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WO2012028732A1 - Nouvelles utilisations d'inhibiteurs de la v-atpase - Google Patents

Nouvelles utilisations d'inhibiteurs de la v-atpase Download PDF

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Publication number
WO2012028732A1
WO2012028732A1 PCT/EP2011/065235 EP2011065235W WO2012028732A1 WO 2012028732 A1 WO2012028732 A1 WO 2012028732A1 EP 2011065235 W EP2011065235 W EP 2011065235W WO 2012028732 A1 WO2012028732 A1 WO 2012028732A1
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atpase inhibitor
inhibitor according
atpase
influenza
group
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Claude Muller
Konstantin MÜLLER
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Centre de Recherche Public de la Sante
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • 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

Definitions

  • the present invention relates to novel uses of benzolactone enamide v-ATPase inhibitors for the treatment of influenza A virus infections.
  • influenza A Virus causes recurrent outbreaks in humans and animals with serious consequences for public health and even the global economy. Rapid accumulation of mutations and reassortment of genomic segments between strains and subtypes enable emerging viruses to acquire new properties including the development of resistance to antiviral drugs.
  • Virus replication depends on a multitude of host cellular functions and proteins.
  • a low pH in the late endosomes triggers fusion between viral and endosomal membranes mediated by hemagglutinin and the release of viral ribonucleoprotein particles into the cytosol. Inhibition of acidification can prevent IAV entry in vitro (Guinea et al, 1995; Ott et al, 1994; Perez et al, 1994).
  • V-ATPases are host cellular proteins which acidify the late endosome by pumping protons across the endosomal membrane.
  • V-ATPases are multisubunit molecular motors consisting of a cytosolic Vi domain and a transmembrane Vo domain.
  • the Vi domain consists of eight different subunits while the Vo domain consists of five subunits (Forgac,
  • V-ATPases are found on membranes of different intracellular organelles as well as in cytoplasmic membranes and are involved in multiple cellular processes that require pH regulation, including intracellular membrane transport, prohormone processing or transport of neurotransmitters (reviewed in Nishi et al, (2002)).
  • the proton translocation domain of cellular vacuolar ATPase provides a target for the treatment of influenza A virus infections.
  • V-ATPase inhibitors were first described in the early 90s. Because of their
  • v-ATPase inhibitors are macrocyclic lactones with a thiazole side chain that are structurally distinct from the plecomacrolides but share, at least partially, a common binding site on the Vo subunit c (Huss et al. , 2005).
  • Archazolid B (ArchB) was reported to be only slightly less toxic in A549 cells than BafA or ConmyA (Huss et al, 2005).
  • Saliphenylhalamide (SaliPhe) is more stable than SaliA.
  • W09621644 discloses a certain class of v-ATPase inhibitors, the indole derivatives, which have antiviral activity that may be useful for the treatment of influenza A virus infection.
  • WO9801436 reveals another class of ATPase inhibitors, heteroaromatic pentadienoic acid derivatives, which have antiviral activity and that may be useful for the treatment of influenza A virus infection.
  • Konig et al Human host factors required for influenza virus replication. Nature (January, 2010) reported that, diphyllin, a lignan v-ATPase inhibitor, showed antiviral activity in an in vitro assay. However, our studies show that the toxicity concentration reported by Konig is lower than reported before and lower than in our hands. Our studies showed that this compound had no antiviral effect at sub-cytotoxic doses.
  • WO0051589 teaches the use of benzolactone v-ATPase inhibitors for viral infections. This document only contains a broad reference to baculoviruses and retroviruses. No mention is made of influenza A and no examples are given. Additionally, EC 5 0 values for the v- ATPase inhibitors are not provided.
  • GONG JIANZHI ET AL ("Potential Targets and Their Relevant Inhibitors in Anti- influenza Fields", CURRENT MEDICINAL CHEMISTRY, vol. 16, no. 28, October 2009 (2009-10), pages 3716-3739, XP0026304563 ISSN: 0929-8673) states that v-ATPase is a possible target for influenza treatment.
  • the cyctotoxic v-ATPase inhibitors Bafilomycin A and Concanamycin A are mentioned, but their toxicity is not addressed.
  • HUSS MARKUS ET AL ("Inhibitors of V-ATPases: old and new players",
  • BOYD M R ET AL ("Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-ATPases.”
  • KUNZE ET AL ("Cruentaren A, a highly cytotoxic benzolactone from Myxobacteria is a novel selective inhibitor of mitochondria] Fl -ATPases", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 581, no. 18, 19 July 2007 (2007-07-19), pages 3523-3527,
  • V-ATPase inhibitors have been found to be cytotoxic so that their use in treatment of disease is only called for when cytotoxicity is required, such as in the treatment of cancer.
  • the inventors investigated inhibition of influenza viruses in more detail and included toxicity testing and found a unexpectedly good selectivity index and propose SaliPhe to be used as a drug for the treatment of viral diseases. This is in contrast to other applications that require high cytotoxicity (anti-cancer).
  • Saliphenylhalamide and its derivatives have a surprisingly good selectivity index in comparison to Bafilomycin and Concanymcin.
  • the present invention provides a benzolactone v-ATPase inhibitor of the general formula
  • v-ATPase inhibitor for use in the treatment of an influenza A virus infection, wherein the v-ATPase inhibitor is administered in sub-cytotoxic amounts, and wherein
  • Z is an optionally substituted linker group, comprising heteroatoms and/or carbon atoms, whose presence completes an 11 to 15 membered ring,
  • X is H, or a halogen atom
  • Ri is OH or
  • P2 is an enamide-containing substituent of the formula
  • R5 is a linear Ci_ 7 alkyl group, a linear Ci_ 7 alkenyl group optionally substituted by a phenyl group or a methoxyimino group, a linear C 1-7 alkynyl optionally
  • Ci_ 7 thioalkyl group Ci_ 7 thioalkyl group.
  • Suitable Z linker groups include
  • R 3 is OH, or
  • R 4 is H or CH 3 ,
  • R 6 is CH 3 or CH 2 OH
  • R s is OH or H.
  • X may be meta, para or ortho substituted with respect to Rl .
  • X is in the meta position with respect to Rl .
  • X may be any halogen selected from the group F, I, Br or CI.
  • X is F.
  • the double bond of the enamide-containing substituent, R 2 may be in the E or Z configuration.
  • the double bond is in the E configuration.
  • E ent ought
  • Z zusammen
  • Suitable R5 alkyl groups include
  • Suitable R5 alkenyl groups include
  • Suitable R5 thioalkyl groups include By 'sub-cytotoxic' is meant an amount that is effective against influenza A in a patient, with no, or negligible, cytotoxity in the patient.
  • the amount of inhibitor may be selected to be sub-cytotoxic on a patient-by-patient basis. This may be achieved by the skilled physician measuring a quantity of a preparation of known concentration.
  • the preparation of the inhibitor may also be provided in aliquots and in known concentration, such that a smaller number of aliquots may be administered to children, and a larger amount to adults.
  • Preparations of inhibitor may also be provided as individual administration packs, such as syringes equipped with adult amounts and syringes equipped with child amounts of preparation of inhibitor.
  • the benzolactone v-ATPase inhibitor may be salicylihalamide or a derivative thereof, apicularen or a derivative thereof, oximidine or a derivative thereof, or lobatamide or a derivative thereof, or mixtures of the foregoing.
  • Anti-IAV activity is measured by the half maximal effective concentration EC 50, which represents the concentration required for obtaining 50% of the maximum effect against a virus.
  • EC 50 represents the concentration required for obtaining 50% of the maximum effect against a virus.
  • the ratio of IC 5 0 to EC 5 0 provides a selectivity index (SI) for compounds tested.
  • Ri is OH
  • R 2 is an enamide-containing substituent of the formula
  • R 8 is OH or H.
  • the double bond of the enamide-containing substituent, R 2 may be in the E or Z configuration.
  • the double bond is in the E configuration.
  • Preferred oximidines are selected from the group consisting of Oximidin I, Oximidin II, Oximidin III, CJ-13357, CJ-12950 and derivatives thereof, as shown in table 1. Particularly preferred oximidines are CJ-12950, CJ-13357, E-Oximidin I, E-Oximidin II, and E-Oximidin III.
  • benzolactone is a lobatamide of the formula
  • Ri is OH
  • R 6 is CH 3 or CH 2 OH.
  • the double bond of the enamide-containing substituent, R 2 may be in the E or Z configuration.
  • the double bond is in the E configuration.
  • Preferred Lobatamides are selected from the group consisting of Lobatamide A, Lobatamide B, Lobatamide C, Lobatamide D, Lobatamide E, Lobatamide F, and derivatives thereof as shown in table 2.
  • v-ATPase inhibitor is a salicylihalamide of the formula:
  • zig-zag line is an optional double bond
  • X is H or a halogen atom
  • Ri is OH or
  • R 3 is OH, or
  • R 4 is H or CH 3 .
  • X may be meta, para or ortho substituted with respect to Rl .
  • X is in the meta position with respect to Rl .
  • X may be any halogen selected from the group F, I, Br or CI.
  • X is F.
  • the double bond of the enamide-containing substituent, R 2 may be in the E or Z configuration.
  • the double bond is in the E configuration.
  • Particularly preferred salicylihalamide derivatives are Salicylihalamide A;
  • Saliphenylhalamide 4- 19F Salicylihalamide A; WY- 1295; WY-2501 ; S-pentyl ⁇ (lE)-3-[(8Z)- 5, 14-dihydroxy-6-methyl- l-oxo-3,4,5,6,7, 10-hexahydro- lH-2-benzoxacyclododecin-3- yl]prop- l-en-l-yl ⁇ carbamothioate; LX-1072; (8Z)-3-[(2E)-3-
  • Salicylihalamide A Saliphenylhalamide
  • WY- 1295 WY-2501
  • LX-1072 and SL3175.
  • R 2 is an enamide-containing substituent of the formula
  • the double bond of the enamide-containing substituent, R 2 may be in the E or Z configuration.
  • the double bond is in the E configuration.
  • Preferred examples may be selected from the group consisting of Apicularen A, Apicularen B, l l-deoxy-apicularen, ApiOpen, ApiAc, ApiCinnamoyl, ApiPhe, ApiEnyne and derivatives thereof, as shown in table 4. Particularly preferred is Apicularen Open (ApiOpen).
  • the inhibitor of the invention is any one of the compounds listed in Tables 1 - 4.
  • the toxicity of the benzolactone v-ATPase inhibitors may be measured by the half maximal inhibitory concentration, IC 5 0, which is the concentration of the v-ATPase inhibitor that causes a 50% reduction in cell survival, whereas the anti-IAV activity is measured by the half maximal effective concentration, EC 5 0, which represents the concentration required for obtaining 50% of the maximum effect in against a virus.
  • IC 5 0 which is the concentration of the v-ATPase inhibitor that causes a 50% reduction in cell survival
  • EC 5 0, half maximal effective concentration
  • the ratio of IC 5 0 to EC 5 0 provides a selectivity index (SI) for each benzolactone v-ATPase inhibitor of the present invention.
  • EC 5 0 is dependent on the different strains of influenza A, and the half maximal inhibitory concentration (IC 5 0) is dependant on the different types of cells involved.
  • the IC 5 0 of the compounds of the present invention is between ⁇ and 10 OOOnM.
  • the IC50 is between ⁇ and 10 OOOnM.
  • the v-ATPase inhibitors of the present invention have a SI of at least 1.
  • the v-ATPase inhibitors of the present invention have a SI of above 5. Even more preferably, the SI is above 30.
  • Particularly preferred are V- ATPases inhibitors with SI values above 35, 40, 45, 50, 50, 60 and 65. Most preferably, the v- ATPase inhibitors have a SI of above 70.
  • the amount of a compound required is determined by biological activity and bioavailability which, in turn, depends on the mode of administration, the physicochemical properties of the compound employed and whether the compound is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the above-mentioned factors, and particularly the half-life of the compound, within the subject being treated.
  • a dose should be given that is effective for delivering a compound at the target site such that the tissue concentration is around the EC 5 0 of the compound used.
  • Daily doses may be given as a single administration (e.g. as a single daily injection).
  • the compound used may require administration twice or more during a day, provided that systemic concentrations do not reach cytotoxic levels.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the strength of the preparation, and the mode of administration. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to establish specific formulations of compositions and precise therapeutic regimes (such as daily doses of the compounds and the frequency of administration).
  • a suitable dose of the one or more compounds of the invention with an SI of more than 1 may be in the range of about 1 mg to about 5000 mg /kg body weight of the subject per day, e.g., 1, 5, 10, 25, 50, 100, 250, 1000, 2500 or 5000 mg/kg per day.
  • the compound(s) is a salt, solvate, prodrug or the like
  • the amount administered may be calculated on the basis of the parent compound and so the actual weight to be used may be increased proportionately.
  • the suitable dose is between lmg/kg to body weight and 80mg/kg body weight of the subject per day. It can be administered in from one to several portions depending on the condition of the patient.
  • the v-ATPase inhibitors are effective against influenza A viruses including H5N1 (For example, A/Chicken/Nigeria/BA211/2006), A/HlNlv (For example,
  • H1N1 oseltamivir resistant H1N1 (For example, A/Luxem Kunststoff/572/2008), and other seasonal influenza A viruses.
  • the v-ATPase inhibitor or composition may be provided in a form suitable for parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal, topical, oral, rectal, transmucosal, intraperitoneal, nasal, intratracheal and pulmonary administration.
  • the v-ATPase inhibitor may be administered intra-peritoneally and may be delivered as a lipid emulsion or aqueous solution, or as a continuous infusion of a non-toxic dose.
  • the v-ATPase may be provided as a dry powder suitable for inhalation or aerosol delivery.
  • Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
  • Formulations in the form of finely comminuted powders may conveniently be presented for use in an inhalation device. Suitable examples include pierceable capsules, and self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent.
  • Suitable liquid propellants are well known in the art.
  • the compounds of the present invention may be in the form of a solution or suspension for nasal drops, or for use in an atomizer or nebuliser. It is also envisaged that the present invention can be used in combination with other treatments for influenza A.
  • the present invention provides a benzolactone v-ATPase inhibitor and a further antiviral agent for use in the treatment of an influenza A virus infection, wherein the amount of v-ATPase inhibitor is selected to be sub- cytotoxic.
  • Suitable further antivirals include inhibitors of the M protein of influenza A, such as amantadine or rimantadine, or inhibitors of the neuraminidase protein of influenza A, such as oseltamivir or zanamivir.
  • Also provided is a method for treating an influenza A infection comprising administering, to a subject in need thereof, a therapeutically effective, non-toxic dose of a benzolactone enamide v-ATPase inhibitor.
  • a “subject” may be a bird, mammal, domestic animal or human being.
  • v-ATPase inhibitors of the invention include pharmaceutically acceptable salts such as acetate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate,
  • glucoheptanoate glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide, hydroiodide, 2-hydroxyethane- sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, or thiocyanate salts of the compounds of the present invention.
  • Archazolid B (ArchB); adenocarcinoma human alveolar basal epithelial cells (A549 cells); Bafilomycin Al (BafA); Concanamycin A (ConmyA); H1N1 oseltamivir resistant (H1N1 OR); Influenza A viruses (IAV); Madin-Darby canine kidney (MDCK); Multiplicity of infection (MOI); Saliphenylhalamide (SaliPhe); Salicylihalamide A (SaliA); Selectivity index (SI); Swine origin pandemic influenza virus (S-OIV); Vacuolar-ATPase (v-ATPase); Virus growth medium (VGM), green fluorescence protein (GFP)
  • VGM green fluorescence protein
  • MDCK cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 20 mM HEPES, 25 mg/ml bovine serum albumin, 100 U/ml penicillin and 100 U/ml streptomycin.
  • A549 cells were cultured in EMEM (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM ultra-glutamine (Lonza), 100 U/ml penicillin and 100 U/ml streptomycin.
  • VGM-MDCK virus growth medium
  • TPCK-trypsin serum-free MDCK culture medium containing 1 ⁇ g/ml TPCK-trypsin. All incubation steps involving cells were performed at 37°C and 5% C0 2 .
  • Influenza A viruses used in this study include swine origin H1N1 2009 (S-OIV) A/Hamburg/01/2009 (kindly provided by S. Becker, Marburg, Germany),
  • H5N1 A/Chicken/Nigeria/BA211/2006 (H5N1) (Ducatez et al., 2007) and PR8-NS 116-GFP virus (H1N1).
  • PR8-NS 116-GFP virus appeared as a spontaneous frame shift/deletion mutant during a rescue experiment as described previously (Kittel et al., 2004).
  • the fusion protein expressed by the chimeric non- structural (NS) segment consists of the N-terminal 104 aa of NS 1 followed by 12 aa derived from the second NS frame and the GFP open reading frame. All viruses were titered both on MDCK and A549 cells to allow an identical MOI for both cell lines.
  • Example 1 In vitro cytotoxicity assay
  • a 96- well plate was seeded with 3,000 cells per well in 99 ⁇ cell culture medium, v- ATPase inhibitors were dissolved in DMSO and 1 ⁇ of 11 steps of a 2-fold serial dilution of 8 replicates was added to a well. The final DMSO concentration was 1% in all wells.
  • Controls included wells with 1% DMSO or medium. After 5 days of incubation 50 ⁇ of freshly prepared XTT reagent (Roche, Mannheim) was added to the wells and the plate was incubated for 2 h at 37°C. The plate was read at 450 and 650 nm on a SpectraMax plus 384 (Molecular Devices, Berkshire).
  • v-ATPase inhibitors In order to determine the in vitro cytotoxicity of the v-ATPase inhibitors, increasing concentrations of the compounds were added to Madin-Darby canine kidney (MDCK) or A549 cells. After 5 days, the half maximal concentrations inhibiting cell growth (IC 50 ) were determined using a standard XTT assay.
  • SaliPhe had the lowest cytotoxicity (IC 50 of 1.74 + 0.2 ⁇ and 1 + 0.19 uM), while the IC 50 of the other established v-ATPase inhibitors ArchB (1.4 + 0.1 nM and 0.16 + 0.02 nM), BafA (2.9 + 0.1 nM and 1.3 + 0.2 nM) and ConmyA (0.56 + 0.09 nM and 0.2 + 0.02 nM) were in the low nM range or below.
  • Example 2 In vitro Anti-IAV efficacy assay 96-well plates containing 70,000 adherent MDCK or A549 cells per well were washed three times with 200 ⁇ of serum-free EMEM and overlaid with 99 ⁇ VGM-MDCK or VGM- A549 containing IAV corresponding to a MOI of 0.03.
  • the v-ATPase inhibitors were dissolved in DMSO and stocks were kept at -80°C. From these stocks 2-fold serial dilutions were prepared in DMSO, 1 ⁇ of each dilution was added to a well containing cells and virus. Dilutions were tested as duplicates or triplicates.
  • NS 1-GFP fusion IAV were performed in ⁇ 8 ⁇ bottom (black) plates (Greiner, Frickenhausen) and read after 24 h of incubation on a Tecan Genios plus Reader (Tecan Group, Austria) using 485 nm excitation and 535 nm emission filters for GFP fluorescence. Representative images were taken on a Leitz DM IL microscope with separate mercury lamp and a Leica DFC480 camera. Unmodified wild type IAVs were tested as follows: After 24h of incubation supernatant was transferred to a second plate; the attached cells in each well were lysed (150 ⁇ lysis solution), recombined with 50 ⁇ of supernatant and extracted with the MagMax total RNA kit
  • B eta- actin- specific RT-qPCRs was performed to normalize for the total amount of extracted RNA and to monitor cell survival within assays.
  • Virus proliferation was expressed in percent of the difference between cultures with virus and the lowest (or zero) drug concentration, and cultures without virus. To control for background noise resulting from surviving virus of the initial inoculate, an identical amount of virus was incubated in cell free medium and tested by qPCR.
  • Fluorescence data were fitted to a hill curve with SigmaStat 9.0 software and EC 5 0 values were calculated. The same fit was applied to c t values obtained from qPCRs for viral matrix gene detection. Statistical significance tests were performed with Students t-test, p ⁇ 0.05 was considered significant.
  • NS 1 is an important virulence factor of IAV and its partial replacement with GFP leads to reduced virus growth (Garcia-Sastre et al., 1998), (Kittel et al., 2004) especially in interferon competent cells such as MDCK or A549. Therefore, antiviral activity of the compounds was also tested against unmodified wt IAV, including pandemic S-OIV, highly pathogenic avian influenza H5N1 virus (A/Chicken/Nigeria/BA211/2006) and an oseltamivir resistant seasonal H1N1 strain (A/Luxembourg/572/2008).
  • MDCK cells were infected with 0.03 MOI of the above wt viruses and serial dilutions of v-ATPase inhibitors were added.
  • Drug-free DMSO-diluent served as a negative drug control.
  • Viral replication was monitored by standard matrix gene qPCR after total RNA extraction and reverse transcription ( Figure 4)
  • Apicularen Open (ApiOpen) was tested for toxicity in A549 cells with the XTT cytotoxicity assay after 5 days of incubation with a dilution series of ApiOpen the IC 5 0 value is 341nM.
  • ApiOpen shows an EC 5 0 value of 48nM which yields a selectivity index of 7, which means that the toxicity is 7 times lower than the anti- viral effect. The results are illustrated in Figure 11.
  • mice were housed in a temperature-controlled environment with 12 h light/dark cycles, and received food and water ad libitum.
  • nine mice per group were inoculated intranasally with 50 ⁇ PBS with or without (mock infection) 4 mouse LD 5 0 of mouse adapted A/PR/8/34 strain.
  • mice were treated or mock-treated with a first dose of v-ATPase inhibitor 4 hours before infection followed by i. p. injections every 8 hours until day 9 post infection.
  • BafA was dosed at 350 ng/kg in 200 ⁇ PBS; an equal volume of PBS only was injected in the mock treatment group.
  • SaliPhe was given at a dose of 7 mg/kg resuspended in 200 ⁇ Lipovenos 20%
  • mice per treatment group were inoculated intranasally with one mouse LD 5 0 in 100 ⁇ PBS of mouse-adapted A/PR/8/34 strain and euthanized 4 days post infection to collect the lungs.
  • Lung homogenates were prepared in 1.5 ml of PBS by using a microhomogenisator. The homogenate was cleared by
  • Endpoint virus titers were determined after 4 days, as described by Reed and Muench (Reed et ah, 1938), by interpolating the dilution that infected 50% of the wells, as assayed by
  • Formalin-fixed lung samples were embedded in paraffin. Serial 4 ⁇ sections were double stained with haematoxylin and the mouse monoclonal anti-IAV matrix protein 2 antibody (14C2, Santa Cruz) and Dako Animal Research Kit on a Dako Autostainer Plus instrument. Representative images were taken on a Zeiss Axio Observer Zl with a Zeiss AxioCam HRc color camera.
  • Antiviral activities of the two v-ATPase inhibitors with the best SI in vitro, SaliPhe and BafA were further tested in a standard mouse model of influenza infection. Mice were challenged with 4 mouse LD 5 0 of mouse adapted A/PR/8/34 virus and treated three times daily with injections of 7 mg/kg SaliPhe in Lipovenos or 350 ng/kg BafA in PBS every 8 hours. The first dose of v-ATPase inhibitor was given 4 hours before infection.
  • mice The weight of the animals was monitored as a measure of disease progression ( Figures 5B and E).
  • SaliPhe treated mice showed only a limited average weight loss of less than 10% during the infection, which was fully recovered 11 days post infection.
  • Lipovenos mock-treated mice experienced a rapid and severe weight loss before dying (or euthanasia).
  • Mice receiving BafA or PBS injections showed a steady loss of weight, and there was no difference up to day 7 post infection between both groups. On later days the weight gain was due to a single mouse recovering from the infection (Figure 5E).
  • mice of each treatment group were sacrificed to determine viral titers in the lung.
  • Lungs of BafA and mock treated mice showed very similar viral titers in both groups.
  • Viral infiltration of lungs was also examined by immunohistochemistry using an anti- matrix antibody.
  • Virus infected and mock-treated mice showed massive viral infections of the bronchiolar epithelial cells at the time of death (or euthanasia) (Figure 6A). No staining was visible in mock infected negative control mice ( Figure 6B). In the surviving SaliPhe- treated mice (sacrificed on day 15) the virus was essentially cleared ( Figure 6C).
  • Diphyllin was inactive against viral infections with PR8-NS 116-GFP up to concentrations of 10 ⁇ . Furthermore, we confirm the IC 50 values of 2.4+0.1 ⁇ published by Lim et al. (Lim et al., 2007) which rules out the antiviral activity of diphyllin published by Konig et al. (Konig et al, 2009) ( Figure 10).
  • an assay was used that allowed monitoring of viral growth by measuring the fluorescence of intracellular GFP encoded by a viral GFP-NS 1 fusion gene.
  • the strength of the assay is that the PR8-NS 116- GFP IAV undergoes a complete natural replication cycle in vitro, involving most interactions between host cell proteins and wt virus proteins except full size NS 1.
  • the assay can be readily adapted to high throughput screening of compound libraries to detect anti-IAV activity.
  • SaliPhe was active against the PR8-NS 116-GFP IAV and wt virus isolates that are of major public health concern. While the cytotoxicity of this v-ATPase inhibitor was in the ⁇ range, antiviral activity ranged from 28 nM to 206 nM.
  • the older v- ATPase inhibitors including ArchB, ConmyA and in particular BafA were also active against influenza viruses (EC 5 0 of 0.2-3 nM) but they were cytotoxic in the nanomolar and subnanomolar range.
  • mice intratracheal instillation of acetylsalicylic acid also partially protected mice against a H7N7 strain.
  • transmembrane proton translocating domain of cellular v-ATPase responsible for endosomal acidification can be selectively targeted by benzolactone enamide v-ATPase inhibitor with reduced toxicity to treat influenza virus infection including multiresistant influenza strains.
  • Treatment strategies against influenza that target cellular proteins are expected to be more resistant to virus mutations than drugs blocking viral proteins.
  • Figure IB The structure of a v-ATPase
  • A Representative fluorescence micrographs of MDCK cell cultures infected with a MOI of 0.03 of PR8-NS 116-GFP incubated with increasing concentrations of v-ATPase inhibitors. Drug concentrations between asterisks in panel B are shown.
  • B,C Anti-IAV activity (open diamonds) of v-ATPase inhibitors against PR8-NS 1116-GFP virus in MDCK (B) and A549 cells (C). The curves represent the percent fluorescence of cultures with the lowest concentration of drug and were used to calculate the EC 5 0. Each data point represents the mean of a triplicate culture of a representative experiment repeated at least twice. The cytotoxicity of the inhibitors is shown for comparison (closed squares). The SI was calculated on the ratio IC 5 0/EC 5 0.
  • H1N1 OR Oseltamivir resistant seasonal H1N1
  • S-OrV swine origin pandemic influenza virus
  • HPAI highly pathogenic H5N1 on MDCK cells.
  • the virus was quantified by quantitative PCR (c t values) and was expressed in percent of Ac t values of the lowest drug concentration and virus-free cultures. Raw data were fitted to hill curves to calculate the EC 5 0 shown in the inserts. Essentially the same results are obtained if calculated in percent of drug free cultures containing the DMSO only.
  • Each data point represents a cell culture triplicate of a representative experiment that was repeated at least twice.
  • the cytotoxicity of the inhibitors is shown for comparison (closed squares).
  • the SI was calculated as the ratio IC 5 0 / EC 5 0. S.D. are occasionally smaller than symbols.
  • A,D Kaplan-Maier-Survival curves of mice challenged with 4 mouse LD 5 0 of mouse adapted PR8 treated with SaliPhe (8 mice) or with Lipovenos only (9 mice) (A) and treated with BafA (10 mice) or PBS only (9 mice) (D).
  • FIG. 7 In vivo toxcitiy of SaliPhe and BafA. Body weight (A,B), body temperature (D,E) and organ weights (C,F) of mock infected mice, treated with standard protocol of SaliPhe (9 animals) (A,D) or BafA (12 animals) (B,E) in comparison to diluant treated mice (Lipovenos, 9 mice; or PBS, 12 mice). Body weights and temperature are expressed in percent of day 0. (C,F) Weights of spleens and livers expressed in percent of body weight. S.D. are occasionally smaller than symbols.
  • Anti-viral effect of WY-2051, WY-1295, LX-1072 against a H5N1 IAV isolates The virus was quantified by quantitative PCR (c t values) and was expressed in percent of Ac t values of the lowest drug concentration and virus-free cultures. Raw data were fitted to hill curves to calculate the EC 5 0 shown in the inserts. Essentially the same results are obtained if calculated in percent of drug free cultures containing the DMSO only. Each data point represents a cell culture triplicate of a representative experiment that was repeated at least twice. The cytotoxicity of the inhibitors is shown for comparison (closed squares). The SI was calculated as the ratio IC 5 0 / EC 5 0. S.D. are occasionally smaller than symbols.
  • the curves represent the percent fluorescence of cultures with the lowest concentration of drug and were used to calculate the EC 5 0.
  • Each data point represents the mean of a triplicate culture of a representative experiment repeated at least twice. The cytotoxicity is shown for comparison (closed squares). It is clear that diphyllin is inactive against IAV at toxic concentrations.
  • Vacuole membrane fusion V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel.
  • the IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139(7): 1243-1254.
  • RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463(7282): 818-822.
  • Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nat Genet 23(4): 447-451.
  • Acetylsalicylic acid blocks influenza virus propagation via its NF-kappaB -inhibiting activity.
  • bafilomycin Al raised lysosomal pH and both prodigiosins and concanamycin A inhibited growth through apoptosis.
  • Bafilomycin Al is a potassium ionophore that impairs mitochondrial functions. Bioenerg Biomembr 39(4): 321-329.
  • Salicylihalamide A inhibits the V0 sector of the v- ATPase through a mechanism distinct from bafilomycin Al. J Biol Chem 279(19): 19755-19763.

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Abstract

La présente invention concerne des benzolactones, inhibitrices de la v-ATPase, destinées à être utilisées dans le traitement d'infections par le virus de la grippe A. Les composés sont administrés en quantités thérapeutiquement efficaces et inférieures aux doses sous-cytotoxiques. La présente invention concerne en outre des procédés de traitement d'infections par le virus de la grippe A à l'aide de benzolactones inhibitrices de la v-ATPase.
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WO1996021644A1 (fr) 1995-01-10 1996-07-18 Smithkline Beecham S.P.A. Derives indole utiles dans le traitement de l'osteoporose
WO1998001436A1 (fr) 1996-07-09 1998-01-15 Smithkline Beecham S.P.A. Derives de l'acide pentadienoique heteroaromatique utilises comme inhibiteurs de la resorption osseuse
WO2000051589A2 (fr) 1999-03-05 2000-09-08 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Composes inhibiteurs d'atpase-(h+) de type vacuolaire, compositions et uitlisations associees
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