Novel antiviral oleic acid-containing compositions

Abstract

The present invention particularly provides a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and mixtures of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, optionally together with an antiviral agent, said liquid pharmaceutical formulation for use as a medicament for topical administration to the lungs or nose in the treatment or prevention of a viral infection or a disease associated with a viral infection.

Description

[Technical field]

[0001] (Technical field) The present invention relates to liquid pharmaceutical formulations suitable for topical administration to the lungs or nose, and related aspects thereof, including canisters containing the formulations, and spray and nebulizer devices, and metered dose inhalers. The present invention also relates to liquid pharmaceutical formulations for use in the treatment or prevention of viral infections and diseases associated with viral infections, such as SARS-CoV-2 or influenza infections, and related methods of treatment. [Background technology]

[0002] BACKGROUND OF THE PRESENTINVENTION Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus and a member of the Betacoronavirus genus in the Coronaviridae family. SARS-CoV-2 is the causative agent of the respiratory disease COVID-19 (coronavirus disease 2019). COVID-19 is characterized by a range of severity from mild upper respiratory tract disease to severe interstitial pneumonia and acute respiratory distress syndrome (ARDS), a life-threatening lung injury caused by fluid leakage into the lungs. Post-COVID19 syndrome is also an important aspect with pulmonary fibrosis and secondary fatal fungal infection / invasion.

[0003] Currently, seven coronaviruses are known to cause human disease. The 229E, OC43, NL63, and HKU1 human coronavirus (hCoVs) species cause mild upper and lower respiratory tract illness and are estimated to account for one-third of “common cold” cases (Ludwig and Zarbock, 2020). However, the high prevalence, wide distribution, genetic diversity, and frequent cross-species transmission of coronavirus species allows novel human pathogens to easily emerge. Thus, severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2 have caused pandemics with high mortality rates.

[0004] The primary mode of transmission of SARS-CoV-2 is the result of viral replication in the airway epithelial lining of the upper and lower respiratory tract. Initial viral infection occurs through inhalation of very fine respiratory droplets and aerosol particles, through direct splashing and spraying of respiratory droplets and particles onto exposed mucous membranes of the mouth, nose, or eyes, and secondary to contact with mucous membranes through hands contaminated directly by virus-containing respiratory fluids or indirectly through contact with virus-bearing surfaces (CDC: https: / / www.cdc.gov / coronavirus / 2019-ncov / science / science-briefs / sars-cov-2-transmission.html). The virus in the early stages of the disease establishes robust levels of viral replication in the upper respiratory tract before migrating into the lungs to cause viral pneumonia and systemic spread to other organs. Available evidence suggests that disease severity is associated with the viral load in the airways and that therapeutic intervention with antiviral agents reduces the viral load and, therefore, disease severity.

[0005] In general, enveloped respiratory viruses attach to receptors on the host cell surface and enter the cell by endocytosis or direct fusion of the viral membrane with the host cell membrane. Infection of upper and lower respiratory epithelial cells by SARS-CoV-2 is facilitated by binding of the viral spike protein to the host cell receptor angiotensin-converting enzyme 2 (ACE2). Other receptors such as AXL, CD147, CD209 / CD209L, neuropilins, and DPP4 have been identified as possible virus-associated coreceptors (Xie et al., Cantuti-Castelvetri et al.). Subsequent activation of the spike protein by host proteases such as TMPRSS at the apical surface is required for the viral spike protein to be processed and for the virion to enter the cell by membrane fusion.

[0006] Recent studies have demonstrated that SARS-CoV-2 can enter human lung epithelial cells as host cells via two distinct pathways. The first is direct membrane fusion on the cell surface (early pathway), which occurs following spike protein activation by transmembrane serine protease 2 (TMPRSS2) or alternative serine proteases. The second is endocytic uptake (late pathway), whereby cathepsin L activates the spike protein within the endosomal-lysosomal compartment. Importantly, the early entry pathway is preferred when TMPRSS2 or alternative serine proteases are expressed, whereas in the absence of these proteases, the virus relies on the late pathway (Murgolo et al., 2021). In the late pathway, it is the decrease in pH and proteolysis that leads to processing and viral release within endosomes, which are necessary to disrupt the viral envelope and release the internal genetic material. PIKfyve is a phosphoinositide kinase that phosphorylates phosphatidylinositol-3-phosphate (PI(3)P) to generate PI(3,5)P2. PIKfyve plays a key role in endosomal membrane maturation, thereby enabling viral membrane fusion and entry into the cytoplasm.

[0007] There are various reports on the possible use of PIKfyve inhibitors, including apilimod, to treat SARS-CoV-2 infection. WO2021 / 211738 discloses an anti-infective composition useful for reducing the likelihood of pathogenic infection or for reducing the transmission of this pathogen, the anti-infective composition comprising a compound selected from the group including apilimod. WO2021 / 158635 discloses PIKfyve inhibitors, such as apilimod, and compositions thereof, for treating or preventing coronavirus infections, such as SARS-Cov-2. WO2016 / 161176 discloses administering to an individual in need thereof a therapeutically effective amount of a compound of the general formula (I); [ka] The present invention discloses a method for treating a viral infection comprising administering a compound of the formula:

[0008] Optimistic predictions regarding the usefulness of drugs to treat SARS-CoV-2 have often been shown to be misplaced in practice. For example, many early screening experiments utilized the Vero E6 cell line to identify SARS-CoV-2 antivirals. Because this cell line lacks TMPRSS2 and has high expression of ACE2, viral entry is dependent on the endocytic pathway and is therefore an incomplete model for predicting infection of human epithelial cells. Furthermore, additional nonspecific endocytic viral uptake mechanisms could facilitate virion entry into Vero E6 cells. Thus, although many molecules that modulate the endosomal and lysosomal systems, such as chloroquine and apilimod, have been identified as potent SARS-CoV-2 therapeutics in Vero E6 cells, these observations may not apply to human lung epithelial cells (Hoffmann et al., 2020).

[0009] Further efforts to identify effective SARS-CoV-2 antivirals have led to the discovery that camostat (as the mesylate salt) can inhibit SARS-CoV-2 infection by blocking TMPRSS2 and related proteases (Hoffmann et al., 2021). Furthermore, it has been established that remdesivir can be redeveloped as a potent SARS-CoV-2 therapeutic (Pruijssers et al., 2020). Furthermore, the anticoagulant nafamostat (as the mesylate salt) has been confirmed to inhibit SARS-CoV-2 infection of compromised cells (Hoffmann et al., 2020). Alternatively, new SARS-CoV-2 antivirals may be designed. For example, molnupiravir, a commercially available oral treatment for SARS-CoV-2 approved for use in the UK Commonwealth in November 2021, inhibits the function of the viral RNA-dependent RNA polymerase (Kabinger et al., 2021). Similarly, nilmatrellvir was developed as an inhibitor of the SARS-CoV-2 3CL protease (Zhao et al., 2021). Combination of nilmatrellvir with the HIV protease inhibitor ritonavir was found to prolong activity due to slowing the metabolism of nilmatrellvir (Zhao et al., 2021). Similarly, there are conflicting reports that lopinavir, another HIV protease inhibitor, may have activity against SARS-CoV-2, especially when used in combination with ritonavir, which increases plasma concentrations of lopinavir (Cattaneo et al., 2020; Ford et al., 2020).

[0010] Influenza viruses include four species, influenza A-D viruses, each of which forms a separate genus, α-, β-, γ-, and δ-influenza viruses, respectively, within the family Orthomyxoviridae. Influenza viruses are enveloped, negative-sense, single-stranded RNA viruses with segmented genomes that cause mild to severe respiratory illness characterized by fever, sore throat, headache, cough, and malaise. Influenza viruses utilize the endocytic pathway for entry. Binding of the viral HA protein to the host sialic acid receptor initiates cell entry via endosomal uptake. Subsequent acidification within the endosome activates cellular proteases, such as cathepsins, which induce a conformational change in HA that allows endosomal membrane fusion, resulting in the release of virions into the cell. The dependence of influenza on the endocytic pathway for entry has led to speculation that PIKfyve inhibitors, such as apilimod, may be able to treat influenza virus infections. WO2021 / 211738 speculates that the anti-infective compositions of the invention, which may include apilimod, may be used to treat influenza infections, but no evidence of anti-influenza activity is presented.

[0011] Umifenovir is an anti-influenza virus drug that is widely used in Russia and China and has significant clinical importance. Umifenovir binds to the viral HA protein and prevents the conformational changes in HA that allow membrane fusion to occur (Kadam and Wilson, 2017). There are conflicting reports that umifenovir may also have activity against SARS-CoV-2 (Huang et al., 2020).

[0012] Replication of SARS-CoV-2 in human nasal and bronchial epithelium causes a rapid loss of epithelial cilia, characterized by impaired mucociliary clearance, reduced epithelial integrity of the airways, and disruption of epithelial tight junctions (Hao et al., 2020; Robinot et al., 2020). It has also been reported that the main tissue barrier affected by SARS-CoV-2 is the blood-gas barrier (BGB) or alveolar-capillary barrier (Shirvaliloo, 2021).

[0013] For cytopathic infection, 1 cm of epithelium 2 2.5~10 points 5 High viral loads of virions are required (Hao et al., 2020). Extensive damage to the ciliated epithelial layer of the upper airways precedes enhanced spread deep into the lung parenchyma.

[0014] Thus, virus-mediated impairment of epithelial junction integrity leads to loss of the host defense barrier, with consequences of inflammation, fluid leakage, and loss of normal pulmonary gas movement, as well as secondary infections with bacteria and fungi.

[0015] Conventional additives used in pharmaceutical compositions for inhalation and intranasal delivery have traditionally been considered to be medicamentously inactive substances that serve to optimize the delivery of active pharmaceutical ingredients, such as antiallergic and anti-inflammatory agents. It has been reported in the literature that certain natural and synthetic surfactants can exhibit antiviral properties against lipid-enveloped viruses that are susceptible to surface activation.

[0016] Kohn et al. (1980) teaches that unsaturated fatty acids such as oleic acid exhibit in vitro virucidal activity against Sendai virus, Newcastle disease virus, influenza A virus, Sindbis virus, West Nile virus, and herpesvirus 1, but not against poliovirus, encephalomyocarditis virus, or simian virus 40. This activity is proposed to be the result of incorporation into the viral lipid envelope causing disruption of viral membrane integrity. However, this publication does not consider the effects of these molecules on host cell membranes, on human lung or nasal epithelial cells, or on coronaviruses or influenza viruses.

[0017] Thormar et al. (1987) investigated the inactivation of viruses in raw milk by fatty acids. They reported that fatty acids such as oleic acid reduced the viral titers after incubation with herpes simplex virus, VSV (vesicular stomatitis virus), and Visna virus, but not with poliovirus. Antiviral fatty acids were reported to disrupt the integrity of the viral envelope, causing leakage and, at high concentrations, complete disintegration of the envelope and viral particles, as well as to cause degradation of the plasma membrane of tissue culture cells, resulting in cell lysis and death. However, the article did not consider the effect of these molecules on human lung or nasal epithelial cells, or on coronaviruses or influenza viruses.

[0018] Anderson et al. (1991) teach that polysorbate 80 enhances the antiviral activity of the compound hypericin, but discloses that polysorbate 80 itself has no direct antiviral activity when tested against vaccinia and herpes simplex virus strains.

[0019] Hilmarsson et al. (2007) teaches that oleic acid significantly reduces the titer of respiratory syncytial virus (RSV) after incubation in milk or fruit juice. However, this study teaches that due to the instability of long-chain unsaturated fatty acids such as oleic acid, other bactericidal compounds such as lauryl alcohol, lauric acid, monolaurin, and monocaprin are more viable as active ingredients in topical formulations against RSV, parainfluenza, or influenza infections. Furthermore, this document does not consider the effect of oleic acid or similar molecules on lung or nasal epithelial cells or on coronaviruses.

[0020] Chen et al. (2019) teaches that polysorbate 80 cannot induce inactivation of enveloped viruses, but can be cleaved to oleic acid, which has been demonstrated to have antiviral activity against pseudorabies virus and xenotropic murine leukemia virus, but not against porcine parvovirus. Thus, polysorbate 80 is suggested to be a viable replacement for Triton X-100 in manufacturing processes. However, this literature is limited to a discussion of the effects of antiviral surfactants in the biopharmaceutical manufacturing field, and does not consider the effects of surfactants on host cell membranes or on coronaviruses or influenza viruses.

[0021] Other findings suggest that oleic acid may actually lead to increased viral load: Raini et al. (2021) teaches that supplementing infected cells with oleic acid leads to increased Zika virus titers, and since lipid droplets are the primary platform for Zika virus replication, the increased titers are likely the result of inducing lipid droplet formation.

[0022] Thus, there is a large amount of conflicting and non-specific literature regarding the antiviral effects of surfactants such as polysorbate 80 and oleic acid.

[0023] There remains a need for the development of pharmaceutical agents for the treatment and prevention of viral infections, particularly those that affect human pulmonary or nasal epithelial cells. Summary of the Invention

[0024] (Summary of the invention) Commonly used additives are generally considered to be pharmacologically inactive. Surprisingly, the present inventors have discovered new biological activities for certain surfactants that have been used as additives in the past.

[0025] Accordingly, the present invention provides a liquid pharmaceutical formulation suitable for local administration to the lungs or nose and having antiviral properties, comprising a particular surfactant(s) and an antiviral agent. The present invention provides a liquid pharmaceutical formulation suitable for local administration to the lungs or nose, comprising (i) a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma-ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, and (ii) an antiviral agent.

[0026] The present invention also provides a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising a surfactant component selected from the group consisting of oleic acid and a mixture of oleic acid and a polyoxyethylene sorbitan fatty acid ester, said liquid pharmaceutical formulation for use as a medicament for topical administration to the lungs or nose in the prevention of a viral infection or a disease associated with a viral infection. [Brief description of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows the protocol of the experiment described in Example 2. [Diagram 2]Figure 2 shows the effect of apical treatment with apilimod (as the mesylate salt), surfactants (0.15% w / w polysorbate 80 and 0.2% w / w oleic acid) and their combinations on SARS-CoV-2 viral load in apical washes from SARS-CoV-2-infected air-liquid interface (ALI)-cultured nasal epithelium. The effects of these treatments are compared to basolateral treatment with remdesivir. [Diagram 3] Figure 3 shows the effect of apical treatment with apilimod (as the mesylate salt), surfactants (0.15% w / w polysorbate 80 and 0.2% w / w oleic acid) and their combination on SARS-CoV-2-induced loss of epithelial integrity as determined by transepithelial electrical resistance (TEER). The effects of these treatments are compared to basolateral treatment with remdesivir. [Figure 4] Figure 4 shows the effect of oseltamivir carboxylate (1 μM), detergent (0.15% polysorbate 80 and 0.2% oleic acid) and their combinations on viral load in apical washes of H1N1- A / Switzerland / 7717739 / 2013 (H1N1)-infected air-liquid interface-cultured nasal epithelium treated on the apical side, compared to vehicle (water)-treated infected controls. H1N1 influenza virus particles were detected by RT-PCR, and genome copy numbers calculated from a standard curve were shown. Oseltamivir carboxylate (10 μM) was also treated in the basolateral chamber as an assay control. [Diagram 5] Figure 5 shows the effect of oseltamivir carboxylate (1 μM), detergent (0.15% polysorbate 80 and 0.2% oleic acid) and their combinations on the H1N1-influenza-induced loss of epithelial integrity compared to vehicle (water)-treated infected controls. Epithelial integrity was determined by TEER (transepithelial electrical resistance). Oseltamivir carboxylate (10 μM) was also administered in the basolateral chamber as an assay control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Invention The present invention is based on the discoveries made by testing the antiviral activity of various antiviral agents in combination with and separately from certain surfactants. The present invention demonstrates that (i) apical administration of a mixture of oleic acid and polysorbate 80, particularly in combination with the antiviral agent apilimod, has a strong effect on reducing viral load in a cultured nasal epithelium infection model with SARS-CoV-2 (see Example 2, Figure 2), with evidence of synergy between the surfactant mixture and apilimod; (ii) apical administration of a mixture of oleic acid and polysorbate 80, optionally in combination with the antiviral agent apilimod, has a strong effect on improving barrier function in a cultured nasal epithelium infection model with SARS-CoV-2 (see Example 2, Figure 3); (iii) apical administration of a mixture of oleic acid and polysorbate 80, optionally in combination with the antiviral agent oseltamivir carboxylate, reduces influenza in cultured nasal epithelium. The present invention is based on the discovery that (i) apical administration of a mixture of oleic acid and polysorbate 80, particularly in combination with the antiviral agent oseltamivir carboxylate, has a potent effect on reducing viral load in an influenza virus H1N1 infection model (see Example 2, Figure 4), with evidence of a synergistic effect between the surfactant mixture and oseltamivir carboxylate; and (iv) apical administration of a mixture of oleic acid and polysorbate 80, particularly in combination with the antiviral agent oseltamivir carboxylate, has a potent effect on improving barrier function of cultured nasal epithelium in an influenza virus H1N1 infection model (see Example 2, Figure 5).

[0029] The liquid formulation of the present invention comprises a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester. Representative polyoxyethylene sorbitan fatty acid esters include polysorbate 80 (e.g., Tween 80) and polysorbate 20. For example, the surfactant component is oleic acid or a pharma- ceutical acceptable salt thereof, particularly oleic acid. More preferably, the surfactant component is a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and polysorbate 20 or polysorbate 80, particularly a mixture of oleic acid or a pharma- ceutical acceptable salt thereof (e.g., oleic acid) and polysorbate 80.

[0030] Typically, the surfactant component may be present in the formulation at a concentration of 10 to 30000 ug / mL, for example, 100 to 20000 ug / mL, for example, 100 to 5000 ug / mL. For example, when the surfactant is oleic acid or a pharma- ceutical acceptable salt thereof, the oleic acid may be present in the formulation at a concentration of 1 to 100 ug / mL. For example, when the surfactant component is a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20 or polysorbate 80, the oleic acid may be present in the formulation at a concentration of 10-30000 ug / mL, such as 100-20000 ug / mL, such as 100-5000 ug / mL, and the polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20 or polysorbate 80, may be present in the formulation at a concentration of 10-20000 ug / mL, such as 100-15000 ug / mL, such as 100-5000 ug / mL. For example, the ratio of the amount of oleic acid or a pharma- ceutical acceptable salt thereof to the amount of polyoxyethylene sorbitan fatty acid ester, each measured in ug / mL, is about 5:1-1:5, such as 2:1-1:2. The above amounts and ratios are based on the equivalent amount of free acid (oleic acid) if the salt form is used. Suitably, the oleic acid is used as oleic acid (i.e., not in salt form).

[0031] Pharmaceutically acceptable salt forms of oleic acid which can be used include the sodium, potassium, ammonium and especially the sodium salt. Most preferably, oleic acid is used as the free acid.

[0032] The liquid pharmaceutical formulation of the present invention may be suitable for local administration to the lungs or nose. The liquid pharmaceutical formulation of the present invention may be administered by inhalation, for example, by oral inhalation to the lungs or by local administration to the nose. It will be understood that the formulation of the present invention suitable for administration to the lungs or nose may also include administration to the pharynx when administered locally to the lungs by oral inhalation or locally to the nose.

[0033] In one embodiment, the liquid pharmaceutical formulation of the present invention comprises an antiviral agent. Antiviral agents include substances that have direct antiviral activity, for example by inhibiting viral replication, as well as those that have indirect antiviral activity, for example by inhibiting viral uptake, for example by modulating the endosomal and lysosomal systems of target cells, or by stimulating the host immune system to produce an antiviral response. In one embodiment, the antiviral agent is apilimod (i.e., the free base) or a pharmaceutically acceptable salt thereof, for example the mesylate salt thereof. In one embodiment, the antiviral agent is camostat or a pharmaceutically acceptable salt thereof, for example the mesylate salt thereof. In one embodiment, the antiviral agent is umifenovir. In one embodiment, the antiviral agent is oseltamivir or a pharmaceutically acceptable salt thereof, in particular oseltamivir phosphate. In one embodiment, the antiviral agent is ribavirin or a pharmaceutically acceptable salt thereof, in particular ribavirin. In one embodiment, the antiviral agent is pirodavir or a pharma- ceutically acceptable salt thereof, in particular pirodavir. In one embodiment, the antiviral agent is remdesivir or a pharma- ceutically acceptable salt thereof, in particular remdesivir. In one embodiment, the antiviral agent is molnupiravir or a pharma- ceutically acceptable salt thereof, in particular molnupiravir. In one embodiment, the antiviral agent is a coronavirus 3CL protease inhibitor, such as nilmatrervir, or a pharma- ceutically acceptable salt thereof, in particular nilmatrervir. In one embodiment, the antiviral agent is lopinavir or a pharma- ceutically acceptable salt thereof, in particular lopinavir. In one embodiment, the antiviral agent is nilmatrervir or a pharma- ceutically acceptable salt thereof, in particular nilmatrervir. In one embodiment, the antiviral agent is nilmatrervir or a pharma- ceutically acceptable salt thereof, in particular nilmatrervir, in combination with ritonavir or a pharma- ceutically acceptable salt thereof, in particular ritonavir. In one embodiment, the antiviral agent is lopinavir or a pharma- ceutically acceptable salt thereof, particularly lopinavir in combination with ritonavir or a pharma- ceutically acceptable salt thereof, particularly ritonavir.In one embodiment, the antiviral agent is baloxavir or a pharma- ceutically acceptable salt thereof, particularly baloxavir marboxil. In one embodiment, the antiviral agent is ensitrevir or a pharma- ceutically acceptable salt thereof, particularly ensitrevir fumarate. In one embodiment, the antiviral agent is favipiravir (T-705) or a pharma- ceutically acceptable salt thereof, particularly favipiravir. In one embodiment, the antiviral agent is zanamivir or a pharma- ceutically acceptable salt thereof, particularly zanamivir.

[0034] In one embodiment, the antiviral agent is oseltamivir or a pharma- ceutically acceptable salt thereof, in particular oseltamivir phosphate. In one embodiment, the antiviral agent is oseltamivir carboxylate or a pharma- ceutically acceptable salt thereof. In one embodiment, the antiviral agent is laninamivir or a pharma- ceutically acceptable salt thereof, in particular laninamivir. In one embodiment, the antiviral agent is laninamivir octanoate. In one embodiment, the antiviral agent is rupintrivir or a pharma- ceutically acceptable salt thereof, in particular rupintrivir.

[0035] When the liquid pharmaceutical formulation of the present invention is for the treatment or prevention of SARS CoV-2 infection or COVID-19 infection, preferably the antiviral agent is selected from apilimod, camostat, nafamostat, umifenovir, remdesivir, molnupiravir, nilmatrevir, lopinavir, and any pharmaceutically acceptable salt thereof. Alternatively, preferably the antiviral agent is selected from ensitrevir, favipiravir, rupintrivir, and any pharmaceutically acceptable salt thereof.

[0036] When the liquid pharmaceutical formulation of the present invention is for treating or preventing influenza virus infection or influenza, the antiviral agent is preferably selected from camostat, nafamostat, umifenovir, and pharma- ceutical acceptable salts thereof, or is preferably selected from baloxavir, favipiravir, oseltamivir, zanamivir, laninamivir, laninamivir octanoate, and pharma- ceutical acceptable salts thereof, in particular oseltamivir or oseltamivir carboxylate, and pharma- ceutical acceptable salts thereof.

[0037] When the liquid pharmaceutical formulation of the present invention is for the treatment or prevention of a respiratory syncytial virus (RSV) infection or a disease associated with RSV infection, preferably the antiviral agent is selected from ribavirin and its pharma- ceutically acceptable salts.

[0038] When the liquid pharmaceutical formulation of the present invention is for the treatment or prevention of a human rhinovirus (HRV) infection or a disease associated with HRV infection, preferably the antiviral agent is selected from pirodavir, rupintrivir, and pharma- ceutical acceptable salts thereof.

[0039] When the liquid pharmaceutical formulation of the present invention is for the treatment or prevention of a coronavirus infection, such as a seasonal coronavirus infection, such as a 229E infection, and the viral infection associated disease is a coronavirus infection associated disease, such as a seasonal coronavirus infection associated disease, such as a 229E infection associated disease, preferably the antiviral agent is selected from apilimod, camostat, ensitrevir, favipiravir, nafamostat, umifenovir, remdesivir, rupintrivir, molnupiravir, nilmatrevir, lopinavir, and a pharmaceutically acceptable salt of any of them.

[0040] Details of the synthesis of related antivirals and ritonavir can be gleaned from the following documents: apilimod: WO2003 / 047516; camostat: Senokuchi et al.; nafamostat: EP0048433B1; umifenovir: Chai et al.; remdesivir: WO2017 / 184668; molnupiravir: US2020 / 0276219; nilmatrevir: Owen et al.; lopinavir: US5914332. , ritonavir: US5541206, oseltamivir: US5763483, pirodavir: US5231184, ribavirin: USRE29835, baloxavir: JPWO2016 / 175224, ensitrevir: Unoh et al., favipiravir: US6800629, rupintrivir: Dragovich et al., and zanamivir: US5360817, the contents of each of which are incorporated herein by reference in their entirety. Nilmatrevir is also known as PF07321332.

[0041] Pharmaceutically acceptable salts of basic compounds which may be used include acid addition salts such as hydrochloride, hydrobromide, acetate, succinate, and mesylate salts.

[0042] Typically, the antiviral agent may be present in the formulation at a concentration of 0.01 to 2000 ug / mL, for example, 0.01 to 200 ug / mL. For example, when the antiviral agent is apilimod (i.e., free base) or a pharma- ceutical acceptable salt thereof (e.g., mesylate), apilimod may be present in the formulation at a concentration of 2 to 200 ug / mL. For example, when the antiviral agent is camostat (i.e., free base) or a pharma- ceutical acceptable salt thereof (e.g., mesylate), camostat may be present in the formulation at a concentration of 1 to 100 ug / mL. For example, when the antiviral agent is nafamostat (i.e., free base) or a pharma- ceutical acceptable salt thereof (e.g., mesylate), nafamostat may be present in the formulation at a concentration of 0.1 to 100 ug / mL. For example, when the antiviral agent is umifenovir or a medicamentarily acceptable salt thereof, umifenovir may be present in the formulation at a concentration of 10 to 10000 ug / mL. For example, when the antiviral agent is remdesivir or a medicamentarily acceptable salt thereof, remdesivir may be present in the formulation at a concentration of 0.01 to 100 ug / mL. For example, when the antiviral agent is molnupiravir or a medicamentarily acceptable salt thereof, molnupiravir may be present in the formulation at a concentration of 0.01 to 10 ug / mL. For example, when the antiviral agent is nirmatrervir or a medicamentarily acceptable salt thereof, nirmatrervir may be present in the formulation at a concentration of 0.01 to 10 ug / mL. For example, when the antiviral agent is lopinavir or a medicamentarily acceptable salt thereof, lopinavir may be present in the formulation at a concentration of 1 to 1000 ug / mL. For example, when the antiviral agent is oseltamivir or a medicament acceptable salt thereof, oseltamivir may be present in the formulation at a concentration of 0.01 to 100 ug / mL. For example, when the antiviral agent is oseltamivir carboxylate or a medicament acceptable salt thereof, oseltamivir carboxylate may be present in the formulation at a concentration of 0.01 to 100 ug / mL. For example, when the antiviral agent is pirodavir or a medicament acceptable salt thereof, pirodavir may be present in the formulation at a concentration of 0.1 to 100 ug / mL. For example, when the antiviral agent is ribavirin or a medicament acceptable salt thereof, ribavirin may be present in the formulation at a concentration of 1 to 1000 ug / mL.For example, when the antiviral agent is baloxavir or a pharma- ceutically acceptable salt thereof, baloxavir may be present in the formulation at a concentration of 0.01 to 10 ug / mL. For example, when the antiviral agent is ensitreruvir or a pharma- ceutically acceptable salt thereof, ensitreruvir may be present in the formulation at a concentration of 0.01 to 100 ug / mL. For example, when the antiviral agent is favipiravir or a pharma- ceutically acceptable salt thereof, favipiravir may be present in the formulation at a concentration of 1 to 1000 ug / mL. For example, when the antiviral agent is zanamivir or a pharma- ceutically acceptable salt thereof, zanamivir may be present in the formulation at a concentration of 0.01 to 100 ug / mL. For example, when the antiviral agent is laninamivir or a pharma- ceutically acceptable salt thereof, laninamivir may be present in the formulation at a concentration of 0.01 to 100 ug / mL. For example, when the antiviral agent is laninamivir octanoate or a pharma- ceutical acceptable salt thereof, laninamivir octanoate may be present in the formulation at a concentration of 0.01 to 100 ug / mL.For example, when the antiviral agent is rupintrivir or a pharma- ceutical acceptable salt thereof, rupintrivir may be present in the formulation at a concentration of 0.01 to 100 ug / mL.

[0043] Ritonavir or a pharma- ceutically acceptable salt thereof is a potent inhibitor of intestinal and hepatic cytochrome p450 3A4 and may be included in liquid pharmaceutical formulations to enhance the effect of another antiviral agent, such as nilmatre- vir or lopinavir (Choy et al.).

[0044] For example, when ritonavir or a pharma- ceutical acceptable salt thereof is included, ritonavir may be present in the formulation at a concentration of 0.005 to 5 ug / mL.

[0045] For example, when the antiviral agent is nilmatrellvir or a pharma- ceutically acceptable salt thereof, used in combination with ritonavir or a pharma- ceutically acceptable salt thereof, nilmatrellvir may be present in the formulation at a concentration of 0.01-10 ug / mL, and ritonavir may be present in the formulation at a concentration of 0.005-5 ug / mL. For example, when the antiviral agent is lopinavir or a pharma- ceutically acceptable salt thereof, used in combination with ritonavir or a pharma- ceutically acceptable salt thereof, lopinavir may be present in the formulation at a concentration of 1-1000 ug / mL, and ritonavir may be present in the formulation at a concentration of 0.25-250 ug / mL. The amounts listed above are based on the equivalent amount of the free form (e.g., base / acid) of the antiviral agent when used in salt form.

[0046] The liquid pharmaceutical formulation may be a solution formulation in which the antiviral agent is incorporated and dissolved. For solution formulations, a soluble form of the antiviral agent will be selected. For example, the mesylate salt of apilimod is soluble in water.

[0047] Alternatively, the aqueous pharmaceutical formulation may be a suspension formulation containing the antiviral agent as a solid in finely divided form, where the solid is in finely divided form, it will be of a size suitable for the intended route of delivery, i.e., to the lungs or nose.

[0048] In some embodiments, the liquid pharmaceutical formulation may be an aqueous formulation in which the antiviral agent may be incorporated and dissolved or suspended. When the antiviral agent is dissolved in an aqueous formulation (i.e., when the formulation is a solution formulation), a soluble form of the antiviral agent may be selected. For example, the mesylate salt of apilimod is soluble in water. Dissolution of the antiviral agent may be aided by including a solvent in the formulation, such as a polar organic solvent, e.g., an alcohol, a polyol, or a polyethylene glycol (PEG), e.g., ethanol. When the antiviral agent is suspended in an aqueous formulation, a relatively insoluble form of the antiviral agent may be selected. For example, apilimod base is relatively insoluble in water. In such a suspension formulation, the antiviral agent may be in a solid, finely divided form with a size suitable for the intended delivery route, i.e., delivery to the lungs or nose. In the case of an aqueous formulation, the water used in the formulation may be sterile water. The aqueous formulation may optionally include other ingredients, such as preservatives, buffers, and osmotic agents. Examples of preservatives include edetic acid and its alkali salts, such as disodium EDTA (also referred to as "disodium edetate" or "disodium edetate salt") and calcium EDTA (also referred to as "calcium edetate"), benzyl alcohol, methylparaben, propylparaben, butylparaben, chlorobutanol, phenylethyl alcohol, benzalkonium chloride, thimerosal, propylene glycol, sorbic acid, and benzoic acid derivatives. Examples of buffering agents include weak organic acid-based buffers, such as citrate / citric acid buffers. Osmotic agents increase the osmolality of the formulation and improve patient comfort. Examples of osmotic agents include polyols, such as sugars and sugar alcohols, for example, xylitol and glycerol. Aqueous formulations suitable for topical administration to the nose may optionally include wetting agents and thickening agents, such as hyaluronic acid or a pharma- ceutically acceptable salt thereof. Aqueous suspension formulations may optionally contain surfactants and suspending agents that aid in maintaining the suspension, such as cellulose derivatives such as microcrystalline cellulose / sodium carboxymethylcellulose (Avicel), carrageenan, and hyaluronic acid or a pharma- ceutically acceptable salt thereof. The surfactant component in the formulation may act as a suspending agent or contribute to the function of a suspending agent.

[0049] The pH of the aqueous liquid pharmaceutical formulation may generally be within a wide range, for example, from 4 to 8.

[0050] In some embodiments, the aqueous liquid pharmaceutical formulation may be a micellar solution in which the hydrophilic "head" regions of the surfactant molecules are in contact with the surrounding solvent and a single hydrophobic tail region is sequestered in the central micellar region formed from the "tail" regions of the surfactant molecules.

[0051] In some embodiments, the liquid pharmaceutical formulation may be a pressurized liquid formulation in which the antiviral agent is incorporated and dissolved or, more preferably, suspended. For pressurized liquid suspension formulations, a relatively insoluble form of the antiviral agent may be selected. For example, the mesylate salt of apilimod and its base form are relatively insoluble in liquids such as pressurized hydrofluoroalkanes. For pressurized liquid solution formulations, dissolution of the antiviral agent may be aided by including a polar organic solvent, such as ethanol, in the formulation. In a suspension formulation, the antiviral agent will be a solid and in finely divided form. A surfactant component in the formulation may act as a suspending agent or contribute to the function of the suspending agent to help maintain the suspension. If the solid is in finely divided form, it will be of a size appropriate for the intended delivery route, i.e., delivery to the lungs or nose.

[0052] Typically, the finely divided solid particles in the formulation may have a mass median aerodynamic diameter (MMAD) in the range of 1-10 μm. Particles of appropriate size may be produced by air jet milling, spray drying, supercritical fluid extraction, or nanomilling. The MMAD of solid particles may be determined using a next generation impactor (NGI) (Marple et al., 2021).

[0053] The pressurized liquid used in the pressurized liquid formulation is suitably a volatile non-polar liquid such as a hydrofluoroalkane (HFA) or hydrofluoroolefin (HFO), such as HFA134a, HFA227, HFA152a, HFO1234ze or HFO1234zf or mixtures thereof, in particular HFA134a, HFA152a, or HFO1234ze.

[0054] In some embodiments, the non-pressurized liquid pharmaceutical formulation may be administered to the nose using a nasal spray device or nasal drop applicator. Nasal spray devices and nasal drop applicators are known in the art and are disclosed in U.S. Patent No. US2577321 and U.S. Patent No. US6000580, the contents of each of which are incorporated herein by reference in their entirety. As used herein, the term "nasal drop applicator" refers to any dispenser suitable for administering nasal drops. Nasal spray devices typically administer a metered volume of liquid, for example, a volume of 50-200 μL, particularly 100 μL. The metered volume is preferably administered to each nostril (e.g., once or twice per nostril).

[0055] In some embodiments, non-pressurized liquid pharmaceutical formulations, particularly aqueous formulations such as aqueous solution formulations, may be administered using a nebulizer device. Nebulizers are typically continuous while switched on or breath-actuated, generating an aerosol for inhalation. Nebulizer devices may be hand-held and portable, or may be for home or hospital use (i.e., stationary). Popular nebulizer products include the Aeroneb® device and the Pari® device, which are disclosed in U.S. Patent No. US9364618, the contents of each of which are incorporated herein by reference in their entirety. Nebulizer devices may be, for example, piezoelectric nebulizer devices, which are known to generate homogeneous aerosols by high frequency vibration of a metal (usually stainless steel) mesh or membrane with small holes (usually micrometer size). The generated aerosol may be directed to the lungs or lungs and nose by inhalation using a suitable mouthpiece or nosepiece. When the formulations of the invention are administered topically to the lungs by oral inhalation or when administered topically to the nose, the formulation may thereby be administered to the pharynx.

[0056] Pressurized formulations will generally be contained within a canister (eg an aluminium canister) closed with a valve (eg a metering valve) and fitted with an actuator provided with a mouthpiece.

[0057] The canister generally includes a container that can withstand the vapor pressure of the HFA propellant, such as a plastic bottle or a glass bottle with a plastic coating, or preferably a metal can, such as an aluminum can optionally with an oxide coating, lacquer coating and / or plastic coating, closed with a metering valve. The canister may be preferably coated with a fluorocarbon polymer, such as a copolymer of polyethersulfone (PES) and polytetrafluoroethylene (PTFE), as described in WO 96 / 32151. Another possible coating polymer is FEP (fluorinated ethylene propylene). The metering valve is designed to deliver a metered amount of the formulation per actuation and incorporate a gasket to prevent leakage of the propellant through the valve. The gasket may include any suitable elastomeric material, such as low density polyethylene, chlorobutyl, black and white butadiene-acrylonitrile rubber, butyl rubber, and neoprene. Thermoplastic elastomer valves as described in WO 92 / 11190 and valves with EPDM rubber as described in WO 95 / 102651 are particularly suitable. Suitable valves are commercially available from manufacturers well known in the aerosol industry, such as Valois, France (e.g. DF10, DF30, DF60), Bespak pic, UK (e.g. BK300, BK356, BK357), and 3M-Neotechnic Ltd, UK (e.g. Spraymiser™). The DF31 valve from Valois, France, is also suitable.

[0058] The valve seals, particularly the gasket seals, will preferably be made of a material that is inert to the contents of the formulation and resistant to extraction by the contents, particularly when the contents include ethanol. The valve material, particularly the material of manufacture of the metering chamber, will preferably be made of a material that is inert to the contents of the formulation and resistant to distortion by the contents, particularly when the contents include ethanol. Materials particularly suitable for use in the manufacture of the metering chamber include polyesters, such as polybutylene terephthalate (PBT), and acetals, particularly PBT. The material of manufacture of the metering chamber and / or valve stem will desirably be fluorinated, partially fluorinated, or impregnated with a fluorine-containing material to prevent drug deposition. The valve chamber will be of a size appropriate to the dose of liquid pharmaceutical formulation to be dispensed and its concentration, for example 25-100 μL, such as 25 μL or 100 μL.

[0059] Conventional bulk manufacturing methods and machinery well known to those skilled in the pharmaceutical aerosol manufacturing industry can be employed to prepare large batches of filled canisters for commercial production. Thus, for example, in one bulk manufacturing method, a metering valve is crimped onto an aluminum can to form an empty canister. A formulation containing the drug, propellant and other formulation ingredients is charged into the container and pressure filled to form the product container. Typically, in a batch prepared for pharmaceutical use, each filled canister is weighed, coded with a batch number, and packaged in a storage tray prior to release testing. In another process, an aliquot of the liquefied formulation is dispensed into an open canister under conditions cool enough to prevent evaporation of the formulation, and then a metering valve is crimped onto the canister. Typically, in a batch prepared for pharmaceutical use, each filled canister is weighed, coded with a batch number, and packaged in a storage tray prior to release testing.

[0060] Each filled canister is conveniently fitted prior to use with a suitable channel device to form a metered dose inhaler for administering medicament to the lungs or nasal cavity of a patient. A suitable channel device may, for example, comprise a valve actuator and a cylindrical or cone-like passageway through which medicament may be delivered from the filled canister via the metering valve to the patient's nose or mouth, for example a mouthpiece actuator. Metered dose inhalers are designed to deliver a fixed unit dose of medicament per actuation or "puff", for example in the range of 10-5000 μg medicament per puff.

[0061] In a typical arrangement, the valve stem is mounted in a nozzle block having an orifice which leads to an expansion chamber. The expansion chamber has an exit orifice which extends into the mouthpiece. Actuator (exit) orifice diameters in the range 0.1 to 0.45 mm are generally suitable, for example 0.15, 0.22, 0.25, 0.30, 0.33, 0.42 mm. The orifice size should not be so small that jet blockage occurs.

[0062] The actuator jet length is typically in the range of 0.30 to 1.7 mm, for example 0.30, 0.65, or 1.50 mm for intraoral buccal administration (ie oral administration).

[0063] The precise shape and dimensions of the actuator may be suitably adapted for local administration to the lungs or nose.

[0064] An aspect of the invention is a liquid pharmaceutical formulation as described herein for use as a medicament for local administration to the lungs or nose, and in particular for use in the treatment or prevention of an infection caused by a virus or a disease associated with an infection with such a virus. In one embodiment, the liquid pharmaceutical formulation is for use in the treatment of a viral infection or a disease associated with a viral infection. In another embodiment, the liquid pharmaceutical formulation is for use in the prevention of a viral infection or a disease associated with a viral infection.

[0065] A further aspect of the invention is a method for treating or preventing a viral infection or a disease associated with infection by or with such a virus, comprising administering locally to the lungs or nose to a subject in need thereof a therapeutically or prophylactically effective amount of a pharmaceutical formulation as described herein. In one embodiment, the method is a method for treating a viral infection or a disease associated with a viral infection. In another embodiment, the method is a method for preventing a viral infection or a disease associated with a viral infection.

[0066] A further aspect of the invention is the use of a liquid pharmaceutical formulation as described herein for the manufacture of a medicament for local administration to the lungs or nose, and in particular for the treatment or prevention of an infection by a virus or a disease associated with infection with such a virus.

[0067] An aspect of the invention is a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, for use as a medicament for topical administration to the lungs (e.g., by oral inhalation) or nose in the treatment or prevention of a viral infection or a disease associated with a viral infection. In one embodiment, the liquid pharmaceutical formulation is for use in the treatment of a viral infection or a disease associated with a viral infection. In another embodiment, the liquid pharmaceutical formulation is for use in the prevention of a viral infection or a disease associated with a viral infection.

[0068] A further aspect of the invention is a method for treating or preventing a viral infection or a disease associated with a viral infection, comprising administering to a subject in need thereof, topically to the lungs (e.g., by oral administration) or nose, a therapeutically or prophylactically effective amount of a pharmaceutical formulation comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester. In one embodiment, the method is a method for treating a viral infection or a disease associated with a viral infection. In another embodiment, the method is a method for preventing a viral infection or a disease associated with a viral infection.

[0069] A further aspect of the invention is the use of a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, for the manufacture of a medicament for use in the prophylaxis of viral infections or diseases associated with viral infections, for topical administration to the lungs (e.g. by oral inhalation) or nose.

[0070] A further aspect of the invention is a liquid pharmaceutical formulation suitable for topical administration to the lungs (e.g. by oral inhalation) or nose, for use in improving epithelial barrier function in vivo (in particular the barrier function of the pulmonary or nasal epithelium), comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma-ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester.

[0071] A further aspect of the invention is a liquid pharmaceutical formulation suitable for topical administration to the lungs (e.g. by oral inhalation) or nose, for use in improving epithelial barrier function in vivo (in particular the barrier function of the pulmonary or nasal epithelium), comprising (i) a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and mixtures of oleic acid or a pharma-ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, and (ii) an antiviral agent.

[0072] A further aspect of the invention is a method of improving epithelial barrier function (particularly the barrier function of the pulmonary or nasal epithelium) in a subject, comprising topically administering to the pulmonary (e.g. by oral inhalation) or nasal epithelium of the subject a liquid pharmaceutical formulation comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and mixtures of oleic acid or a pharma-ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester.

[0073] A further aspect of the invention is a method of improving epithelial barrier function (particularly the barrier function of the pulmonary or nasal epithelium) in a subject, comprising topically administering to the pulmonary (e.g., by oral inhalation) or nasal epithelium of the subject a liquid pharmaceutical formulation comprising: (i) a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and mixtures of oleic acid or a pharma-ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester; and (ii) an antiviral agent.

[0074] Illustratively for the above embodiment, the viral infection is a coronavirus infection. Illustratively for the above embodiment, the viral infection is a SARS-CoV-2 infection and the viral infection-associated disease is COVID-19. Illustratively for the above embodiment, the viral infection is a seasonal coronavirus infection, e.g., a 229E infection, and the viral infection-associated disease is a seasonal coronavirus-associated disease, e.g., a 229E-associated disease. Illustratively for the above embodiment, the viral infection is an influenza virus infection and the viral infection-associated disease is influenza. Illustratively for the above embodiment, the viral infection is a respiratory syncytial virus (RSV) infection and the viral infection-associated disease is a RSV infection-associated disease. Illustratively for the above embodiment, the viral infection is a human rhinovirus (HRV) infection and the viral infection-associated disease is a HRV infection-associated disease.

[0075] "Influenza virus" as used herein includes influenza A virus, influenza B virus, influenza C virus, and influenza D virus, eg, influenza A virus or influenza B virus.

[0076] In methods for preventing viral infections or diseases associated with viral infections, the liquid pharmaceutical formulation may be administered daily to a subject in need thereof, preferably approximately 1 to 3 days prior to exposure that may result in viral infection, for example, up to 1 to 4 times daily.

[0077] In the method of treating a viral infection or a disease associated with a viral infection, the initial administration of the liquid pharmaceutical formulation to a subject in need thereof may be performed preferably within 72 hours, preferably within 48 hours, of exposure to the viral infection. The therapeutic administration may be performed for a period of typically 3 to 10 days, for example 5 to 10 days.

[0078] In the methods described herein, the liquid pharmaceutical formulations may be administered at a frequency of 1 to 4 times per day.

[0079] Suitable doses of the liquid pharmaceutical formulations per administration for use in the methods described herein are therapeutically or prophylactically effective amounts that can be determined by one of skill in the art.

[0080] Suitably, the treatment or prevention described herein is for the treatment or prevention of a viral infection or disease in a mammal, particularly a human.

[0081] Another aspect of the invention relates to a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutically acceptable salt thereof, and a mixture of oleic acid or a pharma- ceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester. Preferably the formulation is an aqueous formulation. Suitable amounts and concentrations of surfactant are described above. Such a formulation may be for use in the treatment or prevention of a viral infection or a disease associated with a viral infection. The invention also provides a method for treating or preventing said infection / disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of said pharmaceutical formulation. The invention also provides the use of the formulation described herein for the manufacture of a medicament for topical administration to the lungs or nose, and in particular for the treatment or prevention of an infection by a virus or a disease associated with an infection with such a virus. Examples of infections / diseases are described above and include SARS-CoV-2 / COVID-19 and influenza virus infection / influenza. The formulation may be administered locally to the lungs, for example by oral inhalation, or locally to the nose.

[0082] As used herein, a "subject" is preferably a human subject.

[0083] "Treatment" of a viral infection includes prevention of disease associated with a viral infection after exposure, as therapeutic treatment of the viral infection after exposure may lead to prevention of the disease.

[0084] In the formulations of the present invention, the surfactant(s) and the antiviral agent have desirable pharmaceutical and biological properties when co-formulated. However, the components of the formulation may also be co-administered in separate formulations. Thus, the present invention also provides a liquid pharmaceutical formulation suitable for local administration to the lungs or nose, comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma- ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, for use in combination with a liquid pharmaceutical formulation suitable for local administration to the lungs or nose, comprising an antiviral agent, whereby the formulation comprising the surfactant component and the formulation comprising the antiviral agent are co-administered. The present invention also provides a method for treating or preventing a viral infection or a disease associated with infection by or with such a virus, the method comprising co-administering (i) a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising a surfactant component selected from the group consisting of oleic acid or a pharma- ceutical acceptable salt thereof, and a mixture of oleic acid or a pharma-ceutical acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, and (ii) a liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising an antiviral agent. The formulations may be administered locally to the lungs, for example by oral inhalation, or locally to the nose. "Co-administration" in this context means that the two formulations are administered at essentially the same time, for example simultaneously or within a few seconds (e.g., 1-3 seconds) of each other. EXAMPLES

[0085] (Example) Abbreviations used herein are defined below (Table 1). Abbreviations not defined are intended to convey their generally accepted meaning. (Table 1: Abbreviations) [Table 1]

[0086] (material) All starting materials and solvents were obtained from commercial sources.

[0087] Example 1A: Example of a Liquid Pharmaceutical Formulation of Apilimod for Nasal Administration Using a Nasal Spray Device The following aqueous suspension formulations may be prepared: [Table 2]

[0088] The formulation may be administered to the nose using a nasal spray device (e.g., 100 μL spray volume, 1-2 sprays per nostril) for the prevention or treatment of viral infections, e.g., infections caused by SARS-CoV-2.

[0089] Example 1B: Example of a Liquid Pharmaceutical Formulation of Zanamivir for Nasal Administration Using a Nasal Spray Device The following aqueous suspension formulations may be prepared: [Table 3]

[0090] The formulation may be administered to the nose using a nasal spray device (eg, 100 μL spray volume, 1-2 sprays per nostril) for the prevention or treatment of viral infections, such as influenza.

[0091] Example 2: Assessment of viral load and cell integrity in air-liquid interface (ALI) cultured human epithelial cells infected with SARS-CoV-2 - a study with apilimod (Experimental Method) Primary human nasal, tracheal, and bronchial epithelial cells can be cultured and differentiated at the air-liquid interface (ALI) to form pseudostratified mucociliary airway epithelium composed of ciliated, goblet, club, and basal cells, with an arrangement that faithfully reflects the cellular organization in vivo. This in vitro model of human airway epithelium (HAE) cultured at the ALI (HAE-ALI) faithfully recapitulates many key features of respiratory virus-host cell interactions observed in vivo in infected upper and lower respiratory tracts and has been used to study many human respiratory viruses, including SARS-CoV-2. Notably, differentiation at the ALI dramatically increases the expression and polarized presentation of the viral receptor ACE2 on the apical membrane, as well as high levels of TMPRSS 2 expression. Therefore, HAE-ALI is an optimal cell culture model to study SARS-CoV-2 infection in vitro.

[0092] ALI-cultured pooled donor human nasal epithelium (provided by Epithelix Sarl, Geneva, Switzerland) was maintained at the air-liquid interface in Costar Transwell inserts (Corning, NY, USA) using MucilAir™ medium according to the manufacturer's instructions and used for SARS-CoV-2 infection (VirNext, University of Lyon) as previously reported (Pizzorno et al., 2020, Cell Rep Med. 1(4):100059). On day 0, 100 μL of SARS-CoV-2 inoculum (BetaCoV / France / IDF0571 / 2020 strain (accession ID EPI_ISL_411218), diluted in MucilAir medium to a final multiplicity of infection MOI of 0.1) was added to the apical surface for 1 h (37°C / 5% CO2). The viral inoculum was removed and the inserts were washed with sterile PBS (containing Ca2+ / Mg2+).

[0093] As shown in Figure 1, ALI cultures were administered apical side with apilimod (as mesylate) (2 mg / ml, 50 μL) dissolved in water, surfactant (0.15% w / w polysorbate 80 and 0.2% w / w oleic acid), or apilimod (as mesylate) and surfactant 10 min prior to virus inoculation and then for 60 min with the virus inoculum on day 0 (and then removed with the virus inoculum as described above), and similarly reapplied to the apical surface for 10 min on day 1 (24 h post-inoculation) before removal. Vehicle treatment (water) was performed on the corresponding apical surface so that each well received the same number of manipulations. Additionally, remdesivir (5 μM) was added to the basolateral chamber on days 0 and 1. On day 2 (48 hours after virus inoculation), sampling was performed by adding 200 μL of OptiMEM™ medium to the apical surface of each well for 10 minutes (stored at −80° C.).

[0094] Viral load was quantified by RT-PCR as reported by Pizzorno et al. (Cell Rep Med. 1(4):100059, 2020). The supernatants were lysed and viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen). Viral RNA was quantified by RT-qPCR (Express One-Step Superscript™ qRT-PCR kit, Invitrogen). SARS-CoV-2 specific primers and probes used for viral genome quantification were as follows: Target ORF1b-nsp14 Forward primer (HKU-ORF1b-nsp14F) [ka] Reverse primer (HKU-ORF1b-nsp14R) [ka] Probe (HKU-ORF1b-nsp141P) [ka] Ct data was determined and the relative changes in gene expression were calculated using -ΔCt The reduction was calculated using the method and expressed as the fold reduction in genome copy number (ORF1b-nsp14 genes of SARS-CoV-2) relative to the mean value of vehicle-treated infected controls.

[0095] Transepithelial electrical resistance (TEER) was measured to investigate the dynamic integrity of tight junctions in pseudostratified epithelium cultured at the air-liquid interface as an indicator of epithelial damage before and after SARS-CoV-2 infection. Chopstick electrodes were placed in the apical and basolateral chambers and TEER was measured using a dedicated volt-ohmmeter (EVOM2 for TEER, Epithelial Volt / Ohm Meter) and expressed in Ohms / cm. 2 It was displayed as.

[0096] (result) High levels of SARS-CoV-2 replication were detected in apical lavage fluids from virus controls 48 hours after inoculation (RT-PCR: Ct=12.1). Comparing the viral load of each treatment to that of the mock control (=1), apilimod alone showed only a small effect on viral load (0.1 Log reduction), whereas surfactant showed a 1.3 Log reduction in viral load. However, the combination of apilimod and surfactant showed a synergistic effect in reducing viral load, leading to a 2.2 Log reduction compared to the virus-infected control (see Figure 2). The assay control, remdesivir (5 μM), showed a 3.4 Log reduction in viral load, as predicted from the literature (Pizzorno et al., 2020, Cell Rep Med. 1(4):100059).

[0097] SARS-CoV-2 infection significantly reduced TEER values ​​48 hours after virus inoculation in vehicle-treated virus-infected controls. Rapid virus-induced epithelial damage was demonstrated by the disappearance of zoneura occludin-1 (ZO-1) expression by immunohistochemistry with no clear tight junctions and by partial loss of cilia (Hao et al., 2020, mBio, 11(6):e02852-20). In contrast, apilimod alone showed partial inhibition of the virus-induced decrease in TEER (48% effect). However, surfactant and the combination of apilimod and surfactant not only fully restored TEER but also further increased TEER levels (see Figure 3). Thus, these treatments protected against virus-induced cell damage and further strengthened the epithelial barrier by cell repair or cell proliferation. The assay control, remdesivir (5 μM), also provided 81% protection, as predicted from the literature (Pizzorno et al., 2020, Cell Rep Med. 1(4):100059).

[0098] The antiviral effects were evaluated using primary human nasal epithelial cells grown in air-liquid culture, which underwent extensive mucociliary differentiation resulting in cultures with morphological characteristics similar to those observed in normal human nasal epithelium. As a result, this cell model closely mimics SARS-CoV-2 infection in the human nasal cavity.

[0099] Example 3: Assessment of viral load and cell integrity in air-liquid interface (ALI) cultured human epithelial cells infected with SARS-CoV-2 - A study with oseltamivir carboxylate (Experimental Method) ALI-cultured pooled donor human nasal epithelium (obtained from Epithelix Sarl, Geneva, Switzerland) was maintained at the air-liquid interface in Costar Transwell inserts (Corning, NY, USA) using MucilAir™ medium according to the manufacturer's instructions. On day 0, an H1N1 inoculum (A / Switzerland / 7717739 / 2013 (H1N1) strain, 100 μL; diluted in MucilAir medium to 4.85E3 genome copies / ml) was added to the apical surface for 1.5 h (34°C / 5% CO2). The viral inoculum was removed and the inserts were washed with sterile medium.

[0100] ALI cultures were administered oseltamivir carboxylate (1 μM), detergent (0.15% polysorbate 80 and 0.2% oleic acid), or oseltamivir carboxylate in detergent on the apical side 10 min prior to virus inoculation and then on day 0 for 90 min with the virus inoculum (and then removed with the virus inoculum as above), and similarly reapplied to the apical surface for 10 min on day 1 (24 h post-inoculation) before removal. Vehicle treatment (water) was performed on the corresponding apical surface so that each well received the same number of runs. Additionally, oseltamivir carboxylate (10 μM) was added to the basolateral chamber on day 0. Sampling was performed on days 1 and 2 (24 and 48 h post-virus inoculation) by adding 200 μL of medium to the apical surface of each well for 20 min (stored at −80° C.). Oseltamivir carboxylate is the active metabolite of oseltamivir, the active ingredient in the marketed drug Tamiflu® (oseltamivir phosphate), and is produced in vivo following administration.

[0101] Supernatants were lysed and viral RNA was extracted using the QIAamp 96 virus QUAcube HT kit (Qiagen). Viral RNA was quantified by RT-qPCR with the qTOWER3 detection system (QuantiTect Probe RT-PCR, Qiagen). Ct data were recorded against a standard curve and expressed as genome copies / ml. Transepithelial electrical resistance (TEER) was measured as described above.

[0102] (result) High levels of H1N1 replication were detected in apical lavage fluids from virus controls 24 hours after inoculation. Comparing the viral load of each treatment to that of the mock control, oseltamivir carboxylate alone showed only a minor effect on viral load (1.0 Log reduction), and detergent showed no reduction in viral load. However, the combination of oseltamivir carboxylate and detergent showed a synergistic effect in reducing viral load, leading to a 2.2 Log reduction compared to the virus-infected control (see Figure 4). The assay control, oseltamivir carboxylate (10 μM in the basolateral chamber), showed a 1.7 Log reduction in viral load.

[0103] H1N1 infection only slightly reduced transepithelial electrical resistance (TEER, as an index of epithelial integrity) values ​​24 hours after infection, but significantly reduced them 48 hours after infection, compared to uninfected controls. At 48 hours after infection, oseltamivir carboxylate alone completely restored the TEER levels reduced by H1N1, detergent alone partially restored the TEER levels, while the combination of oseltamivir carboxylate and detergent further increased the TEER levels (see Figure 5). Thus, these treatments protected against virus-induced cell damage and further strengthened the epithelial barrier by cell repair or cell proliferation. The assay control oseltamivir carboxylate (10 μM, basolateral treatment) also protected against loss of epithelial integrity.

[0104] (Summary of Biological Data) The in vitro antiviral activity of apilimod, surfactants and their combination was demonstrated by the reduction of SARS-CoV-2 viral load in infected nasal epithelium. In this assay system, inhibition of viral replication was detected and quantified by the reduction of viral genome obtained by RT-PCR. Surfactants were highly active, and the combination of apilimod and surfactants had a significant and clearly synergistic antiviral effect. The superior antiviral effect of the combination of oseltamivir and surfactants was also confirmed in influenza virus-infected nasal epithelium.

[0105] In vehicle-treated virus-infected controls, transepithelial electrical resistance (TEER, as an index of epithelial integrity) values ​​after SARS-CoV-2 or influenza infection were also significantly decreased. The use of apilimod partially inhibited the SARS-CoV-2-induced decrease in TEER, and the use of surfactants, especially the combination of apilimod and surfactants, completely inhibited the SARS-CoV-2-induced decrease in TEER or even increased TEER. Oseltamivir carboxylate alone completely restored the TEER levels decreased by influenza virus infection, surfactant alone partially restored the TEER levels, while the combination of oseltamivir carboxylate and surfactant further increased the TEER levels. These results suggest that this treatment protects against virus-induced cell damage and enhances barrier integrity by cell repair and cell proliferation. Thus, both therapeutic and prophylactic effects of this administration are evident.

[0106] The results indicate that the liquid pharmaceutical formulations of the present invention comprising a surfactant component comprising oleic acid as described herein, and in particular, a liquid pharmaceutical formulation comprising a surfactant component comprising oleic acid as described herein and an antiviral agent such as apilimod or oseltamivir carboxylate, when administered locally to the lungs or nose, are predicted to be useful in the treatment or prevention of viral infections, such as SARS-CoV-2, and their associated diseases, such as COVID-19 or influenza.

[0107] In SARS-CoV-2 infection, the combination of surfactants and antivirals, apilimod and oseltamivir carboxylate, was the most potent in reducing viral load and recovering TEER, but surfactants alone also had significant activity in reducing viral load and recovering TEER. Surfactants alone also had significant activity in recovering TEER in influenza virus infection. This effect of recovering TEER (an index of barrier integrity) supports the use of surfactants alone and the combination of surfactants and antivirals to prevent and treat viral infections.

[0108] (References) [Table 4] TIFF2024546179000010.tif242170TIFF2024546179000011.tif237170TIFF2024546179000012.tif145170

[0109] (Comprehensive Declaration) Throughout this specification and the claims that follow, unless otherwise required by context, the term "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of the stated integer, step, group of integers or group of steps, but are not intended to exclude other integers, steps, group of integers or group of steps. All patents, patent applications, and references mentioned throughout the specification of the present invention are incorporated herein by reference in their entirety. The present invention encompasses all combinations of the preferred and more preferred groups, and the suitable and further suitable groups and groups of embodiments listed above.

Claims

1. A liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising: (i) a surfactant component selected from a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester; and (ii) an antiviral agent.

2. 2. The liquid pharmaceutical formulation according to claim 1, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polysorbate 80 or polysorbate 20.

3. 3. The liquid pharmaceutical formulation according to claim 2, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polysorbate 80.

4. 4. The liquid pharmaceutical formulation according to any one of claims 1 to 3, wherein the oleic acid is in the form of a free acid.

5. 4. The liquid pharmaceutical formulation according to claim 1, which is a solution formulation in which the antiviral agent is incorporated and dissolved.

6. 4. The liquid pharmaceutical formulation according to any one of claims 1 to 3, which is a suspension formulation comprising the antiviral agent as a solid in finely divided form.

7. The liquid pharmaceutical formulation according to any one of claims 1 to 3, wherein the liquid pharmaceutical formulation is an aqueous formulation.

8. The liquid pharmaceutical formulation according to any one of claims 1 to 3, wherein the liquid pharmaceutical formulation is a pressurized liquid formulation.

9. 9. A liquid pharmaceutical formulation according to claim 8, comprising a pressurised liquid propellant selected from HFA134a, HFA227, HFA152a, HFO1234ze, HFO1234zf and mixtures thereof.

10. 10. The liquid pharmaceutical formulation of claim 9, comprising HFA134a, HFA152a, or HFO1234ze as a pressurized liquid propellant.

11. 9. A canister containing multiple doses of the liquid pharmaceutical formulation of claim 8 and equipped with a metering valve.

12. 12. A metered dose inhaler comprising a canister according to claim 11 equipped with an actuator adapted to administer the formulation to the lungs.

13. 4. A spray or nebulizer device comprising a reservoir containing multiple doses of a liquid pharmaceutical formulation according to any one of claims 1 to 3 and provided with a channel device for localized delivery of the formulation to the lungs or nose.

14. 4. A liquid pharmaceutical formulation according to any one of claims 1 to 3 for use as a medicament for local administration to the lungs (e.g. by oral inhalation) or to the nose, wherein the medicament is for the treatment or prevention of an infection by a virus or a disease associated with infection with such a virus.

15. The pharmaceutical for the treatment or prevention of SARS-CoV-2 infection and / or COVID-19; or for the treatment or prevention of influenza virus infection and / or influenza; or for the treatment or prevention of a coronavirus infection, such as a seasonal coronavirus infection, such as a 229E infection, and / or a disease associated with a coronavirus infection, such as a disease associated with a seasonal coronavirus infection, such as a disease associated with a 229E infection; or for the treatment or prevention of respiratory syncytial virus (RSV) infection and / or diseases associated with RSV infection; or 15. The liquid pharmaceutical formulation for use according to claim 14, for the treatment or prevention of human rhinovirus (HRV) infection and / or diseases associated with HRV infection.

16. A liquid pharmaceutical formulation suitable for local administration to the lungs or nose, comprising a surfactant component selected from a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, said liquid pharmaceutical formulation being for use as a pharmaceutical for local administration to the lungs or nose in the treatment or prevention of a viral infection or a disease associated with a viral infection.

17. The viral infection is a SARS-CoV-2 infection and the disease associated with the viral infection is COVID-19; or the viral infection is an influenza virus infection and the viral infection-related disease is influenza; or The viral infection is a coronavirus infection, such as a seasonal coronavirus infection, such as a 229E infection, and the viral infection-related disease is a disease related to a coronavirus infection, such as a disease related to a seasonal coronavirus infection, such as a disease related to a 229E infection; or The viral infection is a respiratory syncytial virus (RSV) infection, and the viral infection-related disease is a disease related to RSV infection; or 17. The liquid pharmaceutical formulation for use according to claim 16, wherein the viral infection is a human rhinovirus (HRV) infection and the viral infection-associated disease is an associated disease of HRV infection.

18. A liquid pharmaceutical formulation suitable for topical administration to the lungs or nose, comprising a surfactant component selected from a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester.

19. 19. The liquid pharmaceutical formulation according to claim 18, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polysorbate 80 or polysorbate 20.

20. 20. The liquid pharmaceutical formulation according to claim 19, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polysorbate 80.

21. 21. The liquid pharmaceutical formulation according to any one of claims 18 to 20, wherein the oleic acid is in the form of a free acid.

22. The liquid pharmaceutical formulation according to any one of claims 18 to 20, wherein the liquid pharmaceutical formulation is an aqueous formulation.

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