate by changing the amino acid to a stop codon); and, (iii) a nonstop mutation or readthrough mutation, which occurs when a stop codon is exchanged for an amino acid codon, causing the protein to be longer than specified.
  • the amino acid substitution of M2 protein results from a missense mutation.
  • the amino acid substitution in M2 protein comprises at least one amino acid substitution. Examples for mutations that lead to aminoadamantane resistance are known in the art, such as amino acid substitution at amino acid residue selected from: 27, 30, 31, and 34, as described in Hay AJ et al. "The molecular basis of the specific anti -Influenza Action of amantadine.” EMBO J 1985; 4:3021-3024, herein incorporated by reference in its entirety.
  • the amino acid substitution in M2 protein comprises at least one amino acid substitution selected from: S3 IN, V27A, V27G, V27D, I27S, I27T, 127 A, A30T, A30P, and G34E.
  • Examples for M2 mutations that provide resistance to an 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, herein incorporated by reference in its entirety.
  • an avian strain comprising A/Chicken/Germany/34 or H7N1 Rostock or A/Chicken/Germany/27 or H7N7 Weybridge is known to be resistant to amantadine.
  • H3N2 and H1N1 strains are resistant to amantadine.
  • at least one of the mutations selected from: S3 IN (serine 31 to asparagine) and V27A (valine 27 to alanine) results in aminoadamantane resistance (e.g., in the case of WSN/33 H1N1 strain).
  • the Influenza A disclosed herein comprises an aminoadamantane-resistant variant of HIN1.
  • the aminoadamantane- resistant variant of HIN1 comprises S3 IN amino acid substitution.
  • the present invention provides a method of treating or preventing influenza disease or at least one symptom associated therewith. In some embodiments, the present invention provides a method of treating or preventing a disease induced by an 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: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, or any combination thereof.
  • a molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, or any combination thereof.
  • the method comprising administering to the subject a therapeutically effective amount of a molecule selected from: Fludarabine, a derivative thereof, or a metabolite thereof, Asunaprevir, Ravuconazole, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, or a metabolite thereof, and any combination thereof.
  • Fludarabine derivative is selected from: Vidarabine, Nelarabine, Cordycepin, Clofarabine, Cladribine, or any metabolite thereof.
  • Fludarabine derivative is selected from: Vidarabine, Nelarabine, Cordycepin, or any metabolite thereof.
  • Fludarabine derivative is selected from: Vidarabine, Nelarabine, or any metabolite thereof.
  • Fludarabine derivative is selected from: Vidarabine or any metabolite thereof.
  • a metabolite of Vidarabine is or comprises arainosine.
  • a xanthine derivative is selected from: theobromine, 1 -methyl xanthine, 3-methyl xanthine, caffeine, theophylline, enprofylline, paraxanthine, 7- methylxanthine, or any combination thereof.
  • the xanthine derivative is selected from: theobromine, 1 -methyl xanthine, 3-methyl xanthine, theophylline, 7-methylxanthine, or any combination thereof.
  • the xanthine derivative is or comprises theobromine.
  • the theobromine is a theobromine precursor.
  • the theobromine precursor is or comprises caffeine, as long as it metabolizes to a therapeutically effective amount of theobromine.
  • a vidarabine metabolite comprises or is arainosine.
  • the present invention provides compositions comprising a blocker of the M2 channel from an Influenza A, for treating or preventing Influenza A virulence in a subject.
  • the present invention in some embodiments, provides compositions comprising an M2 protein channel blocker for preventing Influenza A virus cell entry, uncoating and/or release from a cell.
  • the present invention in some embodiments, is based, on the finding that at least one molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, inhibits M2 channel from an Influenza A, and therefore, can be used to treat and/or prevent Influenza A virulence.
  • the Influenza A strain that can be treated or prevented by the at least one of the abovementioned molecules is resistant to an aminoadamantane.
  • matrix protein 2 or M2 protein of Influenza A virus (A/Bellamy/1942 H1N1 strain) is disclosed under GenBank Accession no: ABW75846.1.
  • M2 protein of Influenza A virus comprises the amino acid sequence:
  • the M2 protein of influenza 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.
  • the M2 protein comprises an analog of SEQ ID NO: 1 having homology within the range of: 85-100%, 91-100%, and 96-100%.
  • Each possibility represents a separate embodiment of the invention.
  • analog refers to a polypeptide that is similar, but not identical, to the polypeptide of the invention that still belongs to the Influenza A virus.
  • the analog has resistance to aminoadamantane.
  • An analog may have deletions or mutations that result in an amino acids sequence that is different than the amino acid sequence of the polypeptide of the invention. It should be understood that all analogs of the polypeptide of the invention would still be capable of generating an ion channel. Further, an analog may be analogous to a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the polypeptide of the invention.
  • the invention provides a method of treating or preventing an influenza virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an influenza M2 protein channel blocker, thereby treating or preventing an influenza virulence in the subject.
  • the invention provides a method of preventing an influenza release from a cell, the method comprising contacting the cell with an influenza M2 protein channel blocker, thereby preventing an influenza release from the cell.
  • the invention provides a method of preventing an influenza cell entry, the method comprising contacting the cell with an influenza M2 protein channel blocker, thereby preventing an influenza cell entry.
  • the invention provides a method of preventing an influenza uncoating, the method comprising contacting the cell with an influenza M2 channel blocker, thereby preventing an influenza uncoating.
  • the cell is a cell of a subject.
  • contacting is administering to the subject.
  • the subject is a subject infected or suspected as being infected by an Influenza virus.
  • the Influenza virus comprises Influenza A virus.
  • the Influenza virus comprises H1N1 subtype.
  • the Influenza virus is resistant to aminoadamantanes.
  • the influenza M2 channel blocker is at least one molecule selected from: Fludarabine or a salt thereof, Asunaprevir or a salt thereof, Ravuconazole or a salt thereof, Amikacin or a salt thereof, Theobromine or a salt thereof, Flunisolide or a salt thereof, Alvimopan or a salt thereof, Eliglustat or a salt thereof, CM4620 or a salt thereof, Levamlodipine or a salt thereof, Emamectin or a salt thereof, Grazoprevir or a salt thereof, Isavuconazole or a salt thereof, Voriconazole or a salt thereof, Paritaprevir or a salt thereof, Vidarabine or any metabolite thereof, or a salt thereof, and any combination thereof.
  • the influenza M2 channel blocker is at least one molecule selected from: Fludarabine or a salt thereof, Asunaprevir or a salt thereof, Amikacin or a salt thereof, Theobromine or a salt thereof, Flunisolide or a salt thereof, Alvimopan or a salt thereof, Levamlodipine or a salt thereof, Grazoprevir or a salt thereof, Voriconazole or a salt thereof, Paritaprevir or a salt thereof, Vidarabine or any metabolite thereof or a salt thereof, and any combination thereof.
  • influenza M2 channel blocker comprises treatment with two molecules selected from: Theobromine + Vidarabine or a metabolite thereof, and Vidarabine or a metabolite thereof + Grazoprevir.
  • influenza M2 channel blocker comprises treatment with Vidarabine or a metabolite thereof and at least one molecule selected from Theobromine and Grazoprevir.
  • influenza M2 channel blocker comprises treatment with two molecules selected from: Theobromine + arainosine, and arainosine + Grazoprevir.
  • influenza M2 channel blocker comprises treatment with arainosine and at least one molecule selected from Theobromine and Grazoprevir.
  • the treatment of a subject in need thereof with influenza M2 channel blocker comprises treatment with Theobromine and Vidarabine or a metabolite thereof.
  • the invention provides an M2 channel blocker for use in treating or preventing an influenza virulence in a subject in need thereof.
  • the invention provides an M2 channel blocker for use in preventing influenza release from a cell.
  • the M2 channel blocker is within a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • the invention provides a pharmaceutical composition comprising Fludarabine, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises an H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises fludarabine, an analog or a salt thereof.
  • Fludarabine includes Fludarabine (CAS: 21679-14-1, IUPAC: (2R,3S,4S,5R)-2-(6-amino-2-fluoropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • fludarabine comprises a chemotherapy medication used in the treatment of leukemia and/or lymphoma.
  • the invention provides a pharmaceutical composition comprising Asunaprevir, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Asunaprevir, an analog or a salt thereof.
  • Asunaprevir as used herein, includes Asunaprevir (formerly BMS-650032, CAS: 630420-16-5, IUPAC: 3-Methyl-A- ⁇ [(2-methyl-2-propanyl)oxy]carbonyl]-L-valyl-(4R)-4-[(7- chloro-4-methoxy- 1 -isoquinolinyl)oxy ] -N- ⁇ ( 17?, 2S)- 1 - [(cyclopropylsulfonyl)carbamoyl] -2- vinylcyclopropyl ⁇ -L-prolinamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Ravuconazole, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Ravuconazole, an analog or a salt thereof.
  • Ravuconazole includes Ravuconazole (also named BMS-207147 or ER- 30346, CAS: 182760-06-1, IUPAC: 4-[2-[(2R,3R)-3-(2,4-Difluorophenyl)-3-hydroxy-4-(l,2,4- triazol-l-yl)butan-2-yl]-l,3-thiazol-4-yl]benzonitrile), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Amikacin, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises amikacin, an analog or a salt thereof.
  • Amikacin includes amikacin (CAS: 37517-28-5, IUPAC: (2S)-4-Amino- N-[(2S,3S,4R,5S)-5-amino-2-[(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6- (hydroxymethyl)oxan-2-yl]oxy-4-[(2R,3R,4S,5R,6R)-6-(aminomethyl)-3,4,5-trihydroxy-oxan- 2-yl]oxy-3-hydroxy-cyclohexyl]-2-hydroxybutanamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Theobromine, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Theobromine, an analog or a salt thereof.
  • Theobromine includes Theobromine (CAS: 83-67-0, IUPAC: 3,7- dimethyl-lH-purine-2, 6-dione), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Flunisolide, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Flunisolide, an analog or a salt thereof.
  • Flunisolide includes Flunisolide (CAS: 3385-03-3, IUPAC: (lS,2S,4R,8S,9S,HS,12S,13R,19S)-19-fluoro-ll-hydroxy-8-(2-hydroxyacetyl)-6,6,9,13- tetramethyl-5,7-dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Alvimopan, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • the influenza M2 channel blocker comprises Alvimopan, an analog or a salt thereof.
  • Alvimopan includes Alvimopan (CAS: 156053-89-3, IUPAC: 2-([(2S)- 2-([(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-l-yl]methyl) -3- phenylpropanoyl]amino)acetic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Eliglustat, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Eliglustat, an analog or a salt thereof.
  • Eliglustat includes Eliglustat (CAS: 491833-29-5, IUPAC: N-[(1R,2R)- l-(2,3-Dihydro-l,4-benzodioxin-6-yl)-l-hydroxy-3-(l-pyrrolidinyl)-2-propanyl]octanamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising CM4620, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • the influenza M2 channel blocker comprises CM4620, an analog or a salt thereof.
  • CM4620 includes CM4620 (CAS: 1713240-67-5, IUPAC: N-[5-(6- chloro-2,2-difluoro-l,3-benzodioxol-5-yl)pyrazin-2-yl]-2-fluoro-6-methylbenzamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Levamlodipine, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Levamlodipine, an analog or a salt thereof.
  • Levamlodipine includes Levamlodipine (also named levoamlodipine or S-amlodipine, CAS: 103129-82-4, IUPAC: (S)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4- (2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5-dicarboxylate), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • Levamlodipine also named levoamlodipine or S-amlodipine, CAS: 103129-82-4
  • IUPAC (S)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4- (2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5-dicarboxylate
  • the invention provides a pharmaceutical composition comprising Emamectin, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Emamectin, an analog or a salt thereof.
  • Emamectin includes Emamectin (CAS: 119791-41-2 or 155569-91- 8, also named 4"-Deoxy-4''-epi-methylamino-avermectin Bl, Epi-methylamino-4' '-deoxy- avermectin, MK 243, EMA, or GWN 1972), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Grazoprevir, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • the influenza M2 channel blocker comprises Grazoprevir, an analog or a salt thereof.
  • Grazoprevir includes Grazoprevir (also named MK-5172, CAS: 1350514-68-9, IUPAC: 1R,18R,2OR,24S,27S)-A- ⁇ (1R,2S)-1-
  • the invention provides a pharmaceutical composition comprising Isavuconazole, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Isavuconazole, an analog or a salt thereof.
  • Isavuconazole includes Isavuconazole or Isavuconazonium sulfate (CAS: 742049-41-8, 946075-13-4, or 241479-67-4, IUPAC: 4- ⁇ 2-[(lR,2R)-(2,5-Difluorophenyl)-2- hydroxy-l-methyl-3-(lH-l,2,4-triazol-l-yl)propyl]-l,3-thiazol-4-yl]benzonitrile), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • Isavuconazonium is hydrolyzed by esterases in blood or the gastrointestinal tract to the active form comprising Isavuconazole.
  • the invention provides a pharmaceutical composition comprising Voriconazole, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza A virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • the influenza M2 channel blocker comprises Voriconazole, an analog or a salt thereof.
  • Voriconazole includes Voriconazole (CAS: 137234-62-9, IUPAC: (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-l-(lH-l,2,4-triazol-l-yl)butan-2- ol), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • the invention provides a pharmaceutical composition comprising Paritaprevir, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza A virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises Paritaprevir, an analog or a salt thereof.
  • Paritaprevir includes Paritaprevir (also known as ABT-450, CAS: 1216941-48-8, IUPAC: (2R,6S,12Z,13aS,14aR,16aS)-N-(Cyclopropylsulfonyl)-6- ⁇ [(5-methyl- 2-pyrazinyl)carbonyl] amino ⁇ -5,16-dioxo-2-(6-phenanthridinyloxy)- l,2,3,6,7,8,9,10,l l,13a,14,15,16,16a-tetradecahydrocyclopropa[e]pyrrolo [1,2- a][l,4]diazacyclopentadecine-14a(5H)-carboxamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • Paritaprevir also known as ABT-450, CAS: 1216941-48-8
  • IUPAC (2R,6S,12Z
  • the invention provides a pharmaceutical composition comprising Vidarabine, a metabolite thereof, an analog or a salt thereof, for treating a viral infection.
  • the viral infection comprises an Influenza virus infection.
  • the viral infection comprises an Influenza A virus infection.
  • the Influenza A virus infection comprises a H1N1 virus infection.
  • the viral infection comprises a virus infection resistant to aminoadamantane.
  • the viral infection comprises an infection by a virus comprising an influenza M2 protein.
  • influenza M2 channel blocker comprises vidarabine, a metabolite thereof, an analog or a salt thereof.
  • Vidarabine includes Vidarabine (also known as 9-[3-D- arabinofuranosyladenine or ara-A, CAS: 24356-66-9, IUPAC: (2R,3S,4S,5R)-2-(6-Amino-9H- purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol hydrate), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
  • a metabolite of Vidarabine is or comprises arainosine.
  • treatment encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
  • a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
  • administering refers to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
  • the terms “subject” or “individual” or “animal” or “patient” or “mammal” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
  • a therapeutically effective dose of the composition of the invention is administered.
  • therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • a therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the route of administration of the pharmaceutical compositions will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art.
  • compositions of the invention can be lower than when administered via parenteral injection, by using appropriate compositions it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment.
  • the composition of the invention comprising oral delivery.
  • the composition of the invention comprises an oral composition.
  • the composition of the invention further comprises orally acceptable carrier, excipient, or a diluent.
  • influenza M2 channel blocker is for use at a daily dose of 0.01 to 500 mg/kg.
  • the influenza M2 channel blocker comprises Fludarabine or a salt thereof and is for use at a daily dose between about 0.1 mg/m 2 /day and about 50 mg/m 2 /day, 1 mg/m 2 /day and 30 mg/kg/day, and 0.1 mg/m 2 /day and 10 mg m 2 /day.
  • the influenza M2 channel blocker comprises Asunaprevir or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/day, about 5 mg/kg/day and 60 mg/kg/day, and 20 mg/kg/day and 50 mg/kg/day.
  • the influenza M2 channel blocker comprises Ravuconazole acid or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/kg/day, about 1 mg/kg/day and 50 mg/kg/day, and 5 mg/kg/day and 30 mg/kg/day.
  • the influenza M2 channel blocker comprises Amikacin or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/kg/day, about 5 mg/kg/day and 50 mg/kg/day, and 10 mg/kg/day and 20 mg/kg/day.
  • the influenza M2 channel blocker comprises Theobromine or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 200 mg/kg/day, about 5 mg/kg/day and 100 mg/kg/day, and 10 mg/day and 50 mg/kg/day.
  • the influenza M2 channel blocker comprises Flunisolide or a salt thereof and is for use at a daily dose of between about 0.1 mg/kg/day and about 50 mg/kg/day, about 0.1 mg/kg/day and 20 mg/kg/day, and 0.2 mg/kg/day and 5 mg/kg/day.
  • the influenza M2 channel blocker comprises Alvimopan or a salt thereof and is for use at a daily dose of between about 0.1 mg/kg/day and about 50 mg/kg/day, about 0.1 mg/kg/day and 10 mg/kg/day, and 0.5 mg/kg/day and 5 mg/kg/day.
  • the influenza M2 channel blocker channel blocker comprises Lavamlodipine or a salt thereof and is for use at a daily dose between about 0.01 mg/kg/day and about 10 mg/kg/day, 0.01 mg/kg/day and 5 mg/kg/day, and 0.01 mg/kg/day and 1 mg/kg/day.
  • the influenza M2 channel blocker comprises Grazoprevir or a salt thereof and is for use at a daily dose of between about 0.1 mg/kg/day and about 50 mg/kg/day, about 0.1 mg/kg/day and 10 mg/kg/day, and 0.5 mg/kg/day and 5 mg/kg/day.
  • the influenza M2 channel blocker comprises Voriconazole or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 50 mg/kg/day, about 2 mg/kg/day and 20 mg/kg/day, and 2 mg/kg/day and 10 mg/kg/day.
  • the influenza M2 channel blocker comprises Paritaprevir or a salt thereof and is for use at a daily dose of between about 0.5 mg/kg/day and about 500 mg/day, about 1 mg/kg/day and 300 mg/day, and 0.5 mg/day and 10 mg/day.
  • the influenza M2 channel blocker comprises Vidarabine or a metabolite thereof, or a salt thereof and is for use at a daily dose of between about 0.5 mg/kg/day and about 100 mg/day, about 1 mg/kg/day and 50 mg/day, and 5 mg/day and 40 mg/day.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, or excipient.
  • carrier refers to any component of a pharmaceutical composition that is not the active agent.
  • pharmaceutically acceptable carrier refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
  • sugars such as lactose, glucose and sucrose, starches such as com starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered 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, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline, Ringer's solution; ethyl oleate, Ringer's solution;
  • substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered 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), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations.
  • 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 may be used to formulate the compositions contemplated herein.
  • Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of 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); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety.
  • CTFA Cosmetic, Toiletry, and Fragrance Association
  • Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.
  • compositions may also be contained in artificially created structures such as liposomes, ISCOMS, slow -releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides 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 generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally determined by considerations such as liposome size and stability in the blood.
  • a variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • the carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.
  • kits comprising at least two molecules selected from the group consisting of: Fludarabine or a derivative thereof, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Eevamlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
  • the molecule is selected from a group consisting of: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Eevamlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
  • the kit further comprises instructions for mixing the at least two molecules selected from the group consisting of: Fludarabine or a derivative thereof, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
  • the Fludarabine derivative comprises Vidarabine or a metabolite thereof.
  • the metabolite of Vidarabine is Arainosine.
  • the xanthine derivative is Theobromine.
  • the at least two molecules are packaged within a container.
  • the container is made of a material selected from: thin-walled film or plastic (transparent or opaque), paperboard-based, foil, rigid plastic, metal (e.g., aluminum), glass, etc.
  • the content of the kit is packaged, as described below, to allow for storage of the components until they are needed.
  • kits may be packaged in suitable packaging to maintain sterility.
  • the at least two molecules are stored in separate containers within the main kit containment element e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit.
  • the dosage amount of the at least two molecules provided in a kit may be sufficient for a single application or for multiple applications.
  • the kit may have multiple dosage amounts of the at least two molecules packaged in a single container, e.g., a single tube, bottle, vial, 1.5-2 ml tube, e.g., Eppendorf, and the like.
  • the kit may have multiple dosage amounts of the at least two molecules individually packaged such that certain kits may have more than one container of the at least two molecules.
  • multiple dosage amounts of the at least two molecules may be packed in single separate containers.
  • the kit contains instructions for preparing the composition used therein and for how to practice the methods of the invention.
  • the kit further comprises a measuring utensil such as syringe, measuring spoon or a measuring cup.
  • the instructions may be recorded on a suitable recording medium or substrate.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • a method of screening effectiveness of an agent in treating or preventing an Influenza virus infection comprising providing a cell comprising a membrane permeabilized by the influenza M2 channel, contacting the cell with the agent, and determining effect of the agent on growth of the cell, wherein a substantial effect of the agent on cellular growth is indicative of the agent as being effective for treating or preventing an influenza infection, thereby screening effectiveness of an agent in treating or preventing an influenza infection.
  • the Influenza virus comprises Influenza A virus.
  • the Influenza A virus comprises H1N1 subtype.
  • the Influenza virus is resistant to aminoadamantanes.
  • the method comprises a negative assay.
  • the cell is characterized by growth retardation due to the membrane permeabilized by the influenza M2 channel.
  • an agent that alleviates growth retardation is indicative as being effective for treating or preventing an Influenza virus infection.
  • the method comprises a positive assay.
  • the cell is a K + -uptake deficient cell that is incapable of growth in low [K + ] media but experiences growth due to the channel formed by an influenza M2 channel.
  • an agent that induces growth retardation is indicative as being effective for treating or preventing an influenza infection.
  • the method comprises performing both the negative assay and the positive assay.
  • the method further comprises an acidity assay.
  • the cell is a cell comprising a pH-reporter gene or a protein product thereof, such as, but not limited to, pH-sensitive green fluorescent protein (GFP).
  • GFP pH-sensitive green fluorescent protein
  • H + influx to the cell, grown in media supplemented with an acidic solution, is induced.
  • an agent that blocks the pH change within the cell is suitable for treating or preventing an influenza infection.
  • the method further comprises a validation step, comprising examination of the molecules that positively passed all three bacteria assays in a mammaliancell based assay.
  • the validation step requires infection of the mammalian cell with Influenza A virus and examination of the molecules on cell viability and/or growth.
  • Non-limiting examples for growing a bacterial cell applicable for the screening methods provided herein include: Astrahan, P. et al., Acta 1808, 394-8 (2011); Santner, P. et al. Biochemistry 57, 5949-5956 (2016), and Taube, R., Alhadeff, R., Assa, D., Krugliak, M. & Arkin, I. T. PLoS One 9, el05387 (2014).
  • the assay comprises determining susceptibility of the virus to develop resistance against the agent.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm ⁇ 100 nm.
  • Bacteria cultures were grown overnight and finally diluted 500 fold and again set to grow until their O.D.600 reached 0.2. From the culture 50 l were added in 96-well flat- bottomed plates which was pre-treated with 50 pl of particualr chemicals and the isopropyl-P-d- 1 -thiogalactopyranoside (IPTG) inducer. Induction was achieved by employing different concentration of IPTG.
  • IPTG isopropyl-P-d- 1 -thiogalactopyranoside
  • a multi-plate incubator infinite M200 pro from Tecan Group; Mannedorf, Switzerland or LogPhase 600 from BioTek; Santa Clara, CA, USA was used to incubate the plates for 16 hours at 37 °C at a constant, shaking rate (700 rpm). Bacterial growth was monitored by measuring O.D.600 every 15 min. Duplicates, or triplicates were conducted for every measurement.
  • the positive assay employed the same protocol as the negative assay but in this instance a K + -uptake deficient bacteria strain was used . Additionally, overnight growth was conducted in LB media in which KC1 at different concentrations as noted replaced NaCl.
  • the acidity assay is based on bacteria expressing a chromosomal copy of a pH-sensitive GFP. Overnight bacterial cultures were diluted to 1:500 in LB media and subsequently grown to an O.D.600 of 0.6-0.8. Protein expression was induced by IPTG at different concentrations, as noted. After one hour of induction, the cells were diluted to an O.D.600 of 0.2 and pelleted at 3,500 g for 10 min. Subsequently, the cells were resuspended in McILvaine Buffer, which contains 200 mM Na2HPO4 and 0.9% NaCl adjusted to pH 7.6 with 0.1 M citric acid.
  • a chemical library was purchased from MedChem Express (HY-L035, Monmouth Junction, NJ, USA). At the time, the library contained 2839 repurposed drugs, noting that the number of chemicals changes with time. Each chemical was tested at a final concentration of 100 pM. The final concentration of dimethyl sulfoxide was 2%. All manipulations and growths were conducted on a robotic system (EVO 75 Tecan, Mannedorf, Switzerland) or LogPhase 600 microbiology reader (Agilent, Santa Clara, CA, USA).
  • IPTG Isopropyl-P-d- 1 -thiogalactopyranoside
  • Lysogeny broth was used for most of the cases only LBK was used for positive assay, where NaCl is replaced with KCL at lOgm/lt. All media contained ampicillin at 100 g/ml.
  • Assessment of antiviral activity of compounds was conducted in a BSL-3 facility of the Hebrew University as follows: Madin-Darby canine kidney (MDCK) [ATCC MDCK NBL-2] cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM); (Biological Industries; Beit Haemek, Israel ), supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 10 lU/mL Penicillin, and 10 pg/mL streptomycin, Biomycin-3.
  • MDCK Madin-Darby canine kidney
  • DMEM Modified Eagle Medium
  • Influenza A Virus, A/Wisconsin/629-D02452/2009 (H1N1) pdm09 was deposited by the Center for Disease Control and Prevention and obtained through BEI Resources, NIAID, and NIH. Identical aliquots of virus stock were prepared from the mother stock supplied by BEI resources. For infection 1:1000 times diluted sub stocks were prepared from each aliquot.
  • MDCK cells were seeded in 96-well flat bottom plates in 200 pL of medium at a density of 15,000 cells per well and grown overnight.
  • the dilutions of tested compounds were prepared in MEM with 0.3% BSA, 3 pg/ml TPCK treated trypsin and 50 pL was added to the cells.
  • the effects of the drugs on the metabolic activity of MDCK cells were assessed at 48 h post treatment using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation reagent (Promega; Madison, WI, USA).
  • CellTiter 96 Aqueous Non-Radioactive Cell Proliferation reagent Promega; Madison, WI, USA.
  • cells were infected with the Influenza-A virus of 400 TCID 50/well for two hours followed by treatment with listed drugs.
  • each compound concentration was tested in triplicate and each assay plate contained the following controls: no cells (background control), cells treated with medium (mock infection for normalization), infected/untreated cells and infected/solvent-treated cells (infection control).
  • drug efficacies in control of toxicity were assessed by the CellTiter 96 Aqueous NonRadioactive Cell Proliferation reagent (Promega; Madison, Wisconsin, United States) for 3 h at 37°C in a 5% CO2 atmosphere. Reactions were stopped and the virus inactivated by adding 30 pl of 4% formaldehyde. Absorbance was measured at 492 nm using a Tecan plate reader (Mannedorf, Switzerland). Finally, the data were normalized to the mock-infected control, after which EC50 values were calculated by fitting the data to a Monod equation.
  • mice are infected with the H1N1 virus and are given oral treatment (BID) for five days. After nine days, the amount of virus in the lungs is quantitated by q-RT PCR.
  • BID oral treatment
  • BALB/c mice were chosen. At first the inventors did tolerability and pharmacokinetics studies for corresponding drugs by taking 3 mice per group of a total 5 groups.
  • Drug combinations (1:1 molar ratio) for the groups are as follows: (i) 1.5mg/kg Arainosine and 1 mg/kg Theobromine, (ii) 4.5 mg/kg Arainosine and 3 mg/kg Theobromine, (iii) 15 mg/kg Arainosine and 10 mg/kg Theobromine, (iv) 45 mg/kg Arainosine and 30 mg/kg Theobromine and, (v) 150 mg/kg Arainosine and 100 mg/kg Theobromine.
  • a total of 56 numbers of BALB/c mice were divided into 7 groups, so each group contains 8 mice for this set of experiments.
  • the primary assay that was employed involved the expression of the viral channel at increasing levels in “ordinary” Escherichia coli. At a certain viroporin concentration, growth retardation is observed due to excessive membrane permeabilization that hampers bacterial bioenergetics. This test is therefore eponymously termed a negative assay due to the detrimental impact of the protein on the bacteria.
  • the second assay entailed expressing the viral channel at lower levels in K + -uptake deficient bacteria. Such bacteria are incapable of growing in regular media unless they are supplemented by potassium or when they express a channel capable of K + transport. In this instance, the viral channel impacts the bacteria favorably, and the assay is therefore termed positive assay. It should be noted that at high induction levels, while alleviating the K + shortage in the bacteria, the channel impacts the bacteria negatively due to excessive membrane permeabilization akin to the negative assay discussed above.
  • the final assay that examines channel activity is based on a channel's impact on the cytoplasmic pH of bacteria.
  • a concentrated acid is injected into the media, the cytoplasmic pH drops if the bacteria express a channel capable of H + transport.
  • a change in cytoplasmic pH can be detected by monitoring the fluorescence of a chromosomally-expressed, pH-sensitive GFP.
  • No channel refers to the absence of M2 channel induction by IPTG
  • no drug refers to maximum induction of the M2 channel protein by IPTG, in the absence of a drug.
  • the IPTG concentrations were lOOpM, 20pM, and 50pM in the negative, positive, and acidity assays, respectively ( Figures 4-6).
  • Amantadine (Symmetrel®) is a known channel blocker of the M2 channel and as such was expected to be identified in the screen (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).
  • the inventors next sought to characterize the anti-viral activity of the channel blockers that were identified, in tissue culture cells. For that purpose, the inventors cultured Madin-Darby canine kidney (MDCK) cells and examined their viability after viral infection with aminoadamantane -resistant H1N1 and the ability of the drugs to impact the cell viability thereupon.
  • the viral strain that was used was H1N1 that is known to be resistant to aminoadamantanes due to a mutation of Ser31Asn in its M2 protein.
  • Tamiflu® and Avigan® exhibited potent anti-influenza Activity (thus served as positive controls), while gratifyingly, Rimantadine (Flumadine®) was completely ineffective since the infection was conducted with the H1N1 virus strain that is resistant to aminoadamantanes (thus served as negative control).
  • mice were infected with the H1N1 virus and given oral treatment (BID) for five days, as described above. After nine days, the amount of virus in the lungs was quantitated by q- RT-PCR. The results show that even though the herein disclosed drug combination comprising Theobromine + Arainosine was administered at a significantly lower dosage, it was more effective than oseltamivir, the leading drug on the market (Figure 17).
  • the inventors conducted a structure-activity relationship (SAR) analysis examining the effect of xanthine and derivatives (Figure 18) thereof on cell viability.
  • SAR structure-activity relationship
  • Figure 18 the effect of xanthine and derivatives
  • Figure 19 several close-related xanthine derivatives improved cell survival.
  • theobromine was found to increase cell survival at concentrations of 0.3 pM-10 pM, to a great extent compared to control.
  • caffeine which is very similar to Theobromine, did not improve cell viability compared to no-drug control.
  • a similar trend was observed for Enprophylline, and paraxanthine.
  • Theophylline and 3 -methylxanthine were found to increase cell survival at any of the tested concentrations (0.01 pM-1 pM), to a great extent compared to control. 7-methylxanthine was found to increase cell survival at concentrations of 1 pM-10 pM), to a great extent compared to control.
  • the inventors conducted a S AR analysis examining the effect of Fludarabine and derivatives (Figure 22) thereof on cell viability. The results show that Fludarabine and several close-related derivatives thereof have improved cell survival compared to control ( Figure 22). In that regard, Vidarabine was found to increase cell survival at concentrations of 0.3 pM-10 pM, to a great extent compared to control. Nelarabine and Corycepin improved cell survival at concentrations of 10 pM and 1 pM-10 pM, respectively.
  • combinations comprising Vidarabine at a concentration of 300 nM and 1 -methyl xanthine at a concentration of 10 nM-300 nM increased cell viability by about 33-37%, compared to negative control (no drug).
  • Combinations comprising Vidarabine at a concentration of 300 nM and xanthine at a concentration of 10 nM-300 nM increased cell viability by about 27-48%, compared to control.
  • Combinations comprising Vidarabine at a concentration of 10 nM or 100 mM and 3- methyl xanthine at a concentration of 30 nM increased cell viability by about 63% and 28%, respectively compared to control.
  • Combinations comprising vidarabine at a concentration of 30 nM or 100 mM and 3 -methyl xanthine at a concentration of 100 nM increased cell viability by about 63% and 46%, respectively compared to control.
  • Combinations comprising Vidarabine and Theobromine had a profound positive effect on cell viability.
  • Combinations comprising Vidarabine and Theobromine at any of the tested concentrations e.g., from 10 nM to 300 mM (of each compound) increased cell viability by about 37% to 98%, compared to control.
  • combinations comprising Vidarabine at a concentration of 10 nM to 300 mM and Theobromine at any concentration of 30 nM to 300 nM increased cell viability by about 64% to 98%, compared to control.
  • the inventors examined the effect of Theobromine, and Arainosine, a natural metabolite of Vidarabine, on cell viability.
  • the results show that combinations comprising Arainosine at a concentration of 100 nM or 300 mM and Theobromine at a concentration of 10 nM increased cell viability by about 10% to 75%, compared to control.
  • combinations comprising Arainosine at a concentration of 10 nM to 300 mM and Theobromine at any concentration of 30 nM to 300 nM increased cell viability by about 61% to 100%, compared to control.

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Abstract

Pharmaceutical compositions including an Influenza A M2 protein channel blocker for treating or preventing virulence of an Influenza A, such as, H1N1 subtype resistant to aminoadamantane, in a subject, are provided. Further provided is a pharmaceutical composition comprising a blocker of Influenza A M2 protein channel for preventing Influenza A cell entry, uncoating and/or release from a cell.

Description

VIROPORINS BLOCKERS/INHIBITORS AS ANTI-INFLUENZA AGENTS
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[001] The contents of the electronic sequence listing (VRB-P-004-PCT ST26.xml; size: 2,035 bytes; and date of creation: December 6, 2023) is herein incorporated by reference in its entirety.
CROSS-REFERENCE TO RELATED APPLICATIONS
[002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/432,446, titled “VIROPORINS BLOCKERS/INHIBITORS AS ANTI-INFLUENZA AGENTS”, filed 14 December 2022, the contents of which are incorporated herein by reference in their entirety
FIELD OF INVENTION
[003] The present invention is in the field of anti-viral therapy.
BACKGROUND
[004] Influenza is a segmented, single-stranded negative-sense RNA virus that belongs to the Orthomyxoviridae. It represents one of the leading causes of morbidity and mortality due to an infectious disease. In particular, before COVID- 19, it was the primary communicable cause of death in the western world. Combating influenza is achieved by vaccines and antiviral therapy. However, constant genomic shifts and drifts continuously vitiate vaccine effectiveness and antiviral drug benefits. For example, the annual vaccine protection rate revolves around 40%, and resistance to every anti-flu agent has already been reported. In particular, 98% of currently circulating variants are resistant to aminoadamantanes (Symmetrel® and Flumadine®), the first class of antiviral agents developed against influenza in the 1960s (Davies, W. L. et al. "Antiviral Activity of 1-Adamantanamine (Amantadine)." Science 1964; 144:862-863, hereby incorporated by reference in its entirety).
[005] There is an unmet need for methods and compounds for ameliorating or treating the Influenza virus, particularly influenza variants that are resistant to aminoadamantanes. SUMMARY
[006] According to a first aspect, there is provided a method of treating or preventing an 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, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, thereby treating or preventing an Influenza A virulence in the subject.
[007] According to another aspect, there is provided 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 a group consisting of: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
[008] 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, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
[009] In some embodiments, the molecule is selected from a group consisting of: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
[010] In some embodiments, the kit further comprises instructions for mixing the at least two molecules selected from the group consisting of: Fludarabine or a derivative thereof, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
[Oi l] In some embodiments, the Fludarabine derivative comprises Vidarabine or a metabolite thereof.
[012] In some embodiments, the metabolite of Vidarabine is Arainosine.
[013] In some embodiments, the xanthine derivative is Theobromine.
[014] In some embodiments, the kit is for preparing a medicament for treating a subject afflicted with influenza or infected with an Influenza virus. [015] In some embodiments, the molecule is a M2 protein blocker.
[016] In some embodiments, the Influenza A virus is a H1N1 subtype.
[017] In some embodiments, the Influenza A virus is resistant to aminoadamantanes.
[018] In some embodiments, the molecule is for use at a daily dose of 0.01 to 500 mg/kg per body weight of the subject.
[019] In some embodiments, administering is a therapeutically effective amount of 2 molecules selected from: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
[020] In some embodiments, the 2 molecules are Vidarabine or a metabolite thereof, and a molecule selected from: theobromine and grazoprevir.
[021] According to another aspect there is provided 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 a group consisting of: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and Vidarabine or a metabolite thereof.
[022] In some embodiments, the at least two molecules are (i) Vidarabine or a metabolite thereof, and (ii) a molecule selected from: Theobromine and Grazoprevir.
[023] In some embodiments, a metabolite of Vidarabine is or comprises arainosine.
[024] Unless otherwise defined, 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 the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the 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 are not intended to be necessarily limiting.
[025] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and 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. BRIEF DESCRIPTION OF THE DRAWINGS
[026] Fig. 1 includes a graph showing a negative assay of influenza M2 viroporin, in which protein expression is detrimental to bacterial growth. Growth of DH10B bacteria as a function of different concentrations of the protein inducer Isopropyl-P-D-thiogalactoside (IPTG).
[027] Fig. 2 includes a vertical bar graph showing a positive assay of influenza M2 viroporin. Maximal growth rates of K+-uptake deficient bacteria expressing the influenza M2 viroporin is monitored as a function of different K+ concentrations in the presence (10 pM) or absence of the inducer IPTG.
[028] Fig. 3 includes a graph showing an acidity assay of the influenza M2 viroporin. Cytoplasmic H+ concentration is monitored as a function of time whereby at time 0, an acidic solution is injected into the media. Results are shown from bacteria in which viroporin induction is changed a as function of IPTG concentration as noted.
[029] Fig. 4 include a graph showing results of a negative assay used in a blocker screening against influenza M2 viroporin. Bacteria in which the channel expression was not induced (“no channel”), or the drug was not added (“no drug”) were used as controls.
[030] Fig. 5 includes a graph showing results of a positive assay used in a blocker screening against influenza M2 viroporin. Bacteria in which the channel expression was not induced (“no channel”), or the drug was not added (“no drug”) were used as controls.
[031] Fig. 6 includes a graph showing an acidity assay of the influenza M2 viroporin conducted in the presence of the tested drugs.
[032] Fig. 7 includes a vertical bar graph showing cell survival in the presence of various drugs at a concentration of 10 pM (unless indicated otherwise). Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
[033] Fig. 8 includes a vertical bar graph showing cell survival in the presence of various drugs at a concentration of 3 pM (unless indicated otherwise). Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
[034] Fig. 9 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
[035] Fig. 10 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment. [036] Fig. 11 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
[037] Fig. 12 includes a vertical bar graph showing that the effect of the tested drugs on cell viability is dose-dependent. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
[038] Fig. 13 includes graphs showing the half maximal effective concentration (EC50) values of the tested compounds.
[039] Fig. 14 includes graphs showing the half maximal effective concentration (EC50) values of the tested compounds.
[040] Fig. 15 includes a graph showing drug synergism. All drugs were applied at a concentration of 0.01 pM. Dashed line indicates the viability of cells in the ‘vehicle’ treatment.
[041] Fig. 16 includes a vertical bar graph showing relative cell viability after 48 hours of infection, in the presence of various drugs or a combination thereof. It is observed that a combination of Theobromine and Vidarabine synergistically improves cell viability. Further, the effect attributed to the combination of Theobromine and Vidarabine was even more pronounced than that of the approved drugs Oseltamivir, and favipiravir.
[042] Fig. 17 includes a vertical bar graph showing viral RNA per (%) as determined following an in vivo study in mice wherein animals were treated with the indicated drugs. It is observed that a combination Theobromine and arainosine is more effective in vivo than Oseltamivir, the leading drug on the market, even though it is given at a significantly lower dosage.
[043] Fig. 18 includes chemical structures of xanthine and other closely related compounds having a xanthine backbone and further modifications. Positions of modifications are indicated by dashed lines.
[044] Fig. 19 includes a graph showing structure-activity relationship (SAR) analysis of xanthine derivatives. Cell viability was determined as described. Higher values indicate better protection against viral-induced cell death. The different xanthine derivatives including modifications in positions R1-R3 of xanthine, as shown on top.
[045] Fig. 20 includes chemical structures of xanthine and other closely related compounds having a xanthine backbone and further modifications (top row). Positions of modifications are indicated by dashed lines. Further presented are the antiviral drug vidarabine and its natural metabolite arainosine (bottom row). [046] Fig. 21 includes tables summarizing combination therapies with the indicated drug. Higher values indicate better protection against viral-induced cell death. Compounds were used at several concentrations, as presented.
[047] Fig. 22 includes a graph showing SAR analysis of Fludarabine derivatives. Cell viability was determined as described. Higher values indicate better protection against viral-induced cell death. The different Fludarabine derivatives including modifications in positions R1-R5 of Fludarabine, as shown on top.
DETAILED DESCRIPTION
[048] The present invention, in some embodiments, provides compositions comprising a matrix protein 2 or M2 protein channel blocker for treating or preventing an influenza virulence in a subject. The present invention, in some embodiments, provides compositions comprising an influenza M2 protein channel blocker for preventing influenza cell entry, uncoating and/or release from a cell. As used herein, the term “matrix protein 2” and “M2 protein” are used interchangeably.
[049] The present invention, in some embodiments, is based, on the finding that at least one molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, inhibits M2 channel from an Influenza A, and therefore, can be used to treat and/or prevent Influenza A virulence. In some embodiments, the Influenza A strain that can be treated or prevented by the at least one of the abovementioned molecules is resistant to an aminoadamantane .
[050] In some embodiments, at least one molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, or any combination thereof, can be used to treat and/or prevent Influenza A virulence.
M2 protein of Influenza virus
[051] The M2 protein of the 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 M2 channel function is to equilibrate pH across the influenza membrane during cell entry and across the trans-Golgi membrane of an infected cell during viral maturation. In some embodiments, M2 is vital for viral replication. In some embodiments, M2 is a target for the anti-influenza drugs (e.g., amantadine and rimantadine). In some embodiments, M2 protein belongs to the class of viroporins (e.g., small proteins that form ion channels that increase membrane permeability in virus-infected cells). In some embodiments, M2 of Influenza A (also named AM2) and B (also named BM2) are predominantly proton channels. As used herein, the terms “M2 of Influenza A” and “AM2” are interchangeably used. The terms “M2 of influenza B” and “BM2” are interchangeably used. In some embodiments, M2 proteins in influenza C (CM2) and D (DM2), are especially selective for chloride ions, with possibly some permeability to protons.
[052] In some embodiments, the Influenza virus disclosed herein, comprises an Influenza A virus. In some embodiments, the M2 disclosed herein comprises AM2. In some embodiments, the Influenza virus comprises Influenza A and the M2 disclosed herein comprises AM2.
[053] Type A Influenza viruses are commonly divided into subtypes based on the relationships between the antigens hemagglutinin and neuraminidase in the surface glycoproteins. In some embodiments, Influenza A virus comprises at least 16 hemagglutinin subtypes (named H1-H16) and at least nine neuraminidase subtypes (named N1-N9). In some embodiments, each Influenza A virus has one hemagglutinin and one neuraminidase antigen. In some embodiments, Influenza A virus comprises one antigen selected from H1-H16, and one antigen selected from N1-N9, in any combination thereof. In some embodiments, apart from the antigenic similarity on the surface glycoproteins, the subtypes may contain differences in the rest of the genome. In some embodiments, not all the H1N1 (i.e. having the same hemagglutinin 1 and neuraminidase 1 antigens), have the same characteristics.
[054] In some embodiments, Influenza A virus comprises a H1N1 subtype. In some embodiments, the H1N1 Influenza virus comprises M2 protein.
[055] In some embodiments, Influenza virus is resistant to aminoadamantanes. Amantadine (brand names Gocovri, Symadine, and Symmetrel) comprises the organic compound 1- adamantylamine or 1 -aminoadamantane, which consists of an adamantane backbone with an amino group substituted at one of the four methyne positions. Rimantadine (also named flumadine) comprises an adamantane derivative with similar biological properties. In some embodiments, amantadine and rimantadine target the M2 proton channel of Influenza A virus. In some embodiments, an aminoadamantane comprises amantadine (adamantan-1 -amine hydrochloride) and rimantadine (l-(l-Adamantyl)ethanamine hydrochloride). [056] As used herein the term “resistance” or “resistant” refers to reduced ability of the antiviral drug to prevent or to reduce the viral infection. In some embodiments, resistance to aminoadamantane can be observed when aminoadamantanes are applicated prior or post infection. In some embodiments, the nucleic acid encoding M2 protein of the Influenza virus has a mutation that hinders or reduces the ability of an aminoadamantane to inhibit the M2 protein. In some embodiments, the mutation of the nucleic acid encoding M2 protein results in at least one nonsynonymous amino acid substitution. In some embodiments, the virus is or characterized by resistance or being resistant to the drug.
[057] As used herein, the term “nonsynonymous amino acid substitution” refers to any nucleotide mutation that alters the amino acid sequence of a protein. In some embodiments, the amino acid substitution results from at least one of: (i) a missense mutation, i.e., a nonsynonymous substitution that arises from a point mutation in a single nucleotide, (ii) a nonsense mutation (nonsynonymous substitution that arises when a mutation causes a protein to prematurely terminate by changing the amino acid to a stop codon); and, (iii) a nonstop mutation or readthrough mutation, which occurs when a stop codon is exchanged for an amino acid codon, causing the protein to be longer than specified.
[058] In some embodiments, the amino acid substitution of M2 protein results from a missense mutation. In some embodiments the amino acid substitution in M2 protein comprises at least one amino acid substitution. Examples for mutations that lead to aminoadamantane resistance are known in the art, such as amino acid substitution at amino acid residue selected from: 27, 30, 31, and 34, as described in Hay AJ et al. "The molecular basis of the specific anti -Influenza Action of amantadine." EMBO J 1985; 4:3021-3024, herein incorporated by reference in its entirety. In some embodiments, the amino acid substitution in M2 protein comprises at least one amino acid substitution selected from: S3 IN, V27A, V27G, V27D, I27S, I27T, 127 A, A30T, A30P, and G34E.
[059] Examples for M2 mutations that provide resistance to an 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, herein incorporated by reference in its entirety. In some embodiments, an avian strain comprising A/Chicken/Germany/34 or H7N1 Rostock or A/Chicken/Germany/27 or H7N7 Weybridge is known to be resistant to amantadine. In some embodiments, the human strain A/Singapore/1/57 (H2N2) resistant to amantadine, when the latter is implicated prior infection. In some embodiments, several H3N2 and H1N1 strains are resistant to amantadine. [060] In some embodiments, at least one of the mutations selected from: S3 IN (serine 31 to asparagine) and V27A (valine 27 to alanine), results in aminoadamantane resistance (e.g., in the case of WSN/33 H1N1 strain). In some embodiments, the Influenza A disclosed herein comprises an aminoadamantane-resistant variant of HIN1. In some embodiments, the aminoadamantane- resistant variant of HIN1 comprises S3 IN amino acid substitution.
Methods
[061] In some embodiments, the present invention provides a method of treating or preventing influenza disease or at least one symptom associated therewith. In some embodiments, the present invention provides a method of treating or preventing a disease induced by an Influenza A virus in a subject in need thereof. In some embodiments, the method comprising administering to the subject a therapeutically effective amount of a molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, or any combination thereof.
[062] In some embodiments, the method comprising administering to the subject a therapeutically effective amount of a molecule selected from: Fludarabine, a derivative thereof, or a metabolite thereof, Asunaprevir, Ravuconazole, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, or a metabolite thereof, and any combination thereof. [063] In some embodiments, Fludarabine derivative is selected from: Vidarabine, Nelarabine, Cordycepin, Clofarabine, Cladribine, or any metabolite thereof.
[064] In some embodiments, Fludarabine derivative is selected from: Vidarabine, Nelarabine, Cordycepin, or any metabolite thereof.
[065] In some embodiments, Fludarabine derivative is selected from: Vidarabine, Nelarabine, or any metabolite thereof.
[066] In some embodiments, Fludarabine derivative is selected from: Vidarabine or any metabolite thereof.
[067] In some embodiments, a metabolite of Vidarabine is or comprises arainosine.
[068] In some embodiment, a xanthine derivative is selected from: theobromine, 1 -methyl xanthine, 3-methyl xanthine, caffeine, theophylline, enprofylline, paraxanthine, 7- methylxanthine, or any combination thereof. [069] In some embodiments, the xanthine derivative is selected from: theobromine, 1 -methyl xanthine, 3-methyl xanthine, theophylline, 7-methylxanthine, or any combination thereof.
[070] In some embodiments, the xanthine derivative is or comprises theobromine. In some embodiments, the theobromine is a theobromine precursor. In some embodiments, the theobromine precursor is or comprises caffeine, as long as it metabolizes to a therapeutically effective amount of theobromine.
[071] In some embodiments, a vidarabine metabolite comprises or is arainosine.
[072] In some embodiments, the present invention provides compositions comprising a blocker of the M2 channel from an Influenza A, for treating or preventing Influenza A virulence in a subject. The present invention, in some embodiments, provides compositions comprising an M2 protein channel blocker for preventing Influenza A virus cell entry, uncoating and/or release from a cell.
[073] The present invention, in some embodiments, is based, on the finding that at least one molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, inhibits M2 channel from an Influenza A, and therefore, can be used to treat and/or prevent Influenza A virulence.
[074] In some embodiments, at least one molecule selected from: Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, can be used to treat and/or prevent Influenza A virulence.
[075] In some embodiments, the Influenza A strain that can be treated or prevented by the at least one of the abovementioned molecules is resistant to an aminoadamantane.
[076] In some embodiments, at least one molecule selected from: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof, inhibits the M2 channel from an Influenza A.
[077] In some embodiments, matrix protein 2 or M2 protein of Influenza A virus (A/Bellamy/1942 H1N1 strain) is disclosed under GenBank Accession no: ABW75846.1. [078] According to some embodiments, M2 protein of Influenza A virus comprises the amino acid sequence:
MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIVGILHLILWILDRLFFKCIYRLFKHGLK RGPSTEGVPESMREEYRKEQQSAVDADDSHFVNIEL (SEQ ID NO: 1).
[079] According to some embodiments, the M2 protein of influenza 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 within the range of: 85-100%, 91-100%, and 96-100%. Each possibility represents a separate embodiment of the invention.
[080] The term “analog” as used herein, refers to a polypeptide that is similar, but not identical, to the polypeptide of the invention that still belongs to the Influenza A virus. In some embodiments, the analog has resistance to aminoadamantane. An analog may have deletions or mutations that result in an amino acids sequence that is different than the amino acid sequence of the polypeptide of the invention. It should be understood that all analogs of the polypeptide of the invention would still be capable of generating an ion channel. Further, an analog may be analogous to a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the polypeptide of the invention.
[081] According to some embodiments, the invention provides a method of treating or preventing an influenza virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an influenza M2 protein channel blocker, thereby treating or preventing an influenza virulence in the subject.
[082] According to some embodiments, the invention provides a method of preventing an influenza release from a cell, the method comprising contacting the cell with an influenza M2 protein channel blocker, thereby preventing an influenza release from the cell.
[083] According to some embodiments, the invention provides a method of preventing an influenza cell entry, the method comprising contacting the cell with an influenza M2 protein channel blocker, thereby preventing an influenza cell entry.
[084] According to some embodiments, the invention provides a method of preventing an influenza uncoating, the method comprising contacting the cell with an influenza M2 channel blocker, thereby preventing an influenza uncoating. [085] According to some embodiments, the cell is a cell of a subject. According to some embodiments, contacting is administering to the subject. According to some embodiments, the subject is a subject infected or suspected as being infected by an Influenza virus. In some embodiments, the Influenza virus comprises Influenza A virus. In some embodiments, the Influenza virus comprises H1N1 subtype. In some embodiments, the Influenza virus is resistant to aminoadamantanes.
[086] According to some embodiments, the influenza M2 channel blocker is at least one molecule selected from: Fludarabine or a salt thereof, Asunaprevir or a salt thereof, Ravuconazole or a salt thereof, Amikacin or a salt thereof, Theobromine or a salt thereof, Flunisolide or a salt thereof, Alvimopan or a salt thereof, Eliglustat or a salt thereof, CM4620 or a salt thereof, Levamlodipine or a salt thereof, Emamectin or a salt thereof, Grazoprevir or a salt thereof, Isavuconazole or a salt thereof, Voriconazole or a salt thereof, Paritaprevir or a salt thereof, Vidarabine or any metabolite thereof, or a salt thereof, and any combination thereof.
[087] According to some embodiments, the influenza M2 channel blocker is at least one molecule selected from: Fludarabine or a salt thereof, Asunaprevir or a salt thereof, Amikacin or a salt thereof, Theobromine or a salt thereof, Flunisolide or a salt thereof, Alvimopan or a salt thereof, Levamlodipine or a salt thereof, Grazoprevir or a salt thereof, Voriconazole or a salt thereof, Paritaprevir or a salt thereof, Vidarabine or any metabolite thereof or a salt thereof, and any combination thereof.
[088] According to some embodiments, the treatment of a subject in need thereof with influenza M2 channel blocker comprises treatment with two molecules selected from: Theobromine + Vidarabine or a metabolite thereof, and Vidarabine or a metabolite thereof + Grazoprevir. In some embodiments, influenza M2 channel blocker comprises treatment with Vidarabine or a metabolite thereof and at least one molecule selected from Theobromine and Grazoprevir.
[089] According to some embodiments, the treatment of a subject in need thereof with influenza M2 channel blocker comprises treatment with two molecules selected from: Theobromine + arainosine, and arainosine + Grazoprevir. In some embodiments, influenza M2 channel blocker comprises treatment with arainosine and at least one molecule selected from Theobromine and Grazoprevir.
[090] According to some embodiments, the treatment of a subject in need thereof with influenza M2 channel blocker comprises treatment with Theobromine and Vidarabine or a metabolite thereof. [091] According to some embodiments, the invention provides an M2 channel blocker for use in treating or preventing an influenza virulence in a subject in need thereof.
[092] According to some embodiments, the invention provides an M2 channel blocker for use in preventing influenza release from a cell.
[093] According to some embodiments, the M2 channel blocker is within a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
[094] According to some embodiments, the invention provides a pharmaceutical composition comprising Fludarabine, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises an H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[095] According to some embodiments, the influenza M2 channel blocker comprises fludarabine, an analog or a salt thereof.
[096] Fludarabine, as used herein, includes Fludarabine (CAS: 21679-14-1, IUPAC: (2R,3S,4S,5R)-2-(6-amino-2-fluoropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. In some embodiments, fludarabine comprises a chemotherapy medication used in the treatment of leukemia and/or lymphoma.
[097] According to some embodiments, the invention provides a pharmaceutical composition comprising Asunaprevir, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[098] According to some embodiments, the influenza M2 channel blocker comprises Asunaprevir, an analog or a salt thereof.
[099] Asunaprevir as used herein, includes Asunaprevir (formerly BMS-650032, CAS: 630420-16-5, IUPAC: 3-Methyl-A-{ [(2-methyl-2-propanyl)oxy]carbonyl]-L-valyl-(4R)-4-[(7- chloro-4-methoxy- 1 -isoquinolinyl)oxy ] -N- { ( 17?, 2S)- 1 - [(cyclopropylsulfonyl)carbamoyl] -2- vinylcyclopropyl}-L-prolinamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0100] According to some embodiments, the invention provides a pharmaceutical composition comprising Ravuconazole, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0101] According to some embodiments, the influenza M2 channel blocker comprises Ravuconazole, an analog or a salt thereof.
[0102] Ravuconazole, as used herein, includes Ravuconazole (also named BMS-207147 or ER- 30346, CAS: 182760-06-1, IUPAC: 4-[2-[(2R,3R)-3-(2,4-Difluorophenyl)-3-hydroxy-4-(l,2,4- triazol-l-yl)butan-2-yl]-l,3-thiazol-4-yl]benzonitrile), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0103] According to some embodiments, the invention provides a pharmaceutical composition comprising Amikacin, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0104] According to some embodiments, the influenza M2 channel blocker comprises amikacin, an analog or a salt thereof.
[0105] 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)oxan-2-yl]oxy-4-[(2R,3R,4S,5R,6R)-6-(aminomethyl)-3,4,5-trihydroxy-oxan- 2-yl]oxy-3-hydroxy-cyclohexyl]-2-hydroxybutanamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0106] According to some embodiments, the invention provides a pharmaceutical composition comprising Theobromine, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0107] According to some embodiments, the influenza M2 channel blocker comprises Theobromine, an analog or a salt thereof.
[0108] Theobromine, as used herein, includes Theobromine (CAS: 83-67-0, IUPAC: 3,7- dimethyl-lH-purine-2, 6-dione), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0109] According to some embodiments, the invention provides a pharmaceutical composition comprising Flunisolide, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0110] According to some embodiments, the influenza M2 channel blocker comprises Flunisolide, an analog or a salt thereof.
[0111] Flunisolide, as used herein, includes Flunisolide (CAS: 3385-03-3, IUPAC: (lS,2S,4R,8S,9S,HS,12S,13R,19S)-19-fluoro-ll-hydroxy-8-(2-hydroxyacetyl)-6,6,9,13- tetramethyl-5,7-dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0112] According to some embodiments, the invention provides a pharmaceutical composition comprising Alvimopan, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0113] According to some embodiments, the influenza M2 channel blocker comprises Alvimopan, an analog or a salt thereof. [0114] Alvimopan, as used herein, includes Alvimopan (CAS: 156053-89-3, IUPAC: 2-([(2S)- 2-([(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-l-yl]methyl) -3- phenylpropanoyl]amino)acetic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0115] According to some embodiments, the invention provides a pharmaceutical composition comprising Eliglustat, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0116] According to some embodiments, the influenza M2 channel blocker comprises Eliglustat, an analog or a salt thereof.
[0117] Eliglustat, as used herein, includes Eliglustat (CAS: 491833-29-5, IUPAC: N-[(1R,2R)- l-(2,3-Dihydro-l,4-benzodioxin-6-yl)-l-hydroxy-3-(l-pyrrolidinyl)-2-propanyl]octanamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0118] According to some embodiments, the invention provides a pharmaceutical composition comprising CM4620, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0119] According to some embodiments, the influenza M2 channel blocker comprises CM4620, an analog or a salt thereof.
[0120] CM4620, as used herein, includes CM4620 (CAS: 1713240-67-5, IUPAC: N-[5-(6- chloro-2,2-difluoro-l,3-benzodioxol-5-yl)pyrazin-2-yl]-2-fluoro-6-methylbenzamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0121] According to some embodiments, the invention provides a pharmaceutical composition comprising Levamlodipine, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0122] According to some embodiments, the influenza M2 channel blocker comprises Levamlodipine, an analog or a salt thereof.
[0123] Levamlodipine, as used herein, includes Levamlodipine (also named levoamlodipine or S-amlodipine, CAS: 103129-82-4, IUPAC: (S)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4- (2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5-dicarboxylate), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0124] According to some embodiments, the invention provides a pharmaceutical composition comprising Emamectin, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0125] According to some embodiments, the influenza M2 channel blocker comprises Emamectin, an analog or a salt thereof.
[0126] Emamectin, as used herein, includes Emamectin (CAS: 119791-41-2 or 155569-91- 8, also named 4"-Deoxy-4''-epi-methylamino-avermectin Bl, Epi-methylamino-4' '-deoxy- avermectin, MK 243, EMA, or GWN 1972), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0127] According to some embodiments, the invention provides a pharmaceutical composition comprising Grazoprevir, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0128] According to some embodiments, the influenza M2 channel blocker comprises Grazoprevir, an analog or a salt thereof. [0129] Grazoprevir, as used herein, includes Grazoprevir (also named MK-5172, CAS: 1350514-68-9, IUPAC: 1R,18R,2OR,24S,27S)-A-{(1R,2S)-1-
[(Cyclopropylsulfonyl)carbamoyl] -2-vinylcyclopropyl } -7 -methoxy-24-(2-methyl-2-propanyl)- 22,25-dioxo-2,21-dioxa-4,l l,23,26-tetraazapentacyclo[24.2.1.03’12.05’10.0 18,20]nonacosa-
3,5,7,9,l l-pentaene-27-carboxamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0130] According to some embodiments, the invention provides a pharmaceutical composition comprising Isavuconazole, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0131] According to some embodiments, the influenza M2 channel blocker comprises Isavuconazole, an analog or a salt thereof.
[0132] Isavuconazole, as used herein, includes Isavuconazole or Isavuconazonium sulfate (CAS: 742049-41-8, 946075-13-4, or 241479-67-4, IUPAC: 4-{2-[(lR,2R)-(2,5-Difluorophenyl)-2- hydroxy-l-methyl-3-(lH-l,2,4-triazol-l-yl)propyl]-l,3-thiazol-4-yl]benzonitrile), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. In some embodiments, after oral or intravenous (IV) administration, Isavuconazonium is hydrolyzed by esterases in blood or the gastrointestinal tract to the active form comprising Isavuconazole.
[0133] According to some embodiments, the invention provides a pharmaceutical composition comprising Voriconazole, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza A virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0134] According to some embodiments, the influenza M2 channel blocker comprises Voriconazole, an analog or a salt thereof. [0135] Voriconazole, as used herein, includes Voriconazole (CAS: 137234-62-9, IUPAC: (2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-l-(lH-l,2,4-triazol-l-yl)butan-2- ol), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0136] According to some embodiments, the invention provides a pharmaceutical composition comprising Paritaprevir, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza A virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0137] According to some embodiments, the influenza M2 channel blocker comprises Paritaprevir, an analog or a salt thereof.
[0138] Paritaprevir, as used herein, includes Paritaprevir (also known as ABT-450, CAS: 1216941-48-8, IUPAC: (2R,6S,12Z,13aS,14aR,16aS)-N-(Cyclopropylsulfonyl)-6-{ [(5-methyl- 2-pyrazinyl)carbonyl] amino }-5,16-dioxo-2-(6-phenanthridinyloxy)- l,2,3,6,7,8,9,10,l l,13a,14,15,16,16a-tetradecahydrocyclopropa[e]pyrrolo [1,2- a][l,4]diazacyclopentadecine-14a(5H)-carboxamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0139] According to some embodiments, the invention provides a pharmaceutical composition comprising Vidarabine, a metabolite thereof, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises an Influenza virus infection. In some embodiments, the viral infection comprises an Influenza A virus infection. In some embodiments, the Influenza A virus infection comprises a H1N1 virus infection. In some embodiments, the viral infection comprises a virus infection resistant to aminoadamantane. In some embodiments, the viral infection comprises an infection by a virus comprising an influenza M2 protein.
[0140] According to some embodiments, the influenza M2 channel blocker comprises vidarabine, a metabolite thereof, an analog or a salt thereof.
[0141] Vidarabine, as used herein, includes Vidarabine (also known as 9-[3-D- arabinofuranosyladenine or ara-A, CAS: 24356-66-9, IUPAC: (2R,3S,4S,5R)-2-(6-Amino-9H- purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol hydrate), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. [0142] In some embodiments, a metabolite of Vidarabine is or comprises arainosine.
Pharmaceutical compositions
[0143] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
[0144] As used herein, the terms “administering”, “administration” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
[0145] As used herein, the terms “subject” or “individual” or “animal” or “patient” or “mammal” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
[0146] In some embodiments, a therapeutically effective dose of the 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 “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition.
[0147] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The route of administration of the pharmaceutical compositions will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of peptides administered by other routes can be lower than when administered via parenteral injection, by using appropriate compositions it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
[0148] In some embodiments, the composition of the invention comprising oral delivery. In some embodiments, the composition of the invention comprises an oral composition. In some embodiments, the composition of the invention further comprises orally acceptable carrier, excipient, or a diluent.
[0149] According to some embodiments, the influenza M2 channel blocker is for use at a daily dose of 0.01 to 500 mg/kg.
[0150] According to some embodiments, the influenza M2 channel blocker comprises Fludarabine or a salt thereof and is for use at a daily dose between about 0.1 mg/m2/day and about 50 mg/m2/day, 1 mg/m2/day and 30 mg/kg/day, and 0.1 mg/m2/day and 10 mg m2/day.
[0151] According to some embodiments, the influenza M2 channel blocker comprises Asunaprevir or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/day, about 5 mg/kg/day and 60 mg/kg/day, and 20 mg/kg/day and 50 mg/kg/day.
[0152] According to some embodiments, the influenza M2 channel blocker comprises Ravuconazole acid or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/kg/day, about 1 mg/kg/day and 50 mg/kg/day, and 5 mg/kg/day and 30 mg/kg/day.
[0153] According to some embodiments, the influenza M2 channel blocker comprises Amikacin or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/kg/day, about 5 mg/kg/day and 50 mg/kg/day, and 10 mg/kg/day and 20 mg/kg/day.
[0154] According to some embodiments, the influenza M2 channel blocker comprises Theobromine or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 200 mg/kg/day, about 5 mg/kg/day and 100 mg/kg/day, and 10 mg/day and 50 mg/kg/day.
[0155] According to some embodiments, the influenza M2 channel blocker comprises Flunisolide or a salt thereof and is for use at a daily dose of between about 0.1 mg/kg/day and about 50 mg/kg/day, about 0.1 mg/kg/day and 20 mg/kg/day, and 0.2 mg/kg/day and 5 mg/kg/day.
[0156] According to some embodiments, the influenza M2 channel blocker comprises Alvimopan or a salt thereof and is for use at a daily dose of between about 0.1 mg/kg/day and about 50 mg/kg/day, about 0.1 mg/kg/day and 10 mg/kg/day, and 0.5 mg/kg/day and 5 mg/kg/day. [0157] According to some embodiments, the influenza M2 channel blocker channel blocker comprises Lavamlodipine or a salt thereof and is for use at a daily dose between about 0.01 mg/kg/day and about 10 mg/kg/day, 0.01 mg/kg/day and 5 mg/kg/day, and 0.01 mg/kg/day and 1 mg/kg/day.
[0158] According to some embodiments, the influenza M2 channel blocker comprises Grazoprevir or a salt thereof and is for use at a daily dose of between about 0.1 mg/kg/day and about 50 mg/kg/day, about 0.1 mg/kg/day and 10 mg/kg/day, and 0.5 mg/kg/day and 5 mg/kg/day.
[0159] According to some embodiments, the influenza M2 channel blocker comprises Voriconazole or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 50 mg/kg/day, about 2 mg/kg/day and 20 mg/kg/day, and 2 mg/kg/day and 10 mg/kg/day. [0160] According to some embodiments, the influenza M2 channel blocker comprises Paritaprevir or a salt thereof and is for use at a daily dose of between about 0.5 mg/kg/day and about 500 mg/day, about 1 mg/kg/day and 300 mg/day, and 0.5 mg/day and 10 mg/day.
[0161] According to some embodiments, the influenza M2 channel blocker comprises Vidarabine or a metabolite thereof, or a salt thereof and is for use at a daily dose of between about 0.5 mg/kg/day and about 100 mg/day, about 1 mg/kg/day and 50 mg/day, and 5 mg/day and 40 mg/day.
[0162] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, or excipient.
[0163] As used herein, the term “carrier,” “adjuvant” or “excipient” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as com starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered 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, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some nonlimiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered 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), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. 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 may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of 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); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow -releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides 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 generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
[0164] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.
Kit
[0165] 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, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Eevamlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
[0166] In some embodiments, the molecule is selected from a group consisting of: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Eevamlodipine, Grazoprevir, Voriconazole, Paritaprevir, Vidarabine or a metabolite thereof, and any combination thereof.
[0167] In some embodiments, the kit further comprises instructions for mixing the at least two molecules selected from the group consisting of: Fludarabine or a derivative thereof, Asunaprevir, Amikacin, xanthine or a derivative thereof, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
[0168] In some embodiments, the Fludarabine derivative comprises Vidarabine or a metabolite thereof.
[0169] In some embodiments, the metabolite of Vidarabine is Arainosine.
[0170] In some embodiments, the xanthine derivative is Theobromine.
[0171] In some embodiments of the subject kits, the at least two molecules are packaged within a container.
[0172] In some embodiments, the container is made of a material selected from: thin-walled film or plastic (transparent or opaque), paperboard-based, foil, rigid plastic, metal (e.g., aluminum), glass, etc.
[0173] In some embodiments, the content of the kit is packaged, as described below, to allow for storage of the components until they are needed.
[0174] In some embodiments, some or all components of the kit may be packaged in suitable packaging to maintain sterility. [0175] In some embodiments of the subject kits, the at least two molecules are stored in separate containers within the main kit containment element e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit.
[0176] In some embodiments, the dosage amount of the at least two molecules provided in a kit may be sufficient for a single application or for multiple applications.
[0177] In those embodiments, the kit may have multiple dosage amounts of the at least two molecules packaged in a single container, e.g., a single tube, bottle, vial, 1.5-2 ml tube, e.g., Eppendorf, and the like.
[0178] In some embodiments, the kit may have multiple dosage amounts of the at least two molecules individually packaged such that certain kits may have more than one container of the at least two molecules.
[0179] In some embodiments, multiple dosage amounts of the at least two molecules may be packed in single separate containers.
[0180] In some embodiments, the kit contains instructions for preparing the composition used therein and for how to practice the methods of the invention.
[0181] In some embodiments, the kit further comprises a measuring utensil such as syringe, measuring spoon or a measuring cup.
[0182] 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, etc.
[0183] In some embodiments, the instructions may be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
Screening assays [0184] By another aspect, there is provided a method of screening effectiveness of an agent in treating or preventing an Influenza virus infection. According to some embodiments, the method comprising providing a cell comprising a membrane permeabilized by the influenza M2 channel, contacting the cell with the agent, and determining effect of the agent on growth of the cell, wherein a substantial effect of the agent on cellular growth is indicative of the agent as being effective for treating or preventing an influenza infection, thereby screening effectiveness of an agent 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 H1N1 subtype. In some embodiments, the Influenza virus is resistant to aminoadamantanes.
[0185] In some embodiments, the method comprises a negative assay. In some embodiments, the cell is characterized by growth retardation due to the membrane permeabilized by the influenza M2 channel. In some embodiments, an agent that alleviates growth retardation is indicative as being effective for treating or preventing an Influenza virus infection.
[0186] In some embodiments, the method comprises a positive assay. In some embodiments, the cell is a K+-uptake deficient cell that is incapable of growth in low [K+] media but experiences growth due to the channel formed by an influenza M2 channel. In some embodiments, an agent that induces growth retardation is indicative as being effective for treating or preventing an influenza infection.
[0187] In some embodiments, the method comprises performing both the negative assay and the positive assay.
[0188] In some embodiments, the method further comprises an acidity assay. In some embodiments, the cell is a cell comprising a pH-reporter gene or a protein product thereof, such as, but not limited to, pH-sensitive green fluorescent protein (GFP). In some embodiments, H+ influx to the cell, grown in media supplemented with an acidic solution, is induced. In some embodiments, an agent that blocks the pH change within the cell is suitable for treating or preventing an influenza infection.
[0189] In some embodiments, the method further comprises a validation step, comprising examination of the molecules that positively passed all three bacteria assays in a mammaliancell based assay. In some embodiments, the validation step requires infection of the mammalian cell with Influenza A virus and examination of the molecules on cell viability and/or growth.
[0190] Non-limiting examples for growing a bacterial cell applicable for the screening methods provided herein, include: Astrahan, P. et al., Acta 1808, 394-8 (2011); Santner, P. et al. Biochemistry 57, 5949-5956 (2018), and Taube, R., Alhadeff, R., Assa, D., Krugliak, M. & Arkin, I. T. PLoS One 9, el05387 (2014).
[0191] In some embodiment, the assay comprises determining susceptibility of the virus to develop resistance against the agent.
[0192] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.
[0193] It is noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.
[0194] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having 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 systems that have A alone, B alone, C alone, 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 disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, 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."
[0195] 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 the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub -combination was individually and explicitly disclosed herein.
[0196] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
[0197] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
[0198] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
Materials and Methods
Bacteria-based channel assays
Three bacteria-based channel assays were employed for investigating the influenza channel activity and blockers thereof. In all assays the MBP (maltose binding protein) fusion purification system (New England BioLabs; Ipswich, MA, USA) was used, where the M2 channel from a H1N1 strain that is aminoadamantane-resistant was expressed as chimera by fusion to the carboxy terminal of the maltose binding protein. This system ensures the targeting of the protein to the inner bacterial membrane and was used to express and examine many other viral ion channels successfully. The viral strain that the inventors used was H1N1 that is known to be resistant to aminoadamantanes due to a mutation of Ser31 to Asn in its M2 protein (Hay AJ et al. "The molecular basis of the specific anti-influenza Action of amantadine." EMBO J 1985; 4:3021-3024, hereby incorporated by reference in its entirety), as well as a H1N1 strain that is sensitive to aminoadamantane.
Negative assay
[0199] Bacteria cultures (DH10B) were grown overnight and finally diluted 500 fold and again set to grow until their O.D.600 reached 0.2. From the culture 50 l were added in 96-well flat- bottomed plates which was pre-treated with 50 pl of particualr chemicals and the isopropyl-P-d- 1 -thiogalactopyranoside (IPTG) inducer. Induction was achieved by employing different concentration of IPTG. A multi-plate incubator (infinite M200 pro from Tecan Group; Mannedorf, Switzerland or LogPhase 600 from BioTek; Santa Clara, CA, USA) was used to incubate the plates for 16 hours at 37 °C at a constant, shaking rate (700 rpm). Bacterial growth was monitored by measuring O.D.600 every 15 min. Duplicates, or triplicates were conducted for every measurement.
Positive assay
[0200] The positive assay employed the same protocol as the negative assay but in this instance a K+-uptake deficient bacteria strain was used . Additionally, overnight growth was conducted in LB media in which KC1 at different concentrations as noted replaced NaCl.
Acidity assay
[0201] The acidity assay is based on bacteria expressing a chromosomal copy of a pH-sensitive GFP. Overnight bacterial cultures were diluted to 1:500 in LB media and subsequently grown to an O.D.600 of 0.6-0.8. Protein expression was induced by IPTG at different concentrations, as noted. After one hour of induction, the cells were diluted to an O.D.600 of 0.2 and pelleted at 3,500 g for 10 min. Subsequently, the cells were resuspended in McILvaine Buffer, which contains 200 mM Na2HPO4 and 0.9% NaCl adjusted to pH 7.6 with 0.1 M citric acid. 200 pl of cell suspension were added to a 96-well plate (Nunclon f96 Microwell Black Polystyrene, Thermo Fisher Scientific; Waltham, MA, USA), each well containing 30 pl of McILvaine Buffer. The plate included three wells with McILvaine buffer and three with culture without induction as controls. The fluorescent measurements were carried out at an ambient temperature in a microplate reader (Infinite F200 Pro, Tecan Group; Mannedorf, Switzerland) with the emission fixed at 520 nm and alternating between 390 and 466 nm excitations. At the starting point, 70 pl of 300 mM citric acid was added to the bacterial culture, and a fluorescent read-out was taken for 30 s for each wavelength. Finally, proton concentration was calculated from the ratio of two excitation wavelengths.
Chemical Screening
[0202] A chemical library was purchased from MedChem Express (HY-L035, Monmouth Junction, NJ, USA). At the time, the library contained 2839 repurposed drugs, noting that the number of chemicals changes with time. Each chemical was tested at a final concentration of 100 pM. The final concentration of dimethyl sulfoxide was 2%. All manipulations and growths were conducted on a robotic system (EVO 75 Tecan, Mannedorf, Switzerland) or LogPhase 600 microbiology reader (Agilent, Santa Clara, CA, USA).
[0203] For each growth test, two metrics were measured: maximal growth rate and final bacterial density. However, in practice, visual inspection was far superior in identifying individual hits due to spurious factors influencing the aforementioned metrics, such as compound absorbance, solubility, etc.
Chemicals
[0204] Isopropyl-P-d- 1 -thiogalactopyranoside (IPTG) was purchased from Biochemika-Fluka (Buchs, Switzerland). All other chemicals were purchased from Sigma-Aldrich laboratories (Rehovot, Israel).
Bacterial growth media
[0205] Lysogeny broth (LB) was used for most of the cases only LBK was used for positive assay, where NaCl is replaced with KCL at lOgm/lt. All media contained ampicillin at 100 g/ml. [0206] Assessment of antiviral activity of compounds was conducted in a BSL-3 facility of the Hebrew University as follows: Madin-Darby canine kidney (MDCK) [ATCC MDCK NBL-2] cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM); (Biological Industries; Beit Haemek, Israel ), supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 10 lU/mL Penicillin, and 10 pg/mL streptomycin, Biomycin-3. (Biological Industries; Beit Haemek, Israel). Influenza A Virus, A/Wisconsin/629-D02452/2009 (H1N1) pdm09 was deposited by the Center for Disease Control and Prevention and obtained through BEI Resources, NIAID, and NIH. Identical aliquots of virus stock were prepared from the mother stock supplied by BEI resources. For infection 1:1000 times diluted sub stocks were prepared from each aliquot. Subsequent infection of MDCK cells was carried out in MEM (Biological Industries; Beit Haemek, Israel) containing 0.3% Bovine Serum Albumin, (Sigma: A4503, Lot: SLCK2178) and 3 pg/ml TPCK treated trypsin (Sigma, T8802) and further incubated for 48 h at 35 °C in a 5% CO2 atmosphere. All infection experiments were performed in a BSL-3 facility. A 10 mM stock of listed compounds was prepared in DMSO and stored at -80°C in aliquots until further use. MDCK cells were seeded in 96-well flat bottom plates in 200 pL of medium at a density of 15,000 cells per well and grown overnight. The dilutions of tested compounds were prepared in MEM with 0.3% BSA, 3 pg/ml TPCK treated trypsin and 50 pL was added to the cells. The effects of the drugs on the metabolic activity of MDCK cells were assessed at 48 h post treatment using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation reagent (Promega; Madison, WI, USA). To examine the effect of different drugs, cells were infected with the Influenza-A virus of 400 TCID 50/well for two hours followed by treatment with listed drugs. Each compound concentration was tested in triplicate and each assay plate contained the following controls: no cells (background control), cells treated with medium (mock infection for normalization), infected/untreated cells and infected/solvent-treated cells (infection control). At two days post infection, drug efficacies in control of toxicity were assessed by the CellTiter 96 Aqueous NonRadioactive Cell Proliferation reagent (Promega; Madison, Wisconsin, United States) for 3 h at 37°C in a 5% CO2 atmosphere. Reactions were stopped and the virus inactivated by adding 30 pl of 4% formaldehyde. Absorbance was measured at 492 nm using a Tecan plate reader (Mannedorf, Switzerland). Finally, the data were normalized to the mock-infected control, after which EC50 values were calculated by fitting the data to a Monod equation.
Animal studies
[0207] Mice are infected with the H1N1 virus and are given oral treatment (BID) for five days. After nine days, the amount of virus in the lungs is quantitated by q-RT PCR. For the animal experiment BALB/c mice were chosen. At first the inventors did tolerability and pharmacokinetics studies for corresponding drugs by taking 3 mice per group of a total 5 groups. Drug combinations (1:1 molar ratio) for the groups are as follows: (i) 1.5mg/kg Arainosine and 1 mg/kg Theobromine, (ii) 4.5 mg/kg Arainosine and 3 mg/kg Theobromine, (iii) 15 mg/kg Arainosine and 10 mg/kg Theobromine, (iv) 45 mg/kg Arainosine and 30 mg/kg Theobromine and, (v) 150 mg/kg Arainosine and 100 mg/kg Theobromine. For efficacy studies, a total of 56 numbers of BALB/c mice were divided into 7 groups, so each group contains 8 mice for this set of experiments. These 7 groups were treated in following ways: (i) Vehicle (control), (ii) Oseltamivir 20 mg/kg, (iii) 1.5mg/kg Arainosine and 1 mg/kg Theobromine, (iv) 4.5 mg/kg Arainosine and 3 mg/kg Theobromine, (v) 15 mg/kg Arainosine and 10 mg/kg Theobromine, (vi) 45 mg/kg Arainosine and 30 mg/kg Theobromine and, (vii) 150 mg/kg Arainosine and 100 mg/kg Theobromine. Each group initially infected with influenza by around 1000 viruses per animal with 50 pl by spraying intranasally. All the combined drugs in desired concentrations were dissolved in aqueous solution of 1% Emulphor-EL-620 and Oseltamivir in water. The vehicle is therefore aqueous solution of 1% Emulphor-EL-620 alone. Animals were treated by oral gavage with 10 ml/kg twice daily for 5 days. Each day body weight was monitored for each group and after 9 days animals were sacrificed and lungs were collected for further RNA quantification by q-RT-PCR.
EXAMPLE 1
Bacteria-based assays for assessment of ion channels activity
[0208] In order to evaluate the activity of aminoadamantane -resistant H1N1 strain, the inventors used bacteria-based assays in which the channel’s functionality changes the bacteria’s phenotype. The advantages of the assays are that they are amenable to high-throughput screening, and the ease of genetic manipulations in bacteria enables a rapid transition from one sequence/variant to another. Finally, these assays have been tested on numerous viroporins from various viruses (Assa, D. et al. J Mol Biol 2016, 428, 4209-4217, Astrahan, P. et al. 2011, 1808, 394-398, Taube, R, PLoS One 2014, 9, el05387, 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 assay
[0209] The primary assay that was employed involved the expression of the viral channel at increasing levels in “ordinary” Escherichia coli. At a certain viroporin concentration, growth retardation is observed due to excessive membrane permeabilization that hampers bacterial bioenergetics. This test is therefore eponymously termed a negative assay due to the detrimental impact of the protein on the bacteria.
Positive assay
[0210] The second assay entailed expressing the viral channel at lower levels in K+-uptake deficient bacteria. Such bacteria are incapable of growing in regular media unless they are supplemented by potassium or when they express a channel capable of K+ transport. In this instance, the viral channel impacts the bacteria favorably, and the assay is therefore termed positive assay. It should be noted that at high induction levels, while alleviating the K+ shortage in the bacteria, the channel impacts the bacteria negatively due to excessive membrane permeabilization akin to the negative assay discussed above.
Acidity assay
[0211] The final assay that examines channel activity is based on a channel's impact on the cytoplasmic pH of bacteria. When a concentrated acid is injected into the media, the cytoplasmic pH drops if the bacteria express a channel capable of H+ transport. Subsequently, a change in cytoplasmic pH can be detected by monitoring the fluorescence of a chromosomally-expressed, pH-sensitive GFP.
[0212] The negative, positive, and acidity assay results are shown in Figures 1, 2, and 3, respectively, confirming the M2 channel activity in all the examined assays. In all figures, the protein expression level, i.e. the level of the M2 channel is controlled by the level of isopropyl P-D- 1 -thiogalactopyranoside (IPTG).
Blocker screening
[0213] Following confirmation of channel activity of the M2 channel, the inventors sought to identify drugs that could block its function. The screening proceeded in three stages. Initially, all compounds were screened in the negative assay. Each plate had two controls: the positive control was bacteria without IPTG, i.e., without channel induction. Blank DMSO addition served as a negative control. Subsequently, bacteria that experienced growth enhancement beyond an empirical threshold were reexamined in triplicate. Every compound that passed this assay was then examined in the positive assay in triplicate. Finally, compounds that passed the positive and the negative assays were subjected to a dose-response analysis in duplicate and the pH assay in quadruplicate. Finally, examples of compound screening assays are presented in Figures 4-6. “No channel” refers to the absence of M2 channel induction by IPTG, whereas “no drug” refers to maximum induction of the M2 channel protein by IPTG, in the absence of a drug. The IPTG concentrations were lOOpM, 20pM, and 50pM in the negative, positive, and acidity assays, respectively (Figures 4-6).
[0214] As demonstrated in Figures 4-6, the list of the compounds that exhibited activity in one or more assays is as follows: Amantadine*, Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Theobromine, Flunisolide, Alvimopan, Eliglustat, CM4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, Vidarabine, and Arainosine.
* Amantadine (Symmetrel®) is a known channel blocker of the M2 channel and as such was expected to be identified in the screen (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 analyses in mammalian systems
Tissue culture studies
[0215] The inventors next sought to characterize the anti-viral activity of the channel blockers that were identified, in tissue culture cells. For that purpose, the inventors cultured Madin-Darby canine kidney (MDCK) cells and examined their viability after viral infection with aminoadamantane -resistant H1N1 and the ability of the drugs to impact the cell viability thereupon. The viral strain that was used was H1N1 that is known to be resistant to aminoadamantanes due to a mutation of Ser31Asn in its M2 protein.
Compounds active in tissue culture
[0216] Initially, two concentrations of the drugs were used: 10 pM, shown in Figure 7, and later 3 pM, shown in Figure 8. In both experiments, two available anti-influenza drugs were used as positive controls: Oseltamivir (Tamiflu®) and Favipiravir (Avigan®).
[0217] The results indicate that the following drugs exhibited appreciable anti-influenza in tissue culture at 10 pM: Fludarabine, Asunaprevir, Amikacin, Theobromine, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, and Vidarabine.
[0218] The following compounds were active at 3 pM: Fludarabine, Asunaprevir, Theobromine, Flunisolide, Grazoprevir, Voriconazole, and Vidarabine.
[0219] Finally, as expected, Tamiflu® and Avigan® exhibited potent anti-influenza Activity (thus served as positive controls), while gratifyingly, Rimantadine (Flumadine®) was completely ineffective since the infection was conducted with the H1N1 virus strain that is resistant to aminoadamantanes (thus served as negative control).
[0220] The inventors subsequently conducted dose-response analyses to determine the ability of each of the active compounds to inhibit the virus at various concentrations. The results of these analyses are shown in Figures 9-12. The shaded region in the graphs indicate the vehicle level. In Figures 13 and 14 one can find the fits to EC50 values of each compound. [0221] Following the affinity analyses of each drug, the inventors searched for potential additivity and synergism effects between the positive hits. Results of combination experiments, in which each drug is combined with another drug, at a concentration of O.OlpM are shown in Figure 15. As can be seen, theobromine + vidarabine and vidarabine + grazoprevir exhibit stark synergism. Other combinations exhibit an additivity effect, or no effect, as can be seen in table 1 below. Shaded regions indicate individual compounds.
[0222] Combination experiments were conducted at lower drug concentrations. In Figure 16 one can see that the combination of theobromine and vidarabine is particularly efficacious at 30 nm whereby it provides complete protection from viral induced cellular death.
Table 1. Summary of combination effect between positive hits
[0223] Further mice were infected with the H1N1 virus and given oral treatment (BID) for five days, as described above. After nine days, the amount of virus in the lungs was quantitated by q- RT-PCR. The results show that even though the herein disclosed drug combination comprising Theobromine + Arainosine was administered at a significantly lower dosage, it was more effective than oseltamivir, the leading drug on the market (Figure 17).
[0224] Further, the inventors conducted a structure-activity relationship (SAR) analysis examining the effect of xanthine and derivatives (Figure 18) thereof on cell viability. The results show that several close-related xanthine derivatives improved cell survival (Figure 19). In that regard, theobromine was found to increase cell survival at concentrations of 0.3 pM-10 pM, to a great extent compared to control. Surprisingly, caffeine, which is very similar to Theobromine, did not improve cell viability compared to no-drug control. A similar trend was observed for Enprophylline, and paraxanthine. Theophylline and 3 -methylxanthine were found to increase cell survival at any of the tested concentrations (0.01 pM-1 pM), to a great extent compared to control. 7-methylxanthine was found to increase cell survival at concentrations of 1 pM-10 pM), to a great extent compared to control. [0225] Further, the inventors conducted a S AR analysis examining the effect of Fludarabine and derivatives (Figure 22) thereof on cell viability. The results show that Fludarabine and several close-related derivatives thereof have improved cell survival compared to control (Figure 22). In that regard, Vidarabine was found to increase cell survival at concentrations of 0.3 pM-10 pM, to a great extent compared to control. Nelarabine and Corycepin improved cell survival at concentrations of 10 pM and 1 pM-10 pM, respectively.
[0226] Furter, the inventors conducted combination experiments with xanthine or other closely related compounds thereof (having a xanthine backbone) with the antiviral drug vidarabine or its natural metabolite arainosine (Figure 20). The results are summarized in Figure 21.
[0227] Briefly, combinations comprising Vidarabine at a concentration of 300 nM and 1 -methyl xanthine at a concentration of 10 nM-300 nM increased cell viability by about 33-37%, compared to negative control (no drug).
[0228] Combinations comprising Vidarabine at a concentration of 300 nM and xanthine at a concentration of 10 nM-300 nM increased cell viability by about 27-48%, compared to control.
[0229] Combinations comprising Vidarabine at a concentration of 10 nM or 100 mM and 3- methyl xanthine at a concentration of 30 nM increased cell viability by about 63% and 28%, respectively compared to control. Combinations comprising vidarabine at a concentration of 30 nM or 100 mM and 3 -methyl xanthine at a concentration of 100 nM increased cell viability by about 63% and 46%, respectively compared to control.
[0230] Combinations comprising Vidarabine and Theobromine had a profound positive effect on cell viability. Combinations comprising Vidarabine and Theobromine at any of the tested concentrations, e.g., from 10 nM to 300 mM (of each compound) increased cell viability by about 37% to 98%, compared to control. In particular combinations comprising Vidarabine at a concentration of 10 nM to 300 mM and Theobromine at any concentration of 30 nM to 300 nM increased cell viability by about 64% to 98%, compared to control.
[0231] Further, the inventors examined the effect of Theobromine, and Arainosine, a natural metabolite of Vidarabine, on cell viability. The results show that combinations comprising Arainosine at a concentration of 100 nM or 300 mM and Theobromine at a concentration of 10 nM increased cell viability by about 10% to 75%, compared to control. Further, combinations comprising Arainosine at a concentration of 10 nM to 300 mM and Theobromine at any concentration of 30 nM to 300 nM increased cell viability by about 61% to 100%, compared to control. [0232] Although 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, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS What is claimed is:
1. A method of treating or preventing an Influenza A virus in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a molecule selected from the group consisting of: theobromine, vidarabine, Arainosine, fludarabine, Asunaprevir, Ravuconazole, Amikacin, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, and any combination thereof, thereby treating or preventing an Influenza A virulence in said subject.
2. The method of claim 1, wherein said molecule is selected from a group consisting of: Theobromine, Vidarabine, Arainosine, Fludarabine, Asunaprevir, Amikacin, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
3. The method of claim 1 or 2, wherein said molecule is a M2 protein blocker.
4. The method of any one of claims 1 to 3, wherein said Influenza A virus is a H1N1 subtype.
5. The method of any one of claims 1 to 4, wherein said Influenza A virus is resistant to aminoadamantane .
6. The method of any one of claims 1 to 5, wherein said molecule is for use at a daily dose of 0.01 to 500 mg/kg per body weight of said subject.
7. The method of any one of claims 1 to 6, wherein said administering is a therapeutically effective amount of 2 molecules selected from: Theobromine, Vidarabine, Arainosine, Fludarabine, Asunaprevir, Amikacin, Flunisolide, Alvimopan, Levamlodipine, Grazoprevir, Voriconazole, Paritaprevir, and any combination thereof.
8. The method of claim 7, wherein said 2 molecules is Vidarabine or Arainosine and a molecule selected from: Theobromine and Grazoprevir.
9. The method of claim 7, wherein said 2 molecules is Vidarabine or Arainosine 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 said molecule is selected from a group consisting of: theobromine, Vidarabine, Arainosine, Fludarabine, Asunaprevir, Ravuconazole, Amikacin, Flunisolide, Alvimopan, Eliglustat, Cm4620, Levamlodipine, Emamectin, Grazoprevir, Isavuconazole, Voriconazole, Paritaprevir, and any combination thereof.
11. The pharmaceutical composition for use of claim 10, wherein said molecule is selected from a group consisting of: Theobromine, Vidarabine, Arainosine, Fludarabine, Asunaprevir, Amikacin, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, paritaprevir, and any combination thereof.
12. The pharmaceutical composition for use of claim 10 or 11, wherein said molecule is a M2 protein blocker.
13. The pharmaceutical composition for use of any one of claims 10 to 12, wherein said Influenza A virus is a H1N1 subtype.
14. The pharmaceutical composition for use of any one of claims 10 to 13, wherein said Influenza A virus is resistant to aminoadamantanes.
15. The pharmaceutical composition for use of any one of claims 10 to 14, wherein said molecule comprises Vidarabine or Arainosine and Theobromine or Grazoprevir.
16. The pharmaceutical composition for use of any one of claims 10 to 15, wherein said molecule comprises Vidarabine or Arainosine and theobromine.
17. A combination for use in treatment or prevention of Influenza A virulence in a subject in need thereof, wherein said combination comprises at least two molecules selected from a group consisting of: Theobromine, Vidarabine, Arainosine, Fludarabine, Asunaprevir, Amikacin, Flunisolide, Alvimopan, Lev amlodipine, Grazoprevir, Voriconazole, and Paritaprevir.
18. The combination for use of claim 17, wherein said at least two molecules is (i) Vidarabine and (ii) a molecule selected from: Theobromine and Grazoprevir.
19. The combination for use of claim 17 or 18, wherein said at least two molecules is (i)
Vidarabine or Arainosine and (ii) Theobromine.
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