TECHNICAL FIELD OF THE INVENTION

[001] The present invention relates to an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject. The ergosterol- biosynthesis inhibitor is a compound comprising at least one 1 /-/-1 ,2,4-triazole-1-yl group and is selected from the group consisting of itraconazole, posaconazole, voriconazole, fluconazole or fosfluconazole.

BACKGROUND OF THE INVENTION

[002] Viral infection is not only extremely harmful to human health, but also severely endangers the survival and breeding of various animals, and thus becoming an important research task in medical and relevant fields currently. Investigation and invention of a medicine for treating viral infection has a significant application potential. Therefore, once succeeds, it will generate a huge social and economic benefit.

[003] Acute viral infectious disease of the upper respiratory tract such as influenza (shortened as“flu”) is one of the most common infectious diseases. It has some features, such as a strong infectivity, a rapid spreading ability, a short latent period, and a high morbidity. Influenza is a viral infectious disease commonly suffered in human, avian and livestock.

[004] At date the most reliable prevention measure against influenza infections is vaccination, which is currently the first strategy for preventing human influenza. However, influenza virus has many serotypes, so if the antigenicity between the vaccine strain and epidemic strain does not match each other, the vaccine will lose its function, and may not provide corresponding protection. On the other hand, since influenza virus varies frequently, the speed of vaccine development is far behind that of influenza virus variation.

[005] When a new epidemic strain appears, at least 6 months are required for producing its corresponding vaccine (Kamps et al, 2006), which makes the preparation of vaccines always in a passive position. In this case, neither a traditional inactivated vaccine nor a novel vaccine such as genetic engineering vaccine, a nucleic acid vaccine and the like can provide crossing protection for all types of influenza virus. After preventive vaccination full immunization may only be ensured after about 2 weeks.

[006] Further, due to high replication rates and the frequency of mutations in the virus increasing resistance against antiviral medicaments such as Amantadin, which is largely ineffective due to a mutation in the Neuraminidase gene may be expected. [007] Therefore, the protection period for influenza vaccines is very short, i.e. only half a year to a year, so these vaccines need to be injected every year, which is hard to be accepted by the patients. People not being able to become vaccinated due to previous illnesses or not becoming fully vaccinated due to immunodeficiency have to be excluded anyway.

[008] Due to the relatively poor effect of vaccines on prophylaxis of influenza, the investigation of pharmaceuticals for preventing and treating influenza virus has drawn a lot of attention.

[009] Thus, there is a need in the art for new therapy and prophylaxis treatment of influenza- virus infections.

[010] Therefore, the objective of the present invention is to comply with this need.

[011] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the figures and reflected in the claims.

SUMMARY OF THE INVENTION

[012] Surprisingly it was found, that by repurposing the use of an ergosterol-biosynthesis inhibitor of the present invention being applied for a long time in therapy for other diseases such as fungal diseases, the ergosterol-biosynthesis inhibitor of the present invention are now used in the method of treatment or prophylaxis of an influenza virus infection by administering said ergosterol-biosynthesis inhibitor to a subject. By repurposing of known medicaments such as the ergosterol-biosynthesis inhibitor of the present invention, costs for development and approval may significantly be reduced. Thus, also people being treated with ergosterol- biosynthesis inhibitor as a medicament against other diseases such as fungal diseases may already be protected from suffering from influenza, thereby further reducing the costs for vaccination. The use of said ergosterol-biosynthesis inhibitor of the present invention may be an alternative to neuraminidase inhibitors such as Tamiflu or Relenza, against which influenza virus may become resistant.

[013] Accordingly, in a first aspect, the present invention relates to an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject.

[014] The ergosterol-biosynthesis inhibitor for the use in a method of treatment or prophylaxis of an influenza virus infection in a subject may be a compound comprising at least one 1 H- 1 ,2,4-triazole-1-yl group.

[015] The present invention may further envisage the ergosterol-biosynthesis inhibitor for the use as described elsewhere herein, wherein the compound inhibits the lanosterol-14a- demethylase.

[016] The ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject, wherein the ergosterol-biosynthesis inhibitor may be a compound comprising at least one 1 V-1 ,2,4-triazole-1 -yl group, may be itraconazole, posaconazole, voriconazole, fluconazole or fosfluconazole. Preferably, the compound is itraconazole or posaconazole, more preferably itraconazole.

[017] Further, the present invention may encompass the ergosterol-biosynthesis inhibitor for use as described elsewhere herein, wherein the influenza virus is influenza-A-virus or influenza- B-virus. Preferably, the influenza virus being treated by the ergosterol-biosynthesis inhibitor of the present invention is a influenza-A virus H1 N1 subtype.

[018] Additionally, the present invention may envisage that the subject being treated against influenza virus infection is a mammal suffering from a fungal infection. Preferably, the mammal suffering from a fungal infection is a mouse, a rat, a pig, a horse, a dog, a cat, a bird, a human.

[019] The present invention may also comprise that the ergosterol-biosynthesis inhibitor of the present invention is further for the use of a method of treating or preventing a fungal infection. Preferably, a fungal infection being treated or prevented by the ergosterol-biosynthesis inhibitor of the present invention is an Aspergillus infection, cryptococcal disease, histoplasmosis, a yeast infection, a dermatophyte infection.

BRIEF DESCRIPTION OF THE FIGURES

[020] Fig. 1 : Measuring viral titers in cells infected with H1 N1 influenza-A-virus after treatment with posaconazole or itraconazole.

Itraconazole treatment significantly decreases virus titers in A549 human airway epithelial cells, which were infected with H1 N1 influenza A virus strain PR8 at the indicated multiplicity of infection (MOI) after treatment with posaconazole or itraconazole. On the basis of the initial viral titers of DMSO-treated cells, viral titers are decreased by about 97-98% post infection. Data represent means ± standard error of the mean of four-six independent experiments. One-way AN OVA followed by Dunnett's multiple comparison test, n.s. not significant, ^* p < 0.05, ^** p <

0.01 , ^*** p < 0.001.

[021] Fig. 2: Itraconazole treatment decreases lung virus titers in mice infected with H1 N1 influenza-A-virus.

On the basis of the initial viral titers of the control, viral titers are decreased by about 90% (A and B) and by about 95 % (C) at day 3 (A), 5 (B) and 7 (C) post infection with H1 N1 influenza A virus strain PR8 after the mice has also been fed daily with a single dose of either solvent (control) or itraconazole. Scatter plots show individual values, means ± standard error of the mean of 5 mice/group. Mann-Whitney U test, ** p < 0.01 , *** p < 0.001.

[022] Fig. 3: Itraconazole and posaconazole block influenze-A-virus infection at non- cytotoxic concentration.

(A) Dose-response curve of itraconazole and posaconazole in A549 cells. Cells were treated with either DMSO, posaconazole or itraconazole and subsequently infected with influenza A virus strain PR8M (MOI = 0.1 ). Both drugs efficiently inhibit replication of the H1 N1 strain PR8M (B) Virus titers were converted to percentages, posaconazole and itraconazole concentrations were log-transformed, and EC ₅₀ and ECg ₀ values were calculated from the semi-logarithmic fitted curves. The 50% effective concentrations (EC ₅₀ values) were seen at low micromolar concentrations, and higher doses reduced IAV propagation up to 98%.

[023] Fig. 4: The antiviral activities are not virus, subtype or cell line specific.

A549, A431 cells or HUVECs were treated with either DMSO, posaconazole or itraconazole and subsequently infected with the indicated MOI of (A) influenza A virus strain PR8 (H1 N1 ) or (B) strain PAN (H3N2). (C) Madin-Darby canine kidney (MDCK) cells type II were treated with either DMSO, posaconazole or itraconazole, and subsequently infected with the indicated MOI of the influenza B virus, subtype B/Lee/1940 (Lee). Data represent mean viral titers ± SEM, n= 5, one- way ANOVA with Dunnett’s multiple comparison tests, ^**** p<0.0001 ; ^*** p<0.001 ; ^** p<0.01 ; ^* p<0.05.

[024] Fig. 5: Itraconazole treatment restricts progression of influenza A virus infection in mice.

Cumulative survival rate (A) and loss of liveweight (B) plotted against days post infection. Mortality also includes mice that were euthanized because of a body weight loss of > 20%. n= 20 mice/group, Mantel-Cox log rank test, p= 0.0136 or Mann-WhitneyU-test. Statistical significance of the differences was evaluated by unpaired studentt-test onAACt values. ^* p< 0.1 ,

^** p< 0.01 , ^*** p< 0.001 , ^**** p< 0.0001.

DETAILED DESCRIPTION OF THE INVENTION

[025] In order to overcome some of the short comings of the means described so far in the prior art for influenza virus infection, the inventors provide herein repurposing of the use of an ergosterol-biosynthesis inhibitor, now being used for the treatment or prophlaxis of an influenza virus infection in a subject.

[026] As described above, the present invention relates to an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject.

[027] In this context, the“ergosterol-biosynthesis inhibitor” refers to a compound which is able to inhibit the fungal-mediated ergosterol biosynthesis. Such an ergosterol-biosynthesis inhibitor is applied in the present invention. Ergosterol (ergosta-5,7,22-trien-3 -ol) is a sterol found in cell membranes of fungi and protozoa. It is a provitamin form of vitamin D ₂ and has the same function as cholesterol in animal cells. It is a component of yeast and other fungal cell membranes. After generation of ergosterol, the steroid is able to alter the permeability and rigidity of plasma membranes such as cholesterol does in animals. Thus, the prevention of the production of this key compound in fungal cells by ergosterol-biosynthesis inhibitors has become an important target in drug discovery. In general, ergosterol-biosynthesis inhibitor has been used as drugs in fungal infection or in infection against protozoa. Surprisingly, it was now found that said ergosterol-biosynthesis inhibitor of the present invention being an already approved medicament for other purposes such as treating fungal diseases may be used in the method of treatment or prophylaxis of influenza virus infection in a subject according to the present invention.

[028] The subject, which is treated against influenza virus infection with an ergosterol- biosynthesis inhibitor may be a mammal. The mammal may be a mouse, a rat, a pig, a horse, a dog, a cat, a bird, a human, preferably a human. Thus, the present invention comprises an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a human. A subject as defined elsewhere herein, preferably a mammal, more preferably a mouse, a rat, a pig, a horse, a dog, a cat, a bird, a human, even more preferably a human, may also additionally suffer from a fungal disease. Said subject as indicated elsewhere herein may further suffer from a fungal disease, already suffering from an influenza virus infection, wherein a fungal disease may accompany an influenza virus infection in said subject of the present invention.

[029] The ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject may be a compound comprising at least one 1 H-1 ,2,4- triazole-1-yl group.

[030] In this context, the term“compound” refers to a“small molecule” as used herein and denotes an organic molecule comprising at least two carbon atoms, but preferably not more than 7, 12, 15, 17, 20 or 25 rotatable carbon bonds (excluding carbon-hydrogen bonds), more preferably not more than 7, 12, 15, 17 or 20 rotatable carbon bonds, even more preferably not more than 7, 12, 15 or 17 rotatable carbon bonds, having a molecular weight in the range between 100 and 2000 Dalton (DA), preferably between 100 and 1000 Dalton, more preferably about 700 Dalton and optionally including one or two metal atoms. As merely illustrative examples for small molecules itraconazole, posaconazole, voriconazole, fluconazole or fosfluconazole may be mentioned. In a preferred embodiment posaconazole and/or itraconazole may be illustrative examples for small molecules, such as the compound comprising at least one 1/-/-1 ,2,4-triazole-1-yl group of the present invention.

[031] In the case, the compound refers to a mixture of isomers, preferably two diastereomers and two enantiomers, the rotatable carbon bonds of only one isomer may count for the whole number of rotatable carbon bonds of the mixture of isomers, e.g. not more than 15 rotatable carbon bonds of one isomer of the mixture counts for the total amount of rotatable carbon bonds of the whole mixture.

[032] A 1 H-1 ,2,4-triazole-1-yl group refers to triazole derivative comprising three nitrogen atoms at the relative position 1 , 2, and 4 of the triazole, wherein the nitrogen at position 1 is bound to the remaining part of the molecule such as for example itraconazole, posaconazole, voriconazole, fluconazole or fosfluconazole. The structure of the specific 1/-/-1 ,2,4-triazole-1-yl group of the ergosterol-biosynthesis inhibitor is as follows:

Formula I

[033] In this context, the term “triazole” refers to a heterocyclic compound having the molecular formular C ₂ H ₃ N ₃ , thus having a five-membered ring of two carbon atoms and three nitrogen atoms. There are two sets of isomers 1 ,2,3-triazole and 1 ,2,4-triazole, differing in the relative positions of the three nitrogen atoms. 1 ,2,3-triazole comprises 1 H-1 ,2,3-triazole and 2 H- 1 ,2,3-triazole, differing by which nitrogen has a hydrogen bonded to it. Thereby, 2H-1 ,2,3- triazole is the tautomeric form of 1 H-1 ,2,3-triazole. 1 ,2,4-triazole comprises 1 H-1 ,2,4-triazole and 4H-1 , 2, 4-triazole, also differing by which nitrogen has a hydrogen bonded to it. Thereby, 1H-1 ,2,4-triazole is the tautomeric form of 4H-1 ,2,4-triazole.

[034] The structures of each triazole are depicted as follows: Formula II Formula III

1H-1 ,2,3-Triazol 2HA ,2,3-Triazol

Formula IV Formula V

1 H-1 ,2,4-Triazol 4H-1 ,2,4-Triazol

[035] In this context, the term “residue” or“moiety” may be use interchangeably with the term“group”.

[036] In the present invention the mode of action of the ergosterol-biosynthesis inhibitor, wherein the ergosterol-biosynthesis inhibitor refers to said compound comprising at least one 1 /-/-1 ,2,4-triazole-1 -yl group as described elsewhere herein was demonstrated in the Examples shown by the decrease of viral titers determined by viral Plaque assay. It was shown that said ergosterol-biosynthsis inhibitor of the present invention decreases viral titer in PFU/ml (given as plaque forming units per sample unit volume) post infection with influenza A virus strain (in particular H 1 N1 subtype influenza A virus strain) in a cell culture model for studying respiratory infection (Fig. 1 and 3). The decrease of viral titers by the application of said ergosterol- biosynthesis inhibitor of the present invention for use in a method of treatment or prophylaxis of an influenza virus infection in said subject may be determined by Plaque assay. Standard plaque assays being used are well known in the art.

[037] The term “plaque assay” refers to a standard method used to determine virus concentration in terms of infectious dose. Such an assay determines the number of plaque forming units (PFU) in a sample. It may be performed in petri dishes or multi-well plates. In particular, a confluent monolayer (in particular the madin darby canine kidney (MDCK) monolayer) of host cells may be infected with the virus at varying dilutions and covered with a semi-solid medium, including agar or carboxymethyl cellulose, to prevent the virus infection from spreading. Once a virus may infect a cell within the fixed cell monolayer a viral plaque is formed. Said infected cell may lyse, thereby spreading the infection to other cells in the dish or plate, resulting in a repeat of infection-to-lysis cycle. Said infected cell layer may create a plaque (holes in the cell layer) referring to an area of infection surrounded by uninfected cells. Those plaques can be seen visually or with an optical microscope. The virus plaques may be visualized by staining with neutral red, methylene blue or crystal violet. The plaques are then counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). Thus, virus titers may be indicated as PFU/ml in a sample.

[038] With regard to the above said, decreasing viral titers determined by the Plaque assay by said ergosterol-biosynthesis inhibitor of the present invention as a direct measure may be preferred in the present invention since this read out is explicitly relevant for assessing / evaluating therapeutical antiviral effects in comparison to other read outs known in the prior art such as host cell factor targeting determined by cell viability (%), which only determines the extent of damage of infected cells.

[039] The present inventors have also demonstrated that said antiviral activities of the ergosterol-biosynthesis inhibitor of the present invention as described elsewhere herein are neither influenza virus, influenza virus subtyp nor cell line specific (Fig. 4). It was clearly shown that the ergosterol-biosynthesis inhibitor of the present invention not only has a protective effect and thus blocks infection with different strains of the influenza A virus (Fig. 4A and B), but can also be used in a method of treatment or prophylaxis of influenza B virus infection in a subject according to the present invention (Fig. 4C). Thus, said ergosterol-biosynthsis inhibitor may be used in a method of treatment or prophylaxis of any influenza virus infection in a subject, also being independent of the specific subtype of the particular virus.

[040] It was further shown, that viral titers determined by the Plaque assay after the treatment with said ergosterol-biosynthsis inhibitor were also decreased in a suitable animal model, such as a mice model. Viral titers (PFU/ml), in particular pulmonary (lung) viral titers, were decreased in said mice model after the treatment with said ergosterol-biosynthesis inhibitor after infection with influenza A virus (in particular H1 N1 subtype influenza A virus strain) (Fig. 2). At least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 300, at least about 400, at least about 500 mg/kg/day, such as about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, abiut 300, about 400, about 500 mg/kg/day of said ergosterol-biosynthesis inhibitor, which may decrease viral titers determined by the Plaque assay, may be administered to the subject in need thereof. In a preferred embodiment and as being described in the Examples, about 70 mg/kg/day of said ergosterol-biosynthesis inhibitor is administered to the subject in need thereof. Thereby, a mouse may be fed with a single dose of either solvent (such as propylene glycol hydroxypropyl- -cyclodextrine as a control) or the q

ergosterol-biosynthesis inhibitor of the present invention, being further infected with an influenza virus strain. Mice may then be sacrificed after infection with said influenza virus strain to determine acute-phase virus titers, preferably in the lungs. In particular, viral titers obtained from lung homogenates may be determined by Plaque assay at the indicated day post infection by preparing serial dilutions of lung homogenate samples and by applying further steps of said Plaque assay as described elsewhere herein.

[041] Thus, the present invention provides an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in said subject such as mice, rat, cat, dog, pig, horse, bird or even in humans, wherein said ergosterol-biosynthesis inhibitor decreases viral titers (in PFU/ml) determined by Plaque assay.

[042] “Decrease”,“decreasing” or“decreased” includes 5% or 10% reduction, or 20% or 30%, preferably 40% or 50%, more preferably 60% or 70%, even more preferably 80% or 85% and particularly preferred 90% reduction, most particularly preferred 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% reduction of the initial viral titer of the control (e.g. DMSO- treated cells or mice control group treated with solvents such as propylene glycol hyd roxy p ro py I-b-cyclod extri n e ) after the treatment with said ergosterol-biosynthesis inhibitor of the present invention. Also included by the term“decrease”,“decreasing” or“decreased” are 5% or 10% reduction, or 20% or 30%, preferably 40% or 50%, more preferably 60% or 70%, even more preferably 80% or 85% and particularly preferred 90%, most particularly preferred 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100 % reduction of the initial viral titer of the control (e.g. DMSO-treated cells or mice control group treated with solvents such as propylene glycol hydroxypropyH3-cyclodextrine) between 10 ⁵ to 10 ¹⁰ PFU/ml, or between 10 ⁶ to 10 ¹⁰ PFU/ml, or between 10 ⁷ to 10 ¹⁰ PFU/ml, or between 10 ⁸ to 10 ¹ ° PFU/ml, or between 10 ⁷ to 10 ⁹ PFU/ml, or between 10 ⁷ to 10 ⁸ PFU/ml after the treatment with said ergosterol-biosynthesis- inhibitor. Further, a decrease may also include 5% or 10% reduction, or 20% or 30%, preferably 40% or 50%, more preferably 60% or 70%, even more preferably 80% or 85% and particularly preferred 90%, most particularly preferred 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100 % reduction of the initial viral titer of the control (e.g. DMSO-treated cells or mice control group treated with solvents such as propylene glycol hydroxypropyl-3-cyclodextrine) of 10 ⁵ PFU/ml, 10 ⁶ PFU/ml, 10 ⁷ PFU/ml, 10 ⁸ PFU/ml, 10 ⁹ PFU/ml, or even 10 ¹ ° PFU/ml after the treatment with said ergosterol-biosynthesis-inhibitor. Viral titer may decrease significantly after 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 day(s) after influenza virus infection (in particular after influenza A virus infection) and after the subject has been treated with said ergosterol- biosynthesis-inhibitor.

[043] In line with the inhibitory effects observed in the cell culture models (e.g., Fig. 1 and 3), a clearly reduced mortality ( vice versa a higher survival rate) in mice treated with the ergosterol- biosynthesis inhibitor of the present invention was observed compared to the control not being treated with the ergosterol-biosynthesis inhibitor of the present invention. It was further shown that the improved survival correlated with diminished body-weight loss during the course of influenza virus infection (Fig. 5).

[044] The ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza vius infection in a subject, which is a compound comprising at least one 1 /-/-1 ,2,4- triazole-1 -yl group may inhibit the lanosterol-14a-demethylase.

[045] The term 1anosterol-14a-demethylase” refers to a cytochrome P450 enzyme. It catalyzes the removal of the C-14a-methyl group from lanosterol. Thus, catalyzing the demethylation of lanosterol is important to creat a precursor being converted to ergosterol. Inhibiting said enzyme as the ergosterol-biosynthesis inhibitor (the compound comprising at least one 1 H-1 ,2,4-triazole-1 -yl group) does, results in destroying the fungal cell’s ability to produce ergosterol. This leads to a disruption of the plasma membrane, thereby resulting in a cellular leakage. It may also result in the death of the pathogen as well. Such function of said lanosterol-14a-demethylase is being inhibited by the ergosterol-biosynthesis inhibitor of the present invention. The ergosterol-biosynthesis inhibitor of the present invention may inhibit lanosterol-14a-demethylase and/or decreases the viral titers determined by Plaque assay as indicated elsewhere herein and which is shown in the Examples as well.

[046] Preferably, said ergosterol-biosynthesis inhibitor of the present invention is itraconazole, posaconazole, voriconazole, fluconazole or fosfluconazole, more preferably itraconazole or posaconazole, even more preferably itraconazole.

[047] Preferably, itraconazole and posaconazole are preferred ergosterol-biosynthesis inhibitors. Thus, the present invention may also refer to itraconazole and/or posaconazole for use in a method of treatment or prophylaxis of an influenza virus infection in said subject of the present invention. The present invention may also refer to itraconazole for use in a method of treatment or prophylaxis of an influenza virus infection in said subject.

[048] Itraconazole and posaconazole are preferred ergosterol-biosynthesis inhibitors, which may inhibit lanosterol-14a-demethylase and/or decrease viral titers determined by the Plaque assay. In a preferred embodiment, posaconazole may inhibit lanosterol-14a-demethylase and/or decrease viral titers determined by the Plaque assay as indicated elsewhere herein. In a particular preferred embodiment itraconazole is the preferred ergosterol-biosynthesis inhibitor which inhibits lanosterol-14a-demethylase and/or decreases viral titers determined by the Plaque assay as indicated elsewhere herein and shown in the Examples (Fig. 1 and 2).

[049] Itraconazole (trade name Sporanox; chemical formula: C ₃ H ₈ Cl N _{8 4} ) (short: ICZ) belongs to the triazole group of antifungals with potent lipophilic features, low toxicity, and broad- spectrum antifungal activity against pathogenic yeasts and fungi. ICZ may be given orally as a drug for Cryptococcus neoformans, Histoplasma capsulatum, and several other fungal pathogens. It may also be given intravenously or as a nasal spray (intra-nasally).

[050] The clinical formulation of Itraconazole may be a mixture of isomers, in particular comprising two diastereomers and two enantiomeres in a racemic mixture, in general a 1 : 1 : 1 : 1 mixture (Kunze et al. 2006, Drug Metabolism and Disposition: the Biological Fate of Chemicals. 34 (4): 583-90). One isomer of itraconazole may comprise not more than 15 rotatable carbon bonds and may have a molecular weight of about 705.64 Dalton (g/mol). The racemic mixture of Itraconazole has the following structure:

[051] (2 R, 4 S, 2’F?)-itraconazole may be the enantiomeric form of (2S, 4 R, 2’S)-itraconazole, whereas (2 R, 4S, 2’S)-itraconazole may be the enantiomeric form of (2 S, 4 R, 2’R)-itraconazole. (2S, 4F?, 2’ ?)-itraconazole may be the diastereomeric form of (2S, 4 R, 2’S)-itraconazole, whereas (2 R, 4S, 2’F?)-itraconazole may be the diastereomeric form of (2 R, 4S, 2'S)- itraconazole.

[052] Posaconazole (trade name Noxafil, chemical formula: Cayl-^F _j NgC ) refers to a fluorinated triazole antifungal drug and is structurally related to itraconazole. In general, it may have activity against Candida species, Aspergillus species, Cryptococcus neoformans, the zygomycetes, and other fungi. Posaconazole may be administered orally or intravenously (Greer 2007, Proc (Bayl Univ Med Cent), 20(2): 188-196). Posaconazole may comprise not more than 17 rotatable carbon bonds and may have a molecular weight of about 700.778 Dalton (g/mol). Posaconazole has the following structure:

[053] Voriconazole (trade name Vfend; chemical formula: Ci ₆ H ₁₄ F ₃ N ₅ 0) is also a triazole antifungal agent and is structurally related to fluconazole. In general, it may be indicated for the treatment of invasive aspergillosis, but is not limited to it. It may be given orally or intravenously (Greer 2003, Proc (Bayl Univ Med Cent), 16(2): 241-248). Voriconazole may comprise not more than 10 rotatable carbon bonds and may have a molecular weight of about 349.3 Dalton (g/mol). Voriconazole has the following structure:

[054] Fluconazole (trade name Diflucan, chemical formula: C ₁₃ H ₁₂ F2N ₆ 0) is water soluble, has a longer plasma half-life than other azoles and has relatively low toxicity. It may be currently the most extensively used anti-fungal agent for the treatment of patients with severe candidiasis, but is not limited to it. It may be available in oral capsule, oral solution and saline-based intravenous solution formulations, thus it may be given orally or intravenously. All formulations exhibit predictable pharmacokinetics (Humphrey et al 1985, Antimicrobial Agents and Chemotherapy 28, 648-53 and Debruyne und Ryckelynck 1993, Clinical Pharmacokinetics 24, 10-27). Fluconazole may comprise not more than 8 rotatable carbon bonds and may have a molecular weight of 306.27 Dalton (g/mol). Fluconazole has the following structure:

[055] Fosfluconazole (trade name Prodif; chemical formula: CI ₃ H ₁₃ F2N ₆ 04P) is a phosphate prodrug of fluconazole (FLCZ), which is highly soluble compared to fluconazole. Fosfluconazole is at least 25-fold less potent than fluconazole in vivo, preferably against single isolates of Candida species and Cryptococcus neoformans. In vivo it has similar efficacy to fluconazole in fungal disease. It may be adiminstered intravenously or orally (Sobue et al. 2004, Br J Clin Pharmacol. 2004 Jun; 57(6): 773-784). Fosfluconazole may comprise not more than 11 rotatable carbon bonds and may have a molecular weight of about 386.25 Dalton (g/mol). Fosfluconazole has the following structure:

[056] An “influenza virus infection”, commonly known as "the flu", which is treated in said subject by the use of an ergosterol-biosynthesis inhibitor of the present invention, is an infectious disease caused by an influenza virus, commonly suffered in human, avian and livestock. It is an acute infectious disease of the upper respiratory tract and has some features, such as a strong infectivity, a rapid spreading ability, a short latent period, and a high morbidity. It is the most rampant infectious disease in the world. Symptoms of influenza can start quite suddenly one to two days after infection. Usually the first symptoms are chills or a chilly sensation, but fever is also common early in the infection, with body temperatures ranging from 38-39°C up to 42°C. Many subjects are so ill that they are confined to bed for several days, with aches and pains throughout their bodies, which are worse in their backs and legs. Symptoms of influenza may include fever and extreme coldness (chills shivering, shaking (rigor), cough, nasal congestion, body aches, especially joints and throat, fatigue, headache, irritated, watering eyes, reddened eyes, skin (especially face), mouth, throat and nose, in children, gastrointestinal symptoms such as diarrhea and abdominal pain (may be severe in children with influenza B). The term “influenza viral infectious disease” may be used interchangeably with the term “influenza virus infection”.

[057] In this context, the term “influenza virus” is the pathogen causing influenza, which belongs to Orthomyxoviridae family, and is a negative-strand RNA virus varying frequently. Influenza virus is essentially consisted of envelope and nucleocapsid, wherein the envelope is composed of spike, bilayer lipid membrane and matrix protein (M). The spike is divided into two categories: one is rod-shaped and consisted of trimers of hemagglutinin (HA) protein; the other is mushroom-shaped and consisted of tetramers of neuraminidase (NA). The bilayer lipid membrane is obtained from host cells membrane while the virion is budding. The matrix protein is closely lined up at the inner side of the envelope, and is a structural protein maintaining the viral shape. The nucleocapsid, coated by the matrix protein, is consisted of 8 RNA- nucleoprotein complexes (RNP) arranged in a helix shape. Each RNP is consisted of monomer negative-strand RNA coated with nucleoprotein (NP). The 8 negative-strand RNAs in influenza virus genome thus constitute 8 RNPs.

[058] According to the different antigenicity between nucleoprotein NP and matrix protein M, influenza virus is divided into 3 types, i.e. A, B and C. The 3 types of influenza virus do not share any common antigen, but all infect human. Among them, influenza A virus has the biggest pandemic scale. Each type of influenza virus is further divided into many subtypes according to the different antigenicity of hemagglutinin (HA; shortly as“H”) and neuraminidase (NA; shortly as“N”) at the viral surface. There has been found 16 subtypes of H, 9 subtypes of N in influenza A virus. Subtype H and N may constitute various combinations, such as H1 N1 , H5N1 , H3N2, and H9N2, etc.

[059] Influenza virus is very rampant, since it constantly varies. The reasons causing the variation mainly include: 1 ) influenza virus genome is consisted of negative-strand RNA and RNA replicase lacks of a correction function during the genomic replication, therefore it is very easy for the viral genome to be varied during the replication in host cells, which leads to the variation of the amino acids encoded by it and thereby leads to an antigen shift; 2) influenza virus genome is consisted of 8 of segmented RNAs, and the 8 segments can be randomly reassorted while one cell is infected with various influenza virion. Theoretically, 2 ⁸ (256) of new viral strains with completely different antigenicity and pathogenicity may be formed through the gene reassortment, therefore an antigen shift will occur, which enables influenza virus to escape the monitor of the immune system, to replicate in a large scale in host cells, and to cause new pandemic of influenza. As a result, the antigen of influenza virus varies rapidly and the reassortment rate among different influenza viral strains is very high, which leads to a huge challenge to the prophylaxis and treatment of influenza virus.

[060] In this context, the term“treatment” refers to a measure for treating a subject suffering from influenza virus infection. [061] Additionally, the term“prophylaxis” refers to a prevention / prevention measure for not developing an influenza virus infection in a subject not suffering at the moment or having suffered in the past from influenza virus infection when the prevention measure starts. In other words, preventing a subject from developing influenza virus infection in the near future at the time of prophylaxis is also envisaged by said term.

[062] Further contemplated by the present invention, is an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject, wherein the influenza virus may be an influenza-A-virus or influenza-B-virus, preferably the influenza virus is an influenza-A virus H1 N1 subtype.

[063] Influenza A virus according to the present invention may be based on any influenza A virus such as a bird flu, human flu, swine influenza, equine influenza or a canine influenza. Various different influenza A virus subtypes exist, differing in the nature of the HA and NA glycoproteins on their surface. Influenza A viruses are accordingly usually categorized into subtypes based on the combination of protein forms of Hemagglutinin (HA) and Neuraminidase (NA) present, two proteins on the surface of the viral envelope. In this context, haemagglutinin, which is a lectin causes red blood cells to clump together and which mediates the binding of the virus to target cells and the entry of the viral genome into the target cell. Neuraminidase, in this context, is a type of glycoside hydrolase enzyme which is important for moving the virus particles through the infected cell and is involved in the release of progeny virus from infected cells in the subject. Sixteen Hemagglutinin forms (H1 to H16) and nine Neuraminidase forms (N1 to N9) have been identified.

[064] Suitable virus strains include, but are not limited to H1 N1 , H1 N2, H1 N3, H1 N4, H1 N5, H1 N6, H1 N7, H1 N8, H1 N9, H2N1 , H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8, H2N9,

H3N1 , H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8, H3N9, H4N1 , H4N2, H4N3, H4N5,

H4N6, H4N7, H4N8, H5N1 , H5N2, H5N3, H5N4, H5N6, H5N7, H5N8, H5N9, H6N1 , H6N2,

H6N3, H6N4, H6N5, H6N7, H6N8, H6N9, H7N1 , H7N2, H7N3, H7N4, H7N5, H7N6, H7N7,

H7N8, H7N9, H8N1 , H8N2, H8N3, H8N4, H8N5, H8N6, H8N7, H9N1 , H9N2, H9N3, H9N4,

H9N5, H9N6, H9N7, H9N8, H9N9, H10N1 , H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, H10N9, H11 N1 , H11 N2, H11 N3, H11 N4, H11 N5, H11 N6, H11 N7, H11 N8, H11 N9, H12N1 , H12N2, H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H13N1 , H13N2, H13N3, H13N4, H13N5, H13N6, H13N8, H14N3, H14N5, H14N6, H15N8, H15N9, H16N3. In some embodiments the influenza virus is one of the strains H1 N1 , H1 N2, H2N2, H3N1 , H3N2, H5N1 and H7N7, preferably of the strain H1 N1.

[065] Influenza A (H1 N1 ) virus is the subtype of influenza A virus that was the most common cause of human influenza (flu) in 2009, and is associated with the 1918 outbreak known as the Spanish Flu. It refers to an orthomyxovirus that contains the glycoproteins haemagglutinin (HA) and neuraminidase (NA).

[066] An example of a H1 N1 strain is Influenza A virus strain A/Puerto Rico/8/1934(H1 N1 ) with Gene bank accession number NC 002016, NC 002017, NC 002018, NC 002019, NC 002020, NC 002021 , NC 002022, NC 002023. Further examples of a H1 N1 strain are Influenza A strain A/Brevig Mission/1/1918 H1 N1 ) (Influenza A virus (strain A/South Carolina/1/1918 H1 N1 ), Influenza A strain A/Russia:St. Petersburg/8/2006 H1 N1 , Influenza A strain A/USA:Texas/UR06- 0195/2007 H1 N1 -strain A/Brevig Mission/1/1918 H1 N1 , Influenza A strain A/South Carolina/1/1918 H1 N1 , Influenza A strain A/Swine/Iowa/15/1930 H1 N1 , Influenza A strain A/Wilson-Smith/1933 H1 N1 , Influenza A strain A/WS/1933 H1 N1 , and strain A/USA:Phila/1935 H1 N1. A further example of a H1 N1 strain is Influenza A virus strain A/New Zealand:South Canterbury/35/2000 H1 N1 , preferably a H1 N1 strain is Influenza A virus strain A/Puerto Rico/8/1934(1-11 N1 ).

[067] An example of a H1 N2 strain is Influenza A virus strain A/Xianfeng/3/1989 H1 N2. Two examples of a H1 N3 strain are Influenza A/duck/NZL/160/1976 H1 N3 and strain A/Whale/Pacific ocean/19/1976 H1 N3. An example of a H1 N4 strain is Influenza A virus strain A/mallard/Netherlands/30/2006 H1 N4. An example of a H1 N5 strain is Influenza A virus strain A/pintail duck/ALB/631/1981 H1 N5. An example of a H1 N6 strain is Influenza A virus strain A/murre/Alaska/305/1976 H1 N6. An example of a H1 N7 strain is Influenza A virus A/swine/England/191973/92 H1 N7. An example of a H1 N8 strain is strain A/Egyptian goose/South Africa/A11448/2007. An example of a H2N1 strain is Influenza A virus strain A/Japan/Bellamy/57 H2N1. An example of a H2N2 strain is Influenza A virus strain

A/Korea/426/68 H2N2 with Gene bank accession numbers NC 007366, NC 007367, NC

007368, NC 007369, NC 007370, NC 007374, NC 007375, NC 007376, NC 007377, NC

007378, NC 007380, NC 007381 and NC 007382. Three further examples of a H2N2 strain are Influenza A strain A/Japan/305/1957 H2N2, A/Czech Republic/1/1966 H2N2 and strain A/Singapore/1 /1957 H2N2. An example of a H2N3 strain is Influenza A virus strain

A/mallard/Minnesota/Sg-00692/2008 H2N3. An example of a H2N4 strain is A/mallard/ Alberta/149/2002 H2N4. An example of a H2N5 strain is Influenza A virus strain A/tern/Australia/1/04 H2N5. An example of a H2N6 strain is Influenza A virus strain A/thick-billed murre/Alaska/44145-199/2006 H2N6. An example of a H2N7 strain is Influenza A virus strain A/northern shoveler/California/HKWF1128/2007 H2N7. An example of a H2N8 strain is Influenza A virus strain A/turkey/CA/1797/2008 H2N8. An example of a H2N9 strain is Influenza A virus strain A/duck/Germany/1972 H2N9. An example of a H3N1 strain is Influenza A virus strain A/mallard duck/ALB/26/1976 H3N1 . An example of a H3N2 strain is Influenza A virus strain A/New York/392/2004 H3N2 with Gene bank accession numbers NC 007371 , NC 007372 and NC 007373. Five further example of a H3N2 strain are Influenza A virus strain A/X-31 H3N2, strain A/Hong Kong/5/1983 H3N2, A/Rio/6/69 H3N2, A/Hong Kong/MA/1968 H3N2 and Influenza A virus strain A/Shanghai/N 12/2007 H3N2. An example of a H3N3 strain is Influenza A virus strain A/duck/Hong Kong/22A/1976 H3N3. An example of a H3N4 strain is Influenza A virus strain A/mallard duck/ALB/1012/1979 H3N4. An example of a H3N5 strain is Influenza A virus strain A/northern shoveler/California/HKWF1046/2007 H3N5. An example of a H3N6 strain is Influenza A virus strain A/Chicken/Nanchang/9-220/2000 H3N6. Examples of a H3N8 strain are Influenza A strain A/Equine/Miami/1/1963 H3N8 and strain A/Duck/Ukraine/1 /1963 H3N8. An example of a H3N9 strain is Influenza A virus strain A/swan/Shimane/227/01 H3N9.

[068] An example of a H4N1 strain is Influenza A virus strain A/chicken/ Singapore/1992(H4N1 ). An example of a H4N2 strain is Influenza A virus strain A/duck/Hong Kong/24/1976(H4N2). An example of a H4N3 strain is Influenza A virus strain A/mallard/Sweden/65/2005(H4N3). An example of a H4N4 strain is Influenza A virus strain A/Grey teal/Australia/2/1979 H4N4. An example of a H4N5 strain is Influenza A virus strain A/duck/Hokkaido/1058/2001 (H4N5). Two examples of a H4N6 strain are Influenza A virus strain A/Duck/Czechoslovakia/1956 H4N6 and Influenza A virus strain A/Duck/Alberta/ 28/1976 H4N6. An example of a H4N7 strain is Influenza A virus strain A/duck/Mongolia/583/02 H4N7. An example of a H4N8 strain is Influenza A virus strain A/Chicken/Alabama/1 /1975 H4N8. An example of a H4N9 strain is Influenza A virus strain A/WDk/ST/988/2000(H4N9). An example of a H5N1 strain is Influenza A virus (A/Goose/Guangdong/1 /96(H5N 1 )) with Gene bank accession numbers NC 007357, NC 007358, NC 007359, NC 007360, NC 007362, NC 007363, and NC 007364. Further examples of a H5N1 strain are Influenza A strain A/Duck/Hong Kong/2986.1/2000 H5N1 , Influenza A strain A/Silky Chicken/Hong Kong/SF189/2001 H5N1 , Influenza A strain A/Chicken/Flong Kong/YU562/2001 FH5N 1 , Influenza A strain A/Chicken/Flong Kong/ FY150/2001 H5N1 , Influenza A strain A/Chicken/Hong Kong/715.5/2001 H5N1 , Influenza A strain A/Guinea fowl/Flong Kong/38/2002 H5N1 , Influenza A strain A/Chicken/Flong Kong/31.2/2002 FH5N 1 , Influenza A strain A/Chicken/Flong Kong/37.4/2002 H5N1 , Influenza A strain A/Silky Chicken/Flong Kong/YU 100/2002 FH5N1 , Influenza A strain A/Chicken/Flong Kong/96.1/2002 H5N1 , Influenza A strain A/Chicken/Flong Kong/YU22/ 2002 H5N1 , Influenza A strain A/Teal/China/2978.1/2002 H5N1 , Influenza A strain A/Hong Kong/212/2003 H5N1 , Influenza A strain A/Chicken/Shantou/4231/2003 FH5N 1 , and Influenza A strain A/Goose/Guangxi/345/2005 FH5N 1. An example of a FI5N2 strain is Influenza A strain

A/Chicken/Pennsylvania/1370/1983 H5N2. An example of a H5N3 strain is Influenza A strain A/duck/Malaysia/F119-3/97 H5N3. An example of a H5N4 strain is Influenza A strain

A/environment/New York/200269-18/2002 H5N4. An example of a H5N5 strain is Influenza A strain A/duck/Massachusetts/Sg-00440/2005 H5N5. An example of a H5N6 strain is A/duck/Potsdam/2216-4/1984 H5N6. An example of a H5N7 strain is

A/mallard/Denmark/64650/03 H5N7. An example of a H5N8 strain is strain A/Duck/Ireland/

113/1983 H5N8. Two examples of a H5N9 strain are Influenza A strain A/Turkey/

Ontario/7732/1966 H5N9 and strain A/chicken/ltaly/22A/1998 H5N9.

[069] An example of a H6N1 strain is A/chicken/Taiwan/PF1/02(FI6N1 ). An example of a H6N2 strain is Influenza A strain A/chicken/California/1316/2001 (FI6N2). An example of a H6N5 strain is Influenza A strain A/Shearwater/Australia/1972 H6N5. An example of a H6N8 strain is Influenza A strain A/Turkey/Minnesota/501/1978 H6N8. An example of a H7N1 strain is Influenza A strain A/Fowl plague virus/Rostock/8/1934 H7N1. An example of a H7N2 strain is Influenza A strain A/duck/Hong Kong/293/1978(H7N2). An example of a H7N3 strain is Influenza A strain strain A/Turkey/Oregon/1971 H7N3. Five examples of a H7N7 strain are Influenza A strain A/Equine/C. Detroit/1/1964 H7N7, Influenza A strain

A/Equine/Cambridge/1/1973 H7N7 and Influenza A strain A/Equine/Sao Paulo/1/1976 H7N7, Influenza A virus strain A/Equine/Prague/1 /1956 H7N7 and Influenza A virus strain A/Chicken/Weybridge H7N7. An example of a H8N2 strain is Influenza A strain A/duck/Alaska/702/1991 (H8N2). An example of a H8N4 strain is Influenza A strain A/Turkey/Ontario/6118/1968 H8N4. An example of a H8N4 strain is Influenza A strain A/duck/Tsukuba/255/2005(H8N5). An example of a H8N7 strain is Influenza A strain A/duck/Alaska/702/1991 (H8N7).

[070] An example of a H9N1 strain is Influenza A virus A/Duck/Shantou/ 2030/00(H9N1 ). An example of a H9N2 strain is Influenza A virus A/Hong Kong/1073/ 99(H9N2) with Gene bank accession numbers NC 004905, NC 004906, NC 004907, NC 004908, NC 004909, NC 004910, NC 004911 , and NC 004912. An example of a H9N3 strain is Influenza A virus A/duck/Viet Nam/340/2001 H9N3. An example of a H9N4 strain is Influenza A virus A/shorebird/D E/231 /2003 H9N4. An example of a H9N5 strain is Influenza A virus A/Duck/Hong Kong/702/79 H9N5. An example of a H9N7 strain is A/turkey/Sco tland/70(H9N7). An example of a H9N8 strain is A/chicken/Korea/04164/ 2004(H9N8). An example of a H9N9 strain is A/turkey/France/03295/2003 H9N9. An example of a H10N1 strain is Influenza A virus

A/duck/Hong Kong/938/80 H10N1. An example of a H10N2 strain is Influenza A virus

A/duck/Alaska/658/1991 H10N2. An example of a H10N5 strain is Influenza A virus A/duck/Hong Kong/15/1976 H10N5. Examples of a H10N7 strain are Influenza A strain A/Chicken/Germany/n/1949 H10N7, strain A/Duck/Germany/1949 H10N7, and strain

A/Duck/Manitoba/1 /1953 H10N7. An example of a H10N7 strain is Influenza A virus strain A/Duck/Germany/1949 H10N7. An example of a H11 N1 strain is Influenza A virus strain A/duck/Miyagi/47/1977 H11 N1 . An example of a H11 N2 strain is A/duck/Yangzhou/906/2002 H11 N2. An example of a H11 N3 strain is A/duck/Thailand/CU5388/2009 H11 N3. An example of a H11 N6 strain is Influenza A virus strain A/Duck/England/1 /1956 H11 N6. An example of a H11 N8 strain is strain A/Duck/Ukraine/2/1960 H11 N8. Two examples of a H11 N9 strain are Influenza A strain A/Duck/Ukraine/1 /1960 H11 N9 and Influenza A strain

A/Tern/Australia/G70C/1975 H11 N9. An example of a H12N1 strain is A/mallard duck/Alberta/342/1983(H12N 1 ). An example of a H12N2 strain is A/duck/Primorie/ 3691/02 H12N2. An example of a H12N3 strain is A/whooper swan/Mongolia/ 232/2005 H12N3. An example of a H12N5 strain is Influenza A virus strain A/Duck/ Alberta/60/1976 H12N5. An example of a H12N6 strain is A/mallard/Alberta/221/2006 H12N6. An example of a H12N7 strain is A/duck/Victoria/ 30a/1981 H12N7. An example of a H12N8 strain is A/mallard/Netherlands/20/ 2005 H12N8. An example of a H12N9 strain is A/red-necked stint/Australia/5745/1981 H12N9.

[071] An example of a H13N1 strain is A/bird feces/lllinois/185997-30/2007 H13N1. An example of a H13N2 strain is Influenza A virus strain A/Whale/Maine/ 328/1984 H13N2. An example of a H13N3 strain is A/shorebird/NJ/840/1986 H13N3. Two examples of a H13N6 strain are Influenza A virus strain A/Gull/Maryland/704/1977 H13N6 and strain A/Gull/Minnesota/945/1980 H13N6. An example of a H13N8 strain is A/black-headed gull/Sweden/1/2005 H13N8. An example of a H14N3 strain is A/mallard/Gur/263/82 H14N3. Three examples of a H14N5 strain are A/mallard/Gurjev/263/1982 H14N5, A/mallard/ Astrakhan/266/1982 H14N5 and A/herring gull/Astrakhan/267/1982 H14N5. An example of a H14N6 strain is strain A/Mallard/Gurjev/244/1982 H14N6. An example of a H15N8 strain is A/d u ck/Au stra I i a/341 / 1983 H15N8. An example of a H15N9 strain is A/shearwater/West Australia/2576/79 H15N9. An example of a H16N3 strain is A/black-headed gull/Sweden/ 2/99 H16N3.

[072] Such virus subtypes are distinguishable serologically, which means that antibodies specific for one subtype do not bind to another subtype with comparable high affinity.

[073] An Influenza-B virus according to the present invention may be based on any influenza B virus strain. Suitable virus strains include, but are not limited to Influenza B virus strain B/Maryland/1959, strain B/Yamagata/1/1973, strain B/Victoria/3/1985, strain B/USSR/ 100/1983, strain B/Tokyo/942/1996, strain B/Texas/ 4/1990, strain B/Singapore/222/ 1979, strain B/South Dakota/5/1989, strain B/Paris/329/1990, strain B/Leningrad/179/1986, strain B/Hong Kong/8/1973, strain B/Fukuoka/80/1981 , strain B/Bangkok/163/1990, strain B/Beijing/1/1987, strain B/Switzerland/9359/99, strain B/Wisconsin/6/2006, strain B/West Virginia/01/2009, strain B/Washington/08/2009, strain B/Uruguay/NG/02, strain B/Texas/ 18/2001 , strain B/Taiwan/S117/2005, strain B/Taiwan/3799/2006, strain B/Spain/WV45/ 2002, strain B/Seoul/232/2004, strain B/Rio Grande do Sul/57/2008, strain B/Quebec/517/98, strain B/Philippines/5072/2001 , strain B/Oslo/1871/2002, strain B/Osaka/983/1997, strain B/Milan/05/2006, strain B/Johannesburg/ 116/01 , strain B/Lee/1940 (Lee) or strain B/Arizona/12/2003.

[074] The present invention further comprises an ergosterol-biosynthesis inhibitor for use in a method of treatment or prophylaxis of an influenza virus infection in a subject of the present invention, wherein the ergosterol-biosynthesis inhibitor may be further for the use of a method of treating or preventing a fungal infection. Thus, people being treated with the ergosterol- biosynthesis inhibitor of the present invention such as itraconazole, posaconazole, voriconazole, fluconazole or fosfluconazole as medicament/drug against a fungal disease may already be protected from suffering from influenza as well.

[075] A fungal infection (also called fungal infectious disease) being further treated or prevented by the use of said ergosterol-biosynthesis inhibitor of the present invention may be, but is not be limited to, an Aspergillus infection, cryptococcal disease, histoplasmosis, a yeast infection, a dermatophyte infection.

[076] In a preferred embodiment, the fungal infection being further treated or prevented by the use of said ergosterol-biosynthesis inhibitor of the present invention may be an Aspergillus infection. Aspergillus infection may be caused by the fungi of the genus Aspergillus. An Aspergillus infection may for example include Aspergillosis.

[077] Cryptococcal disease (cryptococcosis) also being further treated or prevented by the use of said ergosterol-biosynthesis inhibitor of the present invention may be caused by one of two species Cryptococcus neoformans und Cryptococcus gattii. There may be three types of infections: wound or cutaneous cryptococcosis, pulmonary cryptococcosis or Cryptococcal meningitis, all caused by C. neoformans. Thus a fungal infection being further treated or prevented by the use of said ergosterol-biosynthesis inhibitor of the present invention may also comprise wound or cutaneous cryptococcosis, pulmonary cryptococcosis or cryptococcal meningitis.

[078] Histoplasmosis also called “Cave disease” or “Darling’s disease” also being further treated or prevented by the use of said ergosterol-biosynthesis inhibitor of the present invention may be caused by the fungus Histoplasma capsulatum. It primarily affects the lungs, other organs may also be affected as well (Ryan and Ray 2004, Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 674-6).

[079] A yeast infection also being further treated or prevented by the use of said ergosterol- biosynthesis inhibitor of the present invention may be caused for example by the pathogenic yeast Candida, resulting in Candidiasis.

[080] A dermatophyte infection (dermatophytoses) also being further treated or prevented by the use of said ergosterol-biosynthesis inhibitor of the present invention may be caused by dermatophytes that require keratin for growth. Those fungi may cause infections of the skin, hair and nails. Dermatophytoses refer to tinea infections and may also name for the body site being involved. It may include tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea pedis (Barry and Hainer 2003, Am Fam Physician ; 67(1 ): 101 -109).

[081] In a further aspect, the present invention envisages a pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier for use in a method of treatment or prophylaxis of an influenza virus infection in a subject. The subject, which is treated against influenza virus infection with a pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier may be a mammal. The mammaly may be a mouse, a rat, a pig, a horse, a dog, a cat, a bird, a human, preferably a human.

[056] In this context, the term "pharmaceutically acceptable" refers to something being approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[057] The term "carrier" refers to a diluent, adjuvant, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical acceptable carriers can be sterile liquids, such as water and oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously or orally. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[082] The present invention may comprise said pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier for use in a method of treatment or prophylaxis of an influenza virus infection in a subject, wherein said method comprises administering the pharmaceutical composition comprising said ergosterol- biosynthesis inhibitor and a pharmaceutical acceptable carrier orally or nasally. The administration may also be performed by injection or by infusion.

[083] The pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier may be injected. This injection of an e.g. saline-based solution formulation may be performed intraperitoneally, intravenously, subcutaneously or intramuscularly. In this context, the term “injection” refers to the administration of a liquid comprising for example the ergosterol-biosynthesis inhibitor by applying a syringe and a hollow needle, which is pierced though the skin to be administered into the body of the subject.

[084] The pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier may also be infused. In this context, the term“infusion” refers to a continuous, most commonly parenteral administration of liquid comprising for example the ergosterol-biosynthesis inhibitor intravenously, e.g. a saline-based intravenous solution formulation.

[085] The pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier may also be taken orally. The oral administration refers to swallowing an oral capsule with water or any other liquid used as a pharmaceutically acceptable carrier or taking an oral solution.

[086] The pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier may also be taken nasally. The nasal administration refers to applying a nasal spray comprising the pharmaceutical composition comprising said ergosterol- biosynthesis inhibitor and a pharmaceutical acceptable carrier. Preferably, said administration of the pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier is performed via intestinal passage, in particular said administration of the pharmaceutical composition comprising an ergosterol-biosynthesis- inhibitor and a pharmaceutical acceptable carrier is performed, orally. Said abministration of the pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor and a pharmaceutical acceptable carrier may also be performed intravenously or nasally.

[087] Said pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor may be administered daily for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In this context, the term “daily” means every day in a week having 7 days. Further, a single dose of said pharmaceutical composition comprising an ergosterol-biosynthesis-inhibitor of at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 300, at least about 400, at least about 500 mg/kg or more may be administered per day. In a preferred embodiment and as being described in the Examples, about 70 mg/kg/day of said pharmaceutical composition comprising an ergosterol- biosynthesis-inhibitor is administered to the subject in need thereof.

[088] The present invention also envisages in another aspect a method of treating or preventing a subject against influenza virus infection comprising administering an effective amount of an ergosterol-biosynthesis inhibitor to said subject in need thereof. The subject in need thereof may be a human.

[089] Also comprised by the present invention is the use of an ergosterol-biosynthesis inhibitor for the manufacture of a medicament for the therapeutic application in influenza virus infection in a subject. The subject may be a human.

[090] All of the abovementioned concerning said ergosterol-biosynthesis inhibitor for the use according to the present invention as described elsewhere herein may be applicable to the aspect of the pharmaceutical composition comprising an ergosterol-biosynthesis inhibitor and a pharmaceutical acceptable carrier for the use, the method of treatment and the use of such ergosterol-biosynthesis inhibitor for the manufacture of a medicament for the therapeutic application in influenza virus infection in a subject. [091] It is noted that as used herein, the singular forms“a”,“an”, and“the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to“a reagent” includes one or more of such different reagents and reference to“the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[092] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. The term“at least one” refers to one or more such as two, three, four, five, six, seven, eight, nine, or ten. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[093] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[094] The term“less than” or in turn“more than” does include the concrete number.

[095] For example, less than 20 means 20 and less than the number indicated. Similarly, more than or greater than means the number indicated and more than or greater than the indicated number.

[096] Throughout this specification and the claims which follow, unless the context requires otherwise, the word“comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term“containing” or“including” or sometimes when used herein with the term“having”. When used herein“consisting of" excludes any element, step, or ingredient not specified.

[097] The term“including” means“including but not limited to”.“Including" and“including but not limited to” are used interchangeably.

[098] The term “about” means plus or minus 10%, preferably plus or minus 5%, more preferably plur or minus 2%, most preferably plus or minus 1 %.

[099] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[100] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[101] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[102] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[103] A better understanding of the present invention and of its advantages will be gained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

EXAMPLES OF THE INVENTION

[104] Example 1 : Measuring viral titers in cells infected with H1 N1 influenza-A-virus after treatment with posaconazole or itraconazole.

[105] A549 human airway / lung epithelial cells, highly utilized as a cell culture model for studying respiratory infections or used for influenza A virus propagation, were treated with either the solvent DMSO, posaconazole (Posa, 1 pg/ml - see Fig. 1 or 2 pg/ml - see Fig. 3; for both Figures posaconazole has been solubilized in DMSO at 2 mg/ml before being used) or itraconazole (Itra, 1 pg/ml - see Fig. 1 or 2 pg/ml - see Fig. 3; for both Figures itraconazole has been solubilized in DMSO at 2 mg/ml before being used) for 16h. Cells were then washed with PBS and were infected with H1 N1 influenza-A-virus strain PR8 at the indicated multiplicity of infection (MOI) of virus diluted in infection-PBS (PBS containing 0.2% bovine serum albumin (BSA), 1 mM MgCI ₂ , 0.9mM CaCI ₂ , 100 U/ml penicillin and 0.1 mg/ml streptomycin) at 37°C for 30 min. Viral titers were determined by Plaque assay at 24 hours post infection.

[106] In detail, for the Plaque assay virus suspension is diluted in various stages and is applied for 30 min on MDCK monolayer in a petri dish. Then, supernatant is removed and the infected cell layer is covered by agar. The number of plaques (holes in the cell layer) is determined 72 h later generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL).

[107] Data represent mean viral titers ± standard error of the mean of four-six independent experiments (Fig. 1) or of three independent experiments (Fig. 3), which clearly demonstrate that both drugs efficiently inhibited the replication of the H1 N1 strain PR8M.

[108] Example 2: Measuring lung virus titers and survival rate in mice infected with H1 N1 influenza-A-virus after treatment with itraconazole.

[109] Itraconazole treatment decreases lung virus titers in eight-to-ten weeks-old male C57BL/6 mice infected with influenza-A-virus. Pulmonary virus titers (given as plaque forming units, PFU) of mice fed daily (intragastrically) with a single dose of either solvent such as propylene glycol hydroxypropyl- -cyclodextrin (control / vehicle) or itraconazole (Itraconazole from Sigma-Aldrich, 70 mg/kg/day) starting at day -1 , and infected intra-nasally with a LD ₅₀ of H1 N1 influenza A virus strain PR8 on day 0. Mice were anesthetized with isoflurane prior to intranasal infection with 500 pfu (plaque forming units) of the IAV strain PR8M. To assess pain and distress during the course of infection, animals were assessed based on a scoring system with sufficiently frequent observation times that assigns numerical values to several criteria of animal conditions that were considered signs of morbidity or moribundity, including changes in body temperature, physical appearance, behaviour, and weight loss. Animals that reached the cumulative threshold score were euthanized. A body weight loss of >20 percent compared to start of the treatment was the cut-off parameter for euthanasia, regardless of the total score. From a total of 40 infected mice (20 itraconazole-treated, 20 vehicle-treated), 18 mice had to be euthanized because body weight loss was more than 20%. As described above, mice were then sacrificed after intranasal infection to determine acute-phase virus titers in the lungs. Viral titers obtained from lung homogenates were determined by Plaque assay at the indicated day post infection (p.i.) by again preparing serial dilutions of lung homogenate samples and apply further steps of Plaque assay as described in Example 1. Scatter plots show individual values, means ± standard error of the mean of 5 mice/group. Mann-Whitney U test, ^* p < 0.1 , ^** p < 0.01 , ^*** p < 0.001 0.0001 (Fig. 2).

[110] In line with the inhibitory effects observed in the cell culture models (e.g., Example 1), a clearly reduced mortality in itraconazole-treated mice (70% survival) was observed compared to the vehicle group (40% survival). The improved survival correlated with diminished body-weight loss during the course of influenza A virus infection (Fig. 5).

[111] Example 3: Antiviral activities of posaconazole or itraconazole are not subtype or cell line specific.

[112] A549 cells (as already being used in Example 1 ), the human skin epithelial cell line A431 , which is also permissive for influenza A virus or primary cells isolated from the umbilical cord vein (HUVEC), which can also be efficiently infected with influenza A virus, were treated with either DMSO (2 pg/ml), posaconazole (2 pg/ml) or itraconazole (2 pg/ml) for 16 h, and subsequently infected with the indicated MOI of various influenza A virus subtypes PR8M (H1 N1 ) and PAN (H3N2) for 24 h. Viral titers were determined by Plaque assay (see Example 1) at 24 hours post infection.

[113] As expected, all viral infections yielded high virus titres 24 h p.i. in DMSO-treated control cells. Importantly, itraconazole pretreatment for 16 h significantly reduced viral titers for influenza A virus. In A549 and A431 cells pretreated with itraconazole, PR8M load 24 h p.i. was reduced up to 98% when cells were infected at 0.05 MOI (Fig. 4A). Reduced titers were also observed when cells were infected with higher viral doses (0.1 MOI), although the effect was less pronounced. Also when non-malignant primary human cells were used (e.g., HUVEC), propagation of the exemplary influenza A virus strains PR8M was also successfully inhibited upon itraconazole treatment, whereas both drugs could block infection with the PAN strain (Fig. 4B).

[114] In a further experiment, Madin-Darby canine kidney (MDCK) cells type II were treated with either DMSO (2 pg/ml), posaconazole (2 pg/ml) or itraconazole (2 pg/ml) for 16 h, and subsequently infected with the indicated MOI (0.05 MOI) of the influenza B virus subtype B/Lee/1940 (Lee). Viral titers were determined again by Plaque assay (see Example 1) at 24 hours post infection. [115] The inventors found that both drugs used (posaconazole or itraconazole) had even a protective effect against influenza B virus strain B/Lee/1940 (Fig. 4C).