WO2021255218A1 - A pharmaceutical combination comprising an anti-viral protonophore and a serine protease inhibitor - Google Patents

Abstract

The invention relates to a pharmaceutical combination, comprising a therapeutically effective amount of an anti-viral protonophore, and a therapeutically effective amount of a serine protease inhibitor. In preferred embodiments the invention relates to a combination of niclosamide and camostat mesylate. The invention further relates to a pharmaceutical composition comprising the combination, and use of the combination or composition in a treatment and/or prevention of a viral infection in a subject and/or a medical condition associated with a viral infection. In preferred embodiments the invention relates to the treatment and/or prevention of a SARS-CoV virus, preferably SARS-CoV-2.

Description

A PHARMACEUTICAL COMBINATION COMPRISING AN ANTI-VIRAL PROTONOPHORE

AND A SERINE PROTEASE INHIBITOR

DESCRIPTION

The invention relates to a pharmaceutical combination, comprising a therapeutically effective amount of an anti-viral protonophore, and a therapeutically effective amount of a serine protease inhibitor. In preferred embodiments the invention relates to a combination of niclosamide and camostat mesylate. The invention further relates to a pharmaceutical composition comprising the combination, and use of the combination or composition in a treatment and/or prevention of a viral infection in a subject and/or a medical condition associated with a viral infection. In preferred embodiments the invention relates to the treatment and/or prevention of a SARS-CoV virus, preferably SARS-CoV-2.

BACKGROUND OF THE INVENTION

Coronaviruses (CoVs) are a large family of viruses that are common in mammals including humans and birds. Whereas alpha- and beta-coronaviruses infect only mammals, infection with gamma- and delta-coronaviruses is mainly found in birds. Coronavirus infections cause respiratory illness in humans and gastroenteritis in animals (Cui, Li, and Shi 2019). Until recently six coronaviruses pathogenic to humans were known. Based on sequence data an animal origin of all human pathogenic coronaviruses can be deduced. There are four endemic human CoVs (HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1) that mainly cause mild respiratory tract infections (Corman, Lienau, and Witzenrath 2019).

The first CoV, highly pathogenic to humans, was SARS-CoV causing an outbreak of severe acute respiratory syndrome (SARS) in 2002 and 2003 in China. Ten years later, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Middle Eastern countries. In December 2019, a novel SARS-coronavirus 2 (SARS-CoV-2) emerged in Wuhan (Hubei province, China). Subsequently, SARS-CoV-2 rapidly spread within China and Asian countries before causing a worldwide pandemic of a new disease called Covid-19. Like SARS-CoV and MERS-CoV, the most recently emerged coronavirus SARS-CoV-2 can cause severe respiratory illness.

The SARS-CoV-2 virus is a beta-coronavirus, like MERS-CoV and SARS-CoV. All three of these viruses have their origins in bats. At the beginning of the year, an epidemic caused by the new coronavirus SARS-CoV-2 causing Covid-19 began in China (Zhu et al. 2020). So far, more than three and a half million cases causing 250,000 deaths have been diagnosed globally. A total of 215 countries have reported infections.

Epidemiological data suggest that 21 % of all patients that are tested positive for SARS-CoV-2 require hospitalization with supplemental oxygen and 5% need intensive medical care including mechanical ventilation. The complete clinical spectrum with regard to Covid-19 is not fully known (Huang et al. 2020). Reported cases have ranged from very mild (including some with no reported symptoms) to severe, including illness resulting in death. While information so far suggests that most Covid-19 courses of disease are mild (81% of cases), severe disease was reported in 14%, a critical disease in 5% of cases (Z. Wu and McGoogan 2020). Older people and people of all ages with severe chronic medical conditions - like heart disease, lung disease and diabetes, for example - seem to be at higher risk of developing serious Covid-19 illness (Z. Wu and McGoogan 2020; F. Zhou et al. 2020).

The virus is mainly transmitted and easily spread from person-to-person via respiratory droplets (Wolfel et al. 2020). The virus starts replicating in the upper respiratory tract and can spread to the lower respiratory tract or target non-respiratory organs and cells. Typical symptoms can be fever, chills, dry cough, dyspnea and diarrhea, although symptoms may first appear 10-14 days post-infection. Clinical investigations show that also liver, kidney, heart, intestine, brain and lymphocytes can, in addition to the lung, be affected.

It is proposed that the virus directly promotes cell damage, whereby a systemic inflammatory response followed by multiple organ injury is triggered. CoVs also cause a dysfunctional renin- angiotensin system, which increases the pulmonary vascular permeability being a factor for the development pulmonary edema. Both systemic inflammatory responses and a dysfunctional renin-angiotensin system contribute to a so-called cytokine storm that triggers an acute respiratory distress syndrome (ARDS). Multi-organ failure and/or the onset of ARDS represent a severe state of SARS patients with a high mortality risk within few weeks or days.

The entry of SARS-CoV-2 into a target cell is mediated by binding of the viral spike protein via its receptor binding domain (RBD) to the human angiotensin converting enzyme-2 (ACE2) target receptor. Anti-viral substances aim blocking viral entry mediated via ACE2, viral replication, and virus release.

Several trials have been set up recently to test various anti-viral substances like Remdesivir (a substance originally developed against Ebola virus) that showed activity against SARS-CoV-2 in vitro and against SARS and MERS-CoV both in vitro and in animal studies (Sheahan et al. 2017; M. Wang et al. 2020).

A recent clinical trial with other anti-viral substances (a combination of Lopinavir and Ritonavir developed against HIV) showed no benefit over standard care in hospitalized adult patients with severe Covid-19 (Cao et al. 2020). To date, no proven pharmacological regime exists that could improve the natural course of the disease.

The entry of SARS-CoV-2 requires S protein priming by host cell proteases. Recently, it has been shown that the human serine protease TMPRSS2 is indispensable for S protein priming and virus entry. This mechanism can be blocked by a clinically approved TMPRSS2 inhibitor. This inhibitor, camostat, is approved in Japan and South Korea for treatment of chronic pancreatitis and reflux esophagitis. Camostat has shown to be effective in a SARS-CoV-1 animal model by blocking the same mechanism of action. Recent in vitro data show that uptake of SARS-CoV-2 is also inhibited in a dose dependent manner. Yet, it has not been studied to date in a prospective randomized clinical trial if camostat treatment could improve clinical outcome of Covid-19 patients.

Niclosamide is an oral anthelminthic drug (Yomesan®) which has been used to treat tapeworm infection for approximately 50 years. Researchers from the Institute of Virology at Charite - Universitatsmedizin Berlin found that highly pathogenic MERS-CoV limits the cellular recycling process (autophagy) via Beclin-1 degradation and that stabilization of the autophagy-initiating Beclin-1 protein effectively reduced propagation of MERS-CoV. Niclosamide was the most promising Beclin-1 -stabilizing drug reducing MERS-CoV growth in vitro up to 28,000-fold. Experiments performed by the inventors confirmed that niclosamide also inhibits SARS-CoV-2 in cell culture.

Despite the known roles of niclosamide and camostat, as anti-viral agents, their combinatorial administration for viral clearance by blocking viral cell entry and inducing autophagy has not been previously investigated in Covid-19 patients.

SUMMARY OF THE INVENTION

In light of the prior art, the technical problem underlying the present invention is to provide alternative or improved means for the treatment of viral infections. The technical problem may also be viewed as the provision of means for the treatment and/or reduction of risk of medical conditions associated with a SARS Coronavirus, more particular with SARS-CoV-2. The technical problem may also be viewed as the provision of means for the prevention and/or treatment of a SARS Coronavirus-associated respiratory disease.

This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to a pharmaceutical combination, comprising:

(a.) a therapeutically effective amount of an anti-viral protonophore, and

(b.) a therapeutically effective amount of a serine protease inhibitor.

The invention also relates to the combination for use in the treatment of a viral infection, such as Coronavirus infection, and/or for the treatment and/or prophylaxis of SARS Coronavirus- associated respiratory disease, and corresponding methods of treatment. The invention also relates to the combined administration of a therapeutically effective amount of an anti-viral protonophore and a therapeutically effective amount of a serine protease inhibitor in such treatment.

The present invention therefore represents a tailored intervention for restriction of virus entry and viral load, targeting in combination endosomal pH and autophagy by the anti-viral protonophore in combination with the serine protease inhibitor, a blocker for a host cell protease required for virus cell entry.

As described at length herein, the pharmaceutical combination comprising the anti-viral protonophore, and the serine protease inhibitor represents a novel and synergistic approach towards developing an effective Coronavirus therapeutic. The novel pharmaceutical combination described herein enables treatment of a disease, for which the medical community - in the context of the 2019-2020 SARS-CoV-2 pandemic - is in desperate need of means to prevent infections and treat patients. At present, there is no effective medication available.

The combined effect of the anti-viral protonophore and the serine protease inhibitor leads to an unexpected synergistic effect in reducing viral growth and load.

The synergy evident in this combined treatment with respect to reduced virus growth and viral load is quantitatively reproducible. By treating human lung cells with the combination of the invention, comprising an anti-viral protonophore and an serine protease inhibitor, virus growth is reduced to a greater extent than the sum of the effects of each compound when administered individually.

Furthermore, this quantitative synergy can be shown when assessed using in vitro experimental systems. The synergy appears therefore to translate into clinical settings, providing effective means in anti-viral treatment of mammalian, preferably human subjects.

In some embodiments, the quantitative synergy is can be observed for multiple anti-viral protonophores and multiple serine protease inhibitors. This supports the inventive synergy for the classes of active agents as described herein, without limitation to the particular agents tested.

The inventors have established that by changing pH homeostasis in endosomes and therefore inducing autophagy, beneficial effects in reducing virus propagation, such as SARS-CoV-2, can be achieved. By combining these effects with an inhibition of a serine protease that appears indispensable for virus entry, regardless of the particular pharmaceutical agents employed, synergistic therapeutic effects greater than the sum of the individual effects are achieved. Further detailed descriptions of the quantitative synergistic effects achieved by the combination are evident in the examples, and in Figure 1 .

In some embodiments, the respective doses of the anti-viral protonophore (preferably niclosamide or derivatives thereof) and the serine protease inhibitor (preferably camostat or derivatives thereof) can be reduced compared to usually administered doses. As shown in the example below, the synergistic effect of the combination of active agents enables lower doses to be administered, for example doses that appear non-efficacious when administered alone show efficacy when administered in the inventive combination. A skilled person could not have derived from common knowledge or the prior art that the inventive combination would allow a more effective and lower dosing of the active agents, thereby potentially maintaining or enhancing efficacy whilst potentially reducing side effects. As is evident from the experimental support provided herein, even low doses of the active agents, for example between 10-65% of the established maximum doses in humans for some active agents, may be employed. Viral clearance increased by 80-99% when administered in the inventive combination. Even when administered in low doses, the desired effect of an inhibition of viral growth remains greater than the sum of the effects of the individually dosed components, thereby supporting a synergistic effect.

In one embodiment, the anti-viral protonophore inhibits infection by a pH dependent virus, preferably a SARS Coronavirus, by increasing the pH in acidic endosomes of a target cell. The anti-viral protonophore, preferably niclosamide, reduces viral growth by each dose administered, as evident in the examples in Figure 1 . Viral clearance is increased by 80-99% when administered in the inventive combination.

Furthermore, the inventors recently found that SARS Coronavirus limits the cellular recycling process (autophagy) via Beclin-1 degradation and that the anti-viral protonophore is a Beclin-1 stabilizing agent, thereby reducing virus growth, such as SARS Coronavirus growth. The inventors have therefore identified a link between the anti-viral protonophore, Beclin-1 initiated autophagy, and virus growth (Hoffmann, Schroeder, et al. 2020). The anti-viral protonophore and autophagy inducer, preferably niclosamide, reduce virus growth by each dose administered, as evident in the examples in Figure 1. Viral clearance increased by 80-99% when administered in the inventive combination.

The serine protease inhibitor reduce infections with a virus, preferably Coronavirus, by blocking viral entry into cells. The inventors recently found that cell entry of Coronavirus, preferably SARS Coronavirus, depends on the viral spike protein priming by a serine protease of the host cell. The serine protease inhibitor, preferably camostat mesylate or GBPA, reduces virus growth by each dose administered, as evident in the examples and in Figures 1 to 3. Viral clearance increased by 85-99% when administered in the inventive combination.

A skilled person would not have expected these two molecular targets for virus growth to interact beyond a cumulative effect. Even if reduced virus growth, achieved by the anti-viral protonophore and the serine protease inhibitor when each is administered alone, has been derived from the art, a synergistic effect could not have been predicted based on the expectation of a skilled person. Typically, targeting two mechanisms with anti-viral effects would not typically lead to synergistic effects, but to no additional or negligible additive effects. Considering, both the anti-viral protonophore and the serine protease inhibitor reduce virus growth, a skilled person would expect the opposite as has been observed in the present invention. Without being bound by theory, a second (combined) factor would typically lead to no additional or at most a small cumulative effect, as targeting two positions in the anti-viral effects or related effects would typically not lead to further quantitative effects, as the virus growth has already been inhibited by the first agent, such that by targeting another position in the same or a related anti-viral effects by a second agent would not have an additional effect, but merely reinforce the effect already achieved by the first agent. Contrary to expectations, a surprising and quantitative synergy has been achieved by the anti-viral protonophore and the serine protease inhibitor.

In one embodiment, the invention relates to a pharmaceutical combination, comprising a. a therapeutically effective amount of an anti-viral protonophore, and b. a therapeutically effective amount of a serine protease inhibitor, wherein the agents a. and b. achieve a synergistic effect in reducing virus growth.

In one embodiment, the agents a. and b. achieve a synergistic effect in reducing viral growth in an in vitro cell culture assay, preferably as described in the example below.

In one embodiment, the agents a. and b. achieve a synergistic effect in reducing viral growth in an in vitro cell culture assay using human cells, preferably Calu-3 human lung cells. In one embodiment, the agents a. and b. achieve a synergistic effect in reducing viral growth in an in vitro cell culture assay using Calu-3 human lung cells infected with Coronavirus, preferably SARS-CoV-2 virus.

In one embodiment, the synergy is determined by quantifying viral growth. In other embodiments, synergy is determined using a quantification of viral genomic material. In other embodiments, synergy is determined by quantification of viral genomic material using real-time RT-PCR. In one embodiment, the synergy is determined by quantifying cell viability. In one embodiment, the synergy is determined by quantifying the viability of cells, preferably mammalian, human, and animal cells, infected with a virus, such as Coronavirus.

The combinations of these properties could not have been predicted by a skilled person and therefore relate to non-obvious, special technical features of the invention.

In one embodiment, the anti-viral protonophore increases the pH in acidic endosomes of a target cell.

Beneficial effects in viral clearance are achieved through the anti-viral protonophore. Anti-viral protonophores translocate protons, such as H+, across lipid bilayers, which are otherwise not able to pass. The simplest model is based on a weak acidic molecule, which in its anionic form is absorbed at the solution/membrane interface on the positive side of the membrane and binds an H+ there. Through interaction with the H+, the molecule becomes neutral and diffuses towards the negative side of the membrane. The H+ leaves the weak acid again, which then electrophoretically moves back in its anionic form to the more positive side of the membrane and the cycle starts again. The necessary driving forces are provided by the membrane potential and the proton concentration on both sides of the membrane. Viral infections are pH dependent if viruses require a pH range for cell entry through cellular compartments, such as endosomes.

(Sell et al. 2014)

The anti-viral protonophore described in this invention accumulates in cellular membranes, e.g. of endosomes. Enveloped viruses can enter the cell with the help of endocytic machinery. The endocyted virus particles undergo an activation step in the endosome that is typically mediated by the acidic endosomal pH. This leads to the fusion of the viral and endosomal membranes and the release of the viral genome into the cytosol. Viral infections are pH dependent if viruses require a pH range for cell entry through cellular compartments, such as endosomes. Targeting the pH in endosomes can therefore prevent growth of a pH dependent virus, whereby increasing the pH in acidic endosomes is an effective method for treating a subject infected with the pH dependent virus. Without being bound by theory, the anti-viral protonophore is intended to inhibit viral infection and growth by increasing the pH in acidic endosomes of a target cell.

In one embodiment, anti-viral protonophore increases the pH from an acidic range (below 7, preferably below 6.5, more preferably below 6) to an essentially neutral pH (about 7, for example between 6.5 and 7.5) or to an alkaline pH range (above 7, preferably above 7.5, more preferably above 8) in endosomes of a target cell. In some embodiments, a target cell is the cell infected with the pH dependent virus, such as endothelial cells, in particular cells in the vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and/or lung cells.

In some embodiments, the anti-viral protonophore inhibits infection of the target cell with the pH dependent virus, such as DNA viruses and RNA viruses including, but not limited to, influenza virus type A, influenza virus type B, human immunodeficiency virus, chikungunya virus, West Nile virus, Zika virus, Ebola virus, avian sarcoma virus, avian leukosis virus, Dengue virus, preferably a SARS Coronavirus, by increasing the pH in acidic endosomes of a target cell. The anti-viral protonophore, preferably niclosamide, reduces virus entry and virus growth by each dose administered, as evident in the examples in Figure 1. Viral clearance increased by 80-99% when administered in the inventive combination.

In some embodiments, the anti-viral protonophore is an agent that inhibits viral infections by pH dependent viruses. The anti-viral protonophore agents include, without limitation, niclosamide, valinomycin, nigericin, 2,4-dinitrophenol, chloroquine, carbonyl cyanide 3-chlorophenylhydrazone, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, tizoxanide, closantel, pyrazinoicacid, clofazimine, TMC207, benzoate, pyrazinocarbazole, clofazimine, bedaquiline, and SQ109, nitazoxanide, tizoxanide, lansoprazole, rabeprazole, omeprazole, and derivatives thereof, wherein any one or more selected from the group can be used in the pharmaceutical composition described herein for the prevention and treatment of viral infections in a subject. The anti-viral protonophore agents include structures as shown in Feng et al. (Feng et al. 2015).

In one embodiment, the anti-viral protonophore is a pharmaceutically acceptable compound.

The present invention also provides the pharmaceutical combination, wherein the anti-viral protonophore, preferably niclosamide, is used in the prevention or treatment of viral infections in a subject.

Additionally, the invention includes a method of monitoring, determining or quantifying whether the anti-viral protonophore, preferably niclosamide, inhibits virus growth, as described in the Example.

In one embodiment, the anti-viral protonophore is an autophagy inducer.

The onset and progression of a variety of viral infections involve certain autophagic processes that has led to the discovery of methods of treating and diagnosing such ailments targeting autophagy. Further, the pharmaceutical combination described herein includes an effective autophagy inducer in the treatment of viral infections in a subject. The present invention is directed to a method for the treatment of viral infections in a subject that are induced, increased or mediated through autophagy as a mechanism.

In one embodiment, an autophagy inducer is an autophagy inducing agent that induces the formation of an autophagosome in the cell. The autophagy inducing agents include niclosamide, rapamycin, perhexiline, amiodarone, rotrin, torinl , tori , P1103 [3- (4- ( 4-Morpholinyl) pyrido [3 ',

2': 4,5] furo [3,2-d] pyrimidin-2-yl) phenol], phenylethyl isothiocyanate, dexamethasone, lithium (lithium), L-690,330 [[1- (4-Hydroxyphenoxy) ethylidene] bisphosphonic acid], carbamazepine, sodium valproate, verapamil, loperamide , nimodipine, nitrendipine, niguldipine, niguldipine, nicardipine, pimozide, calpastatin, calpestatin, clonidine, clonidine, Rilmenidine, 2 ', 5'- dideoxyadenosine, NF449 [4,4', 4 ", 4 '"-[Carbonylbis (imino-5,1 , 3-benzenetriyl-b is (carbonylimino))] tetrakis -1 ,3-benzened isulfonic acid, octasodium salt], minoxidil, penitrem A, trehalose, spermidine, resveratrol, fluspirilene, trifluperazine ), SMER (small-molecule enhancer) 10, SMER 18, SMER 28 and dorsomorphin (dorsomorphin), wherein any one or more selected from the group can be used in the pharmaceutical composition described herein for the prevention and treatment of viral infections in a subject. (Choi, Bowman, and Jung 2018; KR20120131401A - Novel Use of Autophagy Inducer - Google Patents n.d.; Mao et al. 2019)

In one embodiment, the autophagy inducing agent is a pharmaceutically acceptable compound.

The present invention also provides the pharmaceutical combination, wherein the autophagy inducing agent, preferably niclosamide, is used in the prevention or treatment of viral infections in a subject.

Additionally, the invention includes a method of monitoring, determining or quantifying whether the autophagy inducer, preferably niclosamide, inhibits viral growth as described in the Example.

In one embodiment, the anti-viral protonophore is a salicylanilide or derivative thereof.

Salicylanilide, also known as 2-hydroxy-V-phenylbenzamide, is used as a topical antifungal and fungicide (US2485339A - Aqueous fungicidal dispersions of salicylanilide - Google Patents n.d.). Substituted salicylanilides have been shown to have decoupling activity (See SI3 in (Terada 1990)). Derivatives of salicylanilide include niclosamide, oxyclozanide, rafoxanide, dibromsalan, metabromsalan, and tribromsalan.

However, the vast majority of therapeutic development (especially as anthelmintic) occurred with substituted salicylanilides (such as niclosamine, SI3, oxyclozanide and rafoxanide) that were developed as anthelmintic drugs (Swan 1999).

Additionally, the salicylanilide and derivatives thereof are potent and low toxicity anti-viral protonophores

In one embodiment, the salicylanilide or derivative thereof, is a pharmaceutical acceptable compound.

The present invention also provides the pharmaceutical combination, wherein the salicylanilide or derivative thereof, preferably niclosamide, is used in the prevention or treatment of viral infections in a subject.

Additionally, the invention includes a method of monitoring, determining or quantifying whether the salicylanilide or derivative thereof, preferably niclosamide, inhibits viral growth as described in the Example.

In one embodiment, the anti-viral protonophore is niclosamide, or a derivative thereof.

Niclosamide, sold under the trade name Yomesan among others, is a medication used originally to treat tapeworm infestations. Niclosamide is also known as 5-Chloro-/V-(2-chloro-4-nitrophenyl)- 2-hydroxybenzamide. Niclosamide

Niclosamide has been shown to exhibit strong inhibitory effects in viral entry and growth. Niclosamide treatment blocks pH dependent viral entry by increasing acidic pH in endosomes of target cells. Furthermore, niclosamide treatment reduces viral growth by inducing autophagy through stabilizing the autophagy-initiating Beclin-1 protein. The inhibitory effect of niclosamide in virus growth was quantified in the in vitro cell culture experiment using human lung cell infected with SARS-CoV2, as shown in the Example below. (H. Wang et al. 2008) Various derivatives of niclosamide have been shown to be effective in viral growth inhibition, and are encompassed in the present application. The person skilled in the art knows various chemical methods and techniques to render a chemical substance to generate a derivate, which still comprises the chemical basis, such as addition, deletion or substitution of a group or functional group. Derivatives include those described in e.g. WO2012143377A1 (WO2012143377A1 - Niclosamide for the treatment of cancer metastasis - Google Patents n.d.) .

Examples of niclosamide derivatives are, without limitation:

Further examples of niclosamide derivatives are, without limitation: DK419, as disclosed in Wang et al (J. Wang et al. 2018):

Analog 11 or 32, as disclosed in Arend et al (Arend et al. 2016):

Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) of the arylalkanoic acid class that is marketed in the UK & U.S. by Merck as Clinoril. Like other NSAIDs, it is useful in the treatment of acute or chronic inflammatory conditions. In WO2011088474A2, the inventors show that Sulindac decreases viral infection in a subject. (WO2011088474A2 - Sulindac and sulindac derivatives and their uses related to infection - Google Patents n.d.)

Sulindac Calcimycin is an ionophorous, polyether antibiotic from Streptomyces chartreusensis. It binds and transports calcium and other divalent cations across membranes and uncouples oxidative phosphorylation while inhibiting ATPase of rat liver mitochondria. Calcimycin is also known as A23187, Calcium lonophore, Antibiotic A23187 and Calcium lonophore A23187. It is produced at fermentation of Streptomyces chartreusensis. An anti-viral effect has been demonstrated for Calcimycin. (Onishi, Natori, and Yamazaki 1991)

Calcimycin

Phenothiazines, such as trifluoperazine (TFP), have been shown to induce autophagy (Pharmacological Reports. PR, 01 Jan 2012, 64(1):16-23) and to inhibit inhibited SARS-CoV replication (Barnard et al. 2008).

Trifluoperazine

In one embodiment, the serine protease inhibitor is a trypsin-like serine protease inhibitor. A trypsin-like serine protease inhibitor inhibits a serine protease with trypsin-like activity. Serine serves as the nucleophilic amino acid at the enzyme's active site of the serine protease enzyme. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets for inhibition. Viral entry via endocytosis is dependent on target cell proteases, such as serine proteases.

Viral infections are target cell serine protease dependent if the virus requires a target cell serine protease for viral entry and growth.

The target cell serine protease activates the virus Spike protein required for fusion of the viral and target cell membranes and the release of the viral genome into the cytosol of the target cell. Targeting the host cell serine protease can prevent viral growth of an endocytosis dependent virus, whereby inhibiting the target cell serine protease is an effective method for treating a subject infected with the endocytosis dependent virus. The serine protease inhibitor, preferably a trypsin-like serine protease inhibitor, inhibits viral infection and virus growth.

As non-limiting examples, trypsin-like serine protease inhibitors include camostat, aprotinin, benzamidine, gabexate, leupeptin, nafamostat, pepstatin A, ribavirin, sepimostat, ulinastatin, and patamostat.

In some embodiments, the pharmaceutical combination described herein comprises two or more (or at least two) separate compounds. In some embodiments, the serine protease inhibitor may also be is a trypsin-like serine protease inhibitor (in some embodiments to some residual extent). In some embodiments, the trypsin-like serine protease inhibitor may also be a serine protease inhibitor (in some embodiments to some residual extent).

Preferred trypsin-like serine protease inhibitor have an inhibitory effect on virus entry and virus growth.

In some embodiments, preferred trypsin-like serine protease inhibitor reduces or blocks the transmembrane serine protease activity of a target cell.

In some embodiments, a target cell is the cell infected with the endocytosis dependent virus, such as endothelial cells, in particular cells in the vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and/or lung cells.

In some embodiments, the trypsin-like serine protease inhibitor inhibits infection of the target cell by the endocytosis dependent virus, such as DNA viruses and RNA viruses including, but not limited, influenza virus type A, influenza virus type B, human immunodeficiency virus, chikungunya virus, West Nile virus, Zika virus, Ebola virus, avian sarcoma virus, avian leukosis virus, Dengue virus, preferably a SARS Coronavirus, by increasing the pH in acidic endosomes of a target cell. The trypsin-like serine protease inhibitor, preferably camostat mesylate, reduces viral entry and growth by each dose administered, as evident in the examples and in Figures 1 to 3. Viral clearance is increased by 85-99% when administered in the inventive combination.

In some embodiments, the trypsin-like serine protease inhibitor is a pharmaceutically acceptable compound.

The present invention also provides the pharmaceutical combination, wherein the trypsin-like serine protease inhibitor, preferably camostat mesylate, is used in the prevention or treatment of viral infections in a subject. Additionally, the invention includes a method of monitoring, determining or quantifying whether the trypsin-like serine protease inhibitor, preferably camostat mesylate, inhibits viral growth as described in the Example.

In one embodiment, the serine protease inhibitor inhibits transmembrane serine proteases, preferably the TMPRSS2 serine protease.

Recently, it has been shown that the human transmembrane serine protease 2 (TMPRSS2) is indispensable for virus Spike protein priming and virus entry and that this mechanism can be blocked by a TMPRSS2 serine protease inhibitor (Y. Zhou et al. 2015).

Transmembrane proteases (TMPRSS) play a role in the cell entry of coronaviruses (Coronaviridae). The protease TMPRSS2, which is abundant in the respiratory tract and expressed on the cell surface, promotes the entry of SARS-Coronavirus. TMPRSS2 enters into a complex with the ACE2 receptor, which enables efficient entry of the SARS-Coronavirus directly at the cell surface. TMPRSS2 activates the spike protein by cleaving it into the S1 and S2 subunits, thus enabling endosome-independent cell entry at the cell membrane.

As non-limiting examples, transmembrane serine protease inhibitors include camostat, 4-(2- aminoethyl)benzolsulfonylfluorid-hydrochlorid, 3-Amidinophenylalanylderivate, aprotinin, gabexate, leupeptin, nafamostat, pepstatin A, ribavirin, sepimostat, ulinastatin, ovomucoid, patamostat, and derivatives thereof.

In some embodiments, the pharmaceutical combination described herein comprises two or more (or at least two) separate compounds. In some embodiments, the serine protease inhibitor may also be is a trypsin-like serine protease inhibitor (in some embodiments to some residual extent). In some embodiments, the trypsin-like serine protease inhibitor may also be a serine protease inhibitor (in some embodiments to some residual extent).

A preferred trypsin-like serine protease inhibitor has an inhibitory effect on transmembrane serine proteases, preferably TMPRSS2, for blocking virus entry and virus growth.

In some embodiments, the transmembrane serine proteases inhibitor is a pharmaceutically acceptable compound.

The present invention also provides a pharmaceutical combination, wherein the trypsin-like serine protease inhibitor, preferably the transmembrane serine proteases inhibitor, more preferably camostat mesylate, is used in the treatment and/or prevention of a viral infection in a subject.

Additionally, the invention includes a method of monitoring, determining or quantifying whether the transmembrane serine proteases inhibitor, preferably camostat mesylate, inhibits virus growth as described in the Example.

In one embodiment, the serine protease inhibitor is camostat or a salt thereof, preferably camostat mesylate.

Camostat is also known as N,N-dimethyl-carbamoylmethyl-p-(p- guanidinobenzoyloxy)phenylacetate. In some embodiments, a salt of camostat is selected from one or more of camostat mesylate, camostat hydrogen succinate, camostat succinate, camostat phosphate, camostat acetate, camostat hydrogen tartrate hemihydrate, camostat glycolate, camostat glycolate hemihydrate, camostat hippurate, camostat 1-hydroxy-2-naphthoate (xinafoate), camostat adipate, and camostat glutarate.

In some embodiments, camostat mesylate (Foipan®/FOY-305) is the preferred form of camostat in this invention and is a known trypsin-like serine protease inhibitor that has been used to treat symptoms of chronic pancreatitis and reflux esophagitis approved in Japan for nearly 30 years. (PCL 2009)

The inventors demonstrate that uptake of SARS-CoV-2 is also inhibited by Camostat in vitro in a dose dependent manner, as shown in Figures 1 to 3. The inventors performed a detailed analysis of SARS-CoV-2 cell entry mechanism into host cells and identified the membrane angiotensin converting enzyme II (ACE2) as cell entry receptors for SARS-CoV-2. (Hirano and Murakami 2020; Hoffmann, Kleine-Weber, et al. 2020) This mechanism depends on cellular serine protease TMPRSS2. TMPRSS2 for Spike protein activation and the protease is expressed in SARS-CoV-2 target cells throughout the human respiratory tract. SARS-CoV-2 - like SARS-CoV - exploits its spike protein to facilitate viral entry into target cells. Host cell proteases are required for S protein priming entailing S protein cleavage. TMPRSS2 and the endosomal cysteine proteases cathepsin B and L are used for S protein priming. SARS-CoV2 Spike protein driven entry into host cells could be inhibited by clinically proven TMPRSS2 inhibitor, Camostat mesylate.

Camostat mesylate treatment reduced human Calu-3 lung cell line infection with authentic SARS- CoV-2. Camostat mesylate inhibits virus growth > 90% (EC90) at 1mM concentrations, as shown in the Example. The combinatorial synergy with an anti-viral protonophore (such as niclosamide) also represents an unexpected finding, as a combined beneficial effect could not be derived from any suggestion in the art.

In some embodiments, the serine protease inhibitor is a camostat mesylate metabolite.

In the human body, camostat mesylate rapidly hydrolyzes into the active metabolite 4-(4- guanidinobenzoyloxy)phenylacetic acid (GBPA) and into the non-active metabolite 4- guanidinobenzoic acid (GBA). GBPA shows similar inhibitory effects on serine proteases as compared to camostat mesylate.

GBA

In one embodiment, the camostat mesylate metabolite is 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA).

The inventors demonstrate that uptake of SARS-CoV-2 is also inhibited by GBPA in vitro in a dose dependent manner, as shown in Figure 2. GBPA treatment reduced human Calu-3 lung cell line infection with authentic SARS-CoV-2. GBPA inhibits virus growth > 90% (IC90) at 400 nmol/l concentrations, as shown in the Example. The combinatorial synergy with an anti-viral protonophore (such as niclosamide) also represents an unexpected finding, as a combined beneficial effect could not be derived from any suggestion in the art.

In one embodiment, the invention relates to the pharmaceutical combination comprising:

(a.) niclosamide, and

(b.) camostat mesylate.

The pharmaceutical combination of niclosamide with camostat mesylate represent one preferred embodiment, for which synergistic effects have been demonstrated in the example below. Niclosamide treatment combined with camostat mesylate treatment required 2 to 10 times lower dosage for inhibiting virus growth > 90% (EC90). It was entirely surprising that the quantitative synergies in reducing virus entry and virus growth of SARS-CoV-2 could be achieved through this combination.

In one embodiment, the invention relates to the pharmaceutical combination comprising:

(a.) niclosamide, and (b.) GBPA.

The pharmaceutical combination of niclosamide with GBPA represent another preferred embodiment, for which synergistic effects have been demonstrated in the example below. Niclosamide treatment combined with GBPA treatment required about 8 times lower dosage of niclosamide for inhibiting virus growth > 90% (EC90). It was entirely surprising that the quantitative synergies in reducing virus entry and virus growth of SARS-CoV-2 could be achieved through this combination.

In one embodiment, the invention relates to (a.) the anti-viral protonophore and (b.) the serine protease inhibitor, preferably niclosamide and camostat mesylate, act synergistically to inhibit SARS-CoV infection of a target cell and/or replication of a SARS-CoV, preferably SARS-CoV-2. In one embodiment, the invention relates to (a.) the anti-viral protonophore and (b.) the serine protease inhibitor, preferably niclosamide and GBPA, act synergistically to inhibit SARS-CoV infection of a target cell and/or replication of a SARS-CoV, preferably SARS-CoV-2.

In embodiments of the invention, the pharmaceutical combination as described herein may be administered to various patient groups, for example therapeutically (e.g. to patients with SARS- related illnesses, including critically ill patients) or prophylactically (e.g. to patients who are at risk of having contracted a SARS Coronavirus infection, or to patients who are infected with a SARS Coronavirus but have not yet developed serious health issues).

A skilled person is capable, based on the experimental and written support provided herein, to carry out the invention, in combination with common knowledge in the art. By employing the quantitative in vitro assessment described in the examples, or by using alternative quantitative means for determining reductions in viral infection, replication and/or growth, the combination of pharmaceutical agents and their necessary doses can be determined and adjusted in order to obtain synergistic effects due to the inventive combination.

In one embodiment, the pharmaceutical combination described herein is administered to a patient with symptoms of a viral infectious disease.

In one embodiment, the pharmaceutical combination described herein is administered to a patient with symptoms of a viral infection of the respiratory tract.

In one embodiment, the pharmaceutical combination described herein is administered to a patient that is at risk of developing a severe acute respiratory syndrome (SARS).

In one embodiment, the pharmaceutical combination described herein is administered to an asymptomatic patient that shows no specific symptoms of SARS.

In one embodiment, the pharmaceutical combination described herein is administered to a patient that shows mild to moderate symptoms of SARS.

In one embodiment, the pharmaceutical combination described herein is administered to a patient that has or is at risk of developing a severe acute respiratory syndrome (SARS) and has a SARS Coronavirus infection.

In one embodiment, the pharmaceutical combination described herein is administered to a patient that suffers from an infection, such as with a SARS Coronavirus (SARS-CoV). In preferred embodiments, the SARS Coronavirus is SARS-CoV-2.

In some embodiments, (a.) the anti-viral protonophore and (b.) the serine protease inhibitor have relative amounts of 10000: 1 to 1 : 10000 by weight, preferably 10:1 to 1 :1 by weight,

As demonstrated in the example below, the relative concentrations of the combined agents tested show that no loss of synergy occurs when testing the agents at various relative concentrations.

As such, the invention encompasses any relative concentration and/or amount of the two classes of agents disclosed herein. In one embodiment, (a.) the anti-viral protonophore is niclosamide and (b.) the serine protease inhibitor is camostat mesylate, and (a.) and (b.) have relative amounts of 1000:1 to 1 :1 , preferably 100:1 to 1 :1 , more preferably 10:1 to 1 :1 , more preferably about 3.3:1.

In one embodiment, (a.) the anti-viral protonophore is niclosamide and (b.) the serine protease inhibitor is GBPA, and (a.) and (b.) have relative amounts of 1000:1 to 1 :1 , preferably 100:1 to 1 :1 , more preferably 10:1 to 1 :1 , more preferably about 3.3:1.

In one embodiment, the camostat mesylate metabolite GBPA can be administered to a patient in an equivalent amount to camostat mesylate described herein.

In one embodiment, the equivalent amounts of GBPA and camostat mesylate are determined by the weight ratio of the GBPA and camostat mesylate.

The above embodiments, regarding particular relative amounts, are based on clinically allowed or currently trialed doses of the various compounds described herein, being combined into a pharmaceutical combination as described herein.

In preferred embodiments, the agents a. and b. ((a.) the anti-viral protonophore and (b.) the serine protease inhibitor) are administered in concentrations or amounts sufficient to provide a therapeutic effect. A skilled person can determine such an effect without undue effort. This amount relates to a therapeutically effective amount when an agent is used alone, or to a therapeutically effective amount when an agent is used in combination with a second agent. In preferred embodiments, the agents are administered in concentrations or amounts, or according to dosage regimes, already established in the art, such as those for which regulatory approval has been issued (e.g. by the FDA or EMA), or in doses currently being assessed during phase 2 or 3 clinical trials, and/or according to the maximum allowed dose according to a phase I trial.

In such a treatment, agent (a.) and agent (b.) of the combination may be administered simultaneously or sequentially to said patient, agent (a.) being administered before or after agent (b.). Agent (a.) and agent (b.) may also be administered by the same or a different administration route.

In one embodiment, niclosamide is administered to a human subject in an amount of 100 mg to 5000 mg, preferably 500 mg to 3000 mg, more preferably 500 mg to 2000 mg, or 1000 mg to 2000 mg, or 1500 mg to 2500 mg, preferably about 1000 mg, 1500 mg, 2000 mg or 2500 mg per day. In preferred embodiments, the dose of niclosamide is an oral, daily dose of between 1000- 3000 mg, preferably between 1500 mg-2500 mg, more preferably 2000 mg.

In one embodiment, camostat mesylate is administered to a human subject in an amount of 100 mg to 1000 mg, preferably 200 mg to 900 mg, more preferably 300 mg to 600 mg, or 300 mg to 900 mg, or 200 mg to 600 mg, preferably about 200 mg, 300 mg, 600 mg, or 900 mg per day. In preferred embodiments, the dose of camostat mesylate is an oral, daily dose of between 100-900 mg, preferably between 300 mg-900 mg, more preferably 600 mg.

In one embodiment, GBPA is administered to a human subject in an amount of 100 mg to 1000 mg, preferably 200 mg to 900 mg, more preferably 300 mg to 600 mg, or 300 mg to 900 mg, or 200 mg to 600 mg, preferably about 200 mg, 300 mg, 600 mg, or 900 mg per day. In preferred embodiments, the dose of GBPA is an oral, daily dose of between 100-900 mg, preferably between 300 mg-900 mg, more preferably 600 mg.

In one embodiment, (a.) the anti-viral protonophore is in a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, and (b.) the serine protease inhibitor is in a separate pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, or

In one embodiment, (a.) the anti-viral protonophore and (b.) the serine protease inhibitor are present in a kit, in spatial proximity but in separate containers and/or compositions, or

In one embodiment, (a.) the anti-viral protonophore and (b.) the serine protease inhibitor are combined in a single pharmaceutical composition in admixture with a pharmaceutically acceptable carrier.

The invention further relates to the pharmaceutical combination for use in the treatment and/or prevention of a viral infection in a subject and/or a medical condition associated with a viral infection.

The invention therefore relates to a method for the treatment and/or prevention of a viral infection in a subject in need thereof, or a patient at risk of suffering from a viral infection. The method of treatment and/or prevention preferably comprises administering to said subject a therapeutically effective amount of the pharmaceutical combination, or the two agents of the combination, in order to obtain a therapeutic effect.

In one embodiment, the medical condition associated with a viral infection is a SARS Coronavirus infection.

In one embodiment, the invention relates to pharmaceutical combination as described herein for use in the treatment and/or prevention of a medical condition associated with a SARS Coronavirus.

In one embodiment, the medical condition associated with a SARS Coronavirus is COVID-19.

In one embodiment, the medical condition associated with a SARS Coronavirus is a SARS Coronavirus-associated respiratory disease.

The invention therefore relates to corresponding methods of treatment, comprising the administration of the pharmaceutical combination as described herein to a subject in need thereof in order to treat and/or prevent a medical condition associated with a SARS Coronavirus.

A further aspect of the invention therefore relates to an anti-viral protonophore, preferably niclosamide, for use as a medicament in the treatment and/or prevention of a viral infection, preferably for the treatment and/or prevention of Coronavirus infection, or a particular SARS Coronavirus-associated respiratory disease disclosed herein, wherein said treatment and/or prevention comprises the combined administration of a serine protease inhibitor, preferably camostat mesylate or GBPA.

In one embodiment, the virus is a pH-dependent virus, wherein infection of a target cell with the virus is dependent on pH. In some embodiments, the pharmaceutical combination, or the two agents of the combination, for use in the treatment and/or prevention of an infection of the target cell by a pH dependent virus, such as DNA viruses and RNA viruses including, but not limited, influenza virus type A, influenza virus type B, human immunodeficiency virus, chikungunya virus, West Nile virus, Zika virus,

Ebola virus, avian sarcoma virus, avian leukosis virus, Dengue virus, preferably a SARS Coronavirus, by increasing the pH in acidic endosomes of a target cell. The pharmaceutical combination of an anti-viral protonophore, preferably niclosamide, and a serine protease inhibitor, preferably camostat mesylate or GBPA, reduces virus entry and virus growth by each dose administered, as evident in the examples in Figure 1 to 3. Viral clearance increased by 80-99% when administered in the inventive combination. As evident in Figure 2, SARS-Cov-2 RNA copy numbers decreased up to 10.000-fold for niclosamide combined with camostat mesylate at 800 nmol/l concentration. The combinatorial effect with the anti-viral protonophore niclosamide and the serine protease inhibitor camostat mesylate also represents an unexpected finding, as this combined beneficial effect could not be derived from any suggestion in the art.

In some embodiments, a target cell is the cell infected with the pH dependent virus, such as endothelial cells, in particular cells in the vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and/or lung cells.

In embodiments of the invention, the pharmaceutical combination as described herein may be administered to various patient groups, for example therapeutically (e.g. to patients with SARS- related illnesses, including critically ill patients) or prophylactically (e.g. to patients who are at risk of having contracted a SARS Coronavirus infection, or to patients who are infected with a SARS Coronavirus but have not yet developed serious health issues).

In one embodiment, the virus is a SARS-CoV virus, preferably SARS-CoV-2.

In one embodiment, the pharmaceutical combination described herein is administered to a patient with symptoms of an infectious disease.

In one embodiment, the pharmaceutical combination described herein is administered to a patient with symptoms of a viral infection of the respiratory tract.

In one embodiment, the pharmaceutical combination described herein is administered to an asymptomatic patient that shows no specific symptoms of severe acute respiratory syndrome (SARS).

In one embodiment, the pharmaceutical combination described herein is administered to a patient that has or is at risk of developing SARS and has a SARS Coronavirus infection.

In one embodiment, the pharmaceutical combination described herein is administered to a patient that suffers from an infection, such as with a SARS Coronavirus. In preferred embodiments, the SARS Coronavirus is SARS-CoV-2.

The invention therefore relates to corresponding methods of treatment, comprising the administration of the pharmaceutical combination as described herein to a subject in need thereof in order to treat and/or prevent a medical condition associated with a SARS Coronavirus. In a preferred embodiment the pharmaceutical combination for use as a medicament as described herein is characterized in that the treatment of viral infections comprises the treatment of a subject with detectable viral genome in target cells compared to a control, such as healthy controls.

In some embodiments, the target cell for SARS Coronavirus include endothelial cells, in particular cells in the vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and/or lung cells.

Additionally, the pharmaceutical combination of the present invention is used for pre- and/or postexposure prophylaxis of the disease. The pharmaceutical combination described herein can be used as a prophylactic in groups of patients at risk of COVID-19 infection (such as health care workers), persons at risk of infection after proven contact to an infected individual, in patients who have been infected but do not yet show disease symptoms, or patients at high risk of a severe course or adverse event (e.g. patients with cancer or immunosuppression).

In a further aspect, viral infections and drug-induced therapy efficacy can also be detected and quantified by an in vitro method for determining the presence or absence of SARS-CoV-2 viral genomic material in a sample. Detection of viral genomes or parts thereof can be carried out by amplifying the genetic viral material by means of a polymerase chain reaction (PCR), as shown in the Example. By way of example, a method may be employed selected from the group consisting of nucleic acid amplification methods, such as PCR, qPCR, RT-PCR, qRT-PCR.

The invention therefore relates to methods of treatment and/or prevention of viral infections and patient groups described above. The invention also relates to combined methods of diagnostics and treatment based on viral genomes detected and subsequent viral clearance, respectively.

The present invention further relates to a method for the treatment and/or prevention of viral infection, or a particular group of patients infected with a particular virus as described herein, in a human subject, such as a subject with SARS Coronavirus infection as described herein, comprising: i. having a sample, such as a biological fluid or a swab, ii. having an assay conducted on the sample, said assay comprising determining levels of viral genomes in the sample, for example as described for the diagnostic assays described above, and comparing viral genomes levels to a control sample, and iii. when viral genomes are detected, treating the subject in order to clear viral genomes, said treatment comprising the administration of a pharmaceutical combination as described herein.

The features of the invention regarding methods of treatment and diagnosis, and descriptions of the pharmaceutical combination for use in the treatment of various medical conditions, apply to the pharmaceutical combination itself, and vice versa.

DETAILED DESCRIPTION OF THE INVENTION

Pharmaceutical Combination: According to the present invention, a “pharmaceutical combination” preferably relates to the combined presence of an inhibitor of the anti-viral protonophore with a serine protease inhibitor, i.e. in proximity to one another, or the use of the two components in combined administration. In one embodiment, the combination is suitable for combined administration.

In one embodiment, the pharmaceutical combination as described herein is characterized in that the anti-viral protonophore is in a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, and the serine protease inhibitor is in a separate pharmaceutical composition in admixture with a pharmaceutically acceptable carrier. The pharmaceutical combination of the present invention can therefore in some embodiments relate to the presence of two separate compositions or dosage forms in proximity to each other. The agents in combination are not required to be present in a single composition.

In one embodiment, the pharmaceutical combination as described herein is characterized in that the inhibitor of the anti-viral protonophore and the serine protease inhibitor according to any one of the preceding claims are present in a kit, in spatial proximity but in separate containers and/or compositions. The production of a kit lies within the abilities of a skilled person. In one embodiment, separate compositions comprising two separate agents may be packaged and marketed together as a combination. In other embodiments, the offering of the two agents in combination, such as in a single catalogue, but in separate packaging is understood as a combination.

In one embodiment, the pharmaceutical combination as described herein is characterized in that the inhibitor of the anti-viral protonophore and the serine protease inhibitor according to any one of the preceding claims are combined in a single pharmaceutical composition in admixture with a pharmaceutically acceptable carrier. Combination preparations or compositions are known to a skilled person, who is capable of assessing compatible carrier materials and formulation forms suitable for both agents in the combination.

Host cell and target cell:

As used herein, the terms “host cell” and “target cell” refer to a living cell into which an infectious agent (e.g. a virus) has invaded or is capable of invading.

Inhibitors:

According to the present invention, an “inhibitor”, for example in the context of “a serine protease inhibitor”, is considered any agent, substance, compound, molecule or other means leading to a slowing, repressing, blocking or other interfering or negative action on the activity, function, expression of or signaling caused by the named target. In some embodiments, references to the “agents” in the context of the combinations and methods described herein are to be understood as the serine protease inhibitor. Preferred inhibitors are those described herein.

Proteases are enzymes that can cleave proteins or peptides by hydrolysis of the peptide bonds. Proteases are most commonly classified according to their mechanism of action. Serine proteases exhibit a serine in the catalytic site which forms a covalent ester intermediate during the catalytic reaction pathway by a nucleophilic attack on the carboxy carbon of the peptide bond. In the active site of serine proteases, a catalytic triad comprised of an aspartate, a histidine and the above-mentioned serine is found functioning as a charge relay system. The serine protease family includes, for example, the digestive enzymes trypsin and chymotrypsin, components of the complement cascade, enzymes involved in the blood clotting cascade and enzymes involved in the breakdown, reconstruction and maintenance of components of the extracellular matrix. A characteristic of the serine protease family is the broad spectrum of substrate specificity.

Members of the trypase subgroup cleave to arginine or lysine, chymases to phenylalanine or leucine, aspases to aspartate, metases to methionine and serases to serine. Trypsin-like proteases cleave peptide bonds following a positively charged amino acid (lysine or arginine).

This specificity is typically considered to be driven by the residue which lies at the base of the enzyme's S1 pocket (generally a negatively charged aspartic acid or glutamic acid).

A serine-protease inhibitor is a substance that leads to any inhibition in the activity of a serine protease. Suitable methods for determining the inhibition of a serine protease are known in the art. For example, a serine protease inhibitor may affect serine protease function, serine protease expression (transcription or translation) and/or serine protease-mediated signaling, either directly or indirectly.

A trypsin-like serine protease inhibitor is a protease inhibitor that has a function essentially the same or similar to trypsin. The term trypsin-like serine protease inhibitor is a known term in the art. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets. Important examples include thrombin, factor Vila, factorXa, and b-tryptase. Alternatively such agents may be termed chymotrypsin-like. Functional tests are known to a skilled person to determine the activity of a trypsin-like serine protease inhibitor.

Synergy:

To determine or quantify the degree of synergy or antagonism obtained by any given combination, a number of models may be employed. Typically, synergy is considered an effect of a magnitude beyond the sum of two known effects. In some embodiments, the combination response is compared against the expected combination response, under the assumption of non interaction calculated using a reference model (refer Tang J. et al. (Tang, Wennerberg, and Aittokallio 2015))

Commonly utilized reference models include the Highest single agent (HSA) model (Berenbaum 1989), the Loewe additivity model (LOEWE 1953), the Bliss independence model (BLISS 1939), and more recently, the Zero interaction potency (ZIP) model (Yadav et al. 2015). The assumptions being made in these reference models are different from each other, which may produce somewhat inconsistent conclusions about the degree of synergy. Nevertheless, according to the present invention, when any one of these models indicates synergy between the agents in the combination as described herein, it may be assumed synergy has been achieved. Preferably, 2, 3 or all 4 of these models will reveal synergy between any two agents of the combination described herein.

Without limitation, four reference models are preferred, which can produce reliable results: (i)

HSA model, where the synergy score quantifies the excess over the highest single drug response; (ii) Loewe model, where the synergy score quantifies the excess over the expected response if the two drugs are the same compound; (iii) Bliss model, where the expected response is a multiplicative effect as if the two drugs act independently; and (iv) ZIP model, where the expected response corresponds to an additive effect as if the two drugs do not affect the potency of each other.

The most widely used combination reference, and preferred model for determining synergy, is “Loewe additivity”, or the “Loewe model” (Loewe 1928; LOEWE 1953; Loewe and Muischnek 1926), or "dose additivity" which describes the trade-off in potency between two agents when both sides of a dose matrix contain the same compound. For example, if 50% inhibition is achieved separately by 1 uM of drug A or 1 uM of drug B, a combination of 0.5 uM of A and 0.5 uM of B should also inhibit by 50%. Synergy over this level is especially important when justifying the clinical use of proposed combination therapies, as it defines the point at which the combination can provide additional benefit over simply increasing the dose of either agent.

As a further example of determining Loewe Additivity (or dose additivity), let di and <¼ be doses of compounds 1 and 2 producing in combination an effect e. We denote by D_ei and D_e 2 the doses of compounds 1 and 2 required to produce effect e alone (assuming these conditions uniquely define them, i.e. that the individual dose-response functions are bijective). d_ei/D_e2 quantifies the potency of compound 1 relatively to that of compound 2. d2D_ei/D_e2 can be interpreted as the dose of compound 2 converted into the corresponding dose of compound 1 after accounting for difference in potency. Loewe additivity is defined as the situation where di + d2D_ei/D_e2 = D_ei or di/D_ei + d2/D_e2 = 1. Geometrically, Loewe additivity is the situation where isoboles are segments joining the points (D_ei, 0) and (0, D_e 2) in the domain (di, 0/2). If we denote by fi(di), ¾(¾) and the dose-response functions of compound 1 , compound 2 and of the mixture respectively, then dose additivity holds when di/fi¹ (fn (di, c/2)) + d_å/f2 ¹ (fi2 (di, c/2)) = 1.

Combined administration:

According to the present invention, the term “combined administration”, otherwise known as co administration or joint treatment, encompasses in some embodiments the administration of separate formulations of the compounds described herein, whereby treatment may occur together, within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Alternating administration of two agents is considered as one embodiment of combined administration. Staggered administration is encompassed by the term combined administration, whereby one agent may be administered, followed by the later administration of a second agent, optionally followed by administration of the first agent, again, and so forth. Simultaneous administration of multiple agents is considered as one embodiment of combined administration. Simultaneous administration encompasses in some embodiments, for example the taking of multiple compositions comprising the multiple agents at the same time, e.g. orally by ingesting separate tablets simultaneously. A combination medicament, such as a single formulation comprising multiple agents disclosed herein, and optionally additional anti-cancer medicaments, may also be used in order to co-administerthe various components in a single administration or dosage. A combined therapy or combined administration of one agent may occur together or precede or follow treatment with the other agent to be combined, by intervals ranging from minutes to weeks. In embodiments where the second agent and the first agent are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the first and second agents would still be able to exert an advantageously combined synergistic effect on a treatment site. In such instances, it is contemplated that one would contact the subject with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other, with a delay time of only about 12 h being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In the meaning of the invention, any form of administration of the multiple agents described herein is encompassed by combined administration, such that a beneficial additional therapeutic effect, preferably a synergistic effect, is achieved through the combined administration of the two agents.

Protonophore:

The term “protonophore”, also called an ionophore, refers to a compound or other agent that conducts the process by which protons move across lipid bilayers or other type of membranes via an ATP synthase independent pathway. Protons (H+) are generally not feasible to pass lipid bilayers and membranes without an active transporter molecule. This process can be pharmacologically induced by the negatively charged small molecule protonophore, which transfers protons directly to the lipid bilayers passing them to the inner or outer membrane side. The protonophore can diffuse in a neutral and charged state by passive diffusion over the lipid bilayer and enables proton transport. This process creates a pH and an electrochemical gradient across the membrane. The inner- and the outer membrane pH change from acidic to neutral to basic or vice versa (Sell et al. 2014). An anti-viral protonophore relates therefore to a protonophore that shows an inhibition of viral infection, replication or viral growth, when assessed in an appropriate model, such as described in the examples below.

Therapeutically effective:

As used herein to refer to the compounds, compositions, combinations and / or dosage forms suitable for use in contact with a subject that produces a result that in and of itself helps to treat and/or cure a medical condition, or for example show other beneficial effect against a medical condition, prevention of onset of a disease, condition or symptom in a patient, inhibition of a symptom of a condition, prevention of progression of the symptom, amelioration of a symptom of a condition, or induction of regression of the disease and/or symptom. The phrase "therapeutically effective" is intended to include, in some embodiments and within the scope of sound medical judgment, some toxicity, irritation, allergic reactions, and/or other problems or complications, but commensurate with a reasonable benefit/risk ratio.

Endosomes: Endosomes are membrane-bound intracellular transport carriers in eukaryotic cells and play key roles in the cellular infection cycles of many viruses. Viruses, in particular enveloped viruses, can productively enter cells following endocytosis and transport to endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease plus low pH.

Autophaqy:

Autophagy is used as a term for killing and regeneration of intracellular organelles, endosomes, and proteins by the cell itself. Degenerative proteins or organelles, whose lifespan is shortened or whose function is impaired, are isolated in vesicles called autophagosomes within the cells, and these vesicles are in turn lysosomes. In the lysosomes, it is broken down by digestive enzymes. Autophagy is an essential function in maintaining the quality of intracellular proteins and has been implicated in many diseases, including viral infections. As intracellular parasites, during the course of an infection, intriguing viruses encounter autophagy and interact with the proteins that execute this process by directly eliminating them in autophagosomes.

Agents that modulate autophagy can be determined a person skilled in the art without undue effort. Cell cultures are one of the most frequently used experimental setup for the investigation of autophagy. Orhon et al (Cells. 2017 Sep; 6(3): 20) summarize established methods that are most frequently used to assess autophagy induction and progression, and include monitoring the degradation of a group of autophagosomal cargoes, autophagosome formation through the measurement of LC3-II levels is also possible.

TMPRSS2:

"TMPRSS2" refers to the gene and its products (e.g. RNA and protein) commonly known as transmembrane protease serine 2. The mRNA sequences of several TMRPSS2 variants are known and include e.g. the GenBank accession numbers NMJD01199.1 (transcript variant 1) and NMJD05656.3 (transcript variant 2) (see e.g. Han et al., 2009; Paoloni-Giacobino et al., 1997). TMPRSS2 enables infection with the human coronaviruses SARS-CoV and SARS-CoV-2 via two independent mechanisms, the proteolytic cleavage of the ACE2 receptor that promotes virus uptake and the cleavage of coronavirus spike glycoproteins that activate the glycoprotein for entry into the host cell (Heurich et al. 2014; Hoffmann, Kleine-Weber, et al. 2020). TMPRSS2 proteolytically cleaves and activates the spike glycoproteins of human coronavirus 229E (HCoV- 229E) and human coronavirus EMC (HCoV-EMC) and the fusion glycoproteins F0 of Sendai virus (SeV), human metapneumovirus (HMPV), human parainfluenza viruses 1 , 2, 3, 4a and 4b (HPIV). TMPRSS2 is essential for the spread and pathogenesis of influenza A virus (strains H 1 N 1 , H3N2 and H7N9); involved in the proteolytic cleavage and activation of the haemagglutinin (HA) protein essential for viral infectivity. The entry of SARS-CoV-2 requires S protein priming by host cell proteases. Recently, it has been shown that TMPRSS2 is indispensable for S protein priming and virus entry. TMPRSS2 for S protein activation and the protease are expressed in SARS-CoV-2 target cells throughout the human respiratory tract. SARS-CoV-2 - like SARS-CoV - exploits its spike (S) protein (called SARS-S-2 in SARS-CoV-2) to facilitate viral entry into target cells. Host cell proteases are required for S protein priming entailing S protein cleavage. TMPRSS2 and the endosomal cysteine proteases cathepsin B and L (CatB/L) are used for S protein priming.

Camostat: The entry of SARS-CoV-2 requires S protein priming by host cell proteases. Recently it has been shown that the human serine protease TMPRSS2 is indispensable for S protein priming and virus entry and that this mechanism can be blocked by a clinically approved TMPRSS2 inhibitor. (Y. Zhou et al. 2015) The TMPRSS2 inhibitor referred to is “Camostat” (Foipan®/FOY-305) approved in Japan for treatment of chronic pancreatitis and reflux esophagitis for 30 years. Camostat has been shown to effectively block viral replication in a SARS-CoV-1 animal mode (PCL 2009; Y. Zhou et al. 2015). Recent in vitro data show that uptake of SARS-CoV-2 is also inhibited by Camostat in a dose dependent manner (Hirano and Murakami 2020; Hoffmann, Kleine-Weber, et al. 2020). The authors performed a detailed analysis of SARS-CoV-2 cell entry mechanism into host cells and identified the membrane protein and inactivator of angiotensin 2 ACE2 (angiotensin-converting enzyme II) as cell entry receptors for SARS-CoV-2. This mechanism depends on cellular serine protease TMPRSS2. TMPRSS2 for S protein activation and the protease is expressed in SARS-CoV-2 target cells throughout the human respiratory tract. SARS- CoV-2 - like SARS-CoV - exploits its spike (S) protein (called SARS-S-2 in SARS-CoV-2) to facilitate viral entry into target cells. Host cell proteases are required for S protein priming entailing S protein cleavage. TMPRSS2 and the endosomal cysteine proteases cathepsin B and L (CatB/L) are used for S protein priming. SARS-2-S-driven entry into host cells could be inhibited by clinically proven TMPRSS2 inhibitor, camostat mesylate.

GBPA:

GBPA is a metabolite of camostat. It has been recently shown that camostat mesylate rapidly hydrolyzes into the active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and into non-active 4-guanidinobenzoic acid (GBA) in patients.

In one embodiment, the hydrolyzed active moiety of camostat mesylate, GBPA, can be administered in an equivalent amount to camostat mesylate which is determined by the weight ratio of the two molecules.

The inhibition of recombinant TMPRSS2 by camostat mesylate, GBPA and GBA was studied by Hoffman et al. 2021 (Hoffmann M, Hofmann-Winkler H, Smith JC, Kruger N, S0rensen LK, S0gaard OS, Hasselstram JB, Winkler M, Hempel T, Raich L, Olsson S, Yamazoe T, Yamatsuta K, Mizuno H, Ludwig S, Noe F, Sheltzer JM, Kjolby M, Pohlmann S. camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity. EBioMedicine. 2021 Mar 3;:103255)

FOY-251 , a methanesulfonate of GBPA, showed a 10-fold reduced capacity to inhibit TMPRSS2 as compared to camostat mesylate, although both compounds completely suppressed TMPRSS2 activity at 1 mM or higher. FOY- 251 blocks TMPRSS2 activity but with reduced efficiency as compared to camostat mesylate. In order to obtain insights into the reduced inhibitory activity of FOY-251 , we investigated TMPRSS2 inhibition by GBPA on the molecular level. For this, a combination of extensive all-atom molecular dynamics simulations and Markov modeling of the TMPRSS2-GBPA complex was used. Guanidinobenzoate-containing drugs such as camostat mesylate and GBPA inhibit TMPRSS2 by first forming a noncovalent precomplex which is then catalyzed to form a long-lived covalent complex that is the main source of inhibition. The population of the short-lived precomplex directly relates to the inhibitory activity. By computing the TMPRSS2- GBPA binding kinetics, it was found that (i) the noncovalent TMPRSS2-GBPA complex is metastable, rendering it suitable to form a covalent inhibitory complex, and (ii) its population is 40% lower compared to camostat at equal drug concentrations, consistent with the finding that FOY-251 is a viable but less potent inhibitor. Structurally, GBPA binds in the same manner as camostat. The main stabilizing interaction is its guanidinium group binding into TMPRSS2’s S1 pocket, which is stabilized by a transient salt bridge with Asp-435. The GBPA ester group can interact with the catalytic Ser-441 , making it prone for catalysis and formation of the catalytic complex. The slightly lower stability of the GBPA compared to the camostat mesylate-TMPRSS2 complex is consistent with GBPA’s shorter tail, which has less possibilities to interact with the hydrophobic patch on the TMPRSS2 binding site. Thus, camostat mesylate and GBPA exerted roughly comparable antiviral activity, likely due to conversion of camostat mesylate into GBPA.

Niclosamide:

Niclosamide is an approved drug for the treatment of tapeworm infection for more than 50 years. The highest approved dose of Niclosamide is 2000 mg on Day 1 , followed by 1000 mg/d up to six days. In addition to its current use as a treatment of tapeworm infection, Niclosamide has also found a novel role in cancer therapy and as an anti-viral agent (Xu et al. 2020).

Drug-induced modulation of cellular pathways like autophagy were shown to broadly affect virus growth (Lundin et al. 2014; Pfefferle et al. 2011). The inventors recently found that highly pathogenic MERS-CoV limits the cellular recycling process (autophagy) via Beclin-1 degradation and that stabilization of the autophagy-initiating Beclin-1 protein effectively reduced propagation of MERS-CoV (Gassen et al. 2019). Niclosamide was the most promising Beclin-1 -stabilizing drug reducing MERS-CoV growth in vitro up to 28,000-fold. In ensuing experiments, it was shown that SARS-CoV-2, similar to MERS-CoV, limits autophagy. Furthermore, Niclosamide reduced SARS-CoV-2 propagation up to 16,000-fold in cell cultures. The IC50 of Niclosamide was 0.17 mM (R2= 0.63) at 48 hours post SARS-CoV-2 infection, being well below the observed Niclosamide serum levels of 0.76-18.35 pM upon oral uptake of 2000 mg. These observations were recently confirmed and pre-published by a Korean research team (Jeon et al. 2020). In a recent study performed by inventors it was confirmed that Niclosamide also potently inhibits SARS-CoV-2 (Hoffmann, Schroeder, et al. 2020; Jeon et al. 2020). In some embodiments, the anti-viral protonophore is niclosamide, or pharmaceutically effective salts, esters or derivatives thereof.

Viral infection and viral load:

As used herein, the term "viral infection" describes a disease state in which a virus invades healthy cells. Viruses hijack use the cell's reproductive machinery to multiply or replicate and finally dissolves the cell, leading to cell death, the release of viral particles and infection of other cells by the newly produced progeny viruses. The term “viral load” refers to a quantitative measure of viral genomes per invaded cell. The determination of viral genomic material may be employed in such a method. A latent infection by certain viruses is also a possible consequence of a viral infection. The term “viral growth” relates to the infection and replication of a virus and the production of viral particles during and after the infection of a host cell. The Coronavirus Spike protein, also known as S protein, is a glycoprotein trimer, wherein each monomer of the trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively. As described in Xiuyuan Ou et al (Ou et al. 2020), Coronaviruses (CoV) use the Spike glycoprotein to bind ACE2 and mediate membrane fusion and virus entry. In the S protein structure, N- and C- terminal portions of S1 fold as two independent domains, N-terminal domain (NTD) and C-terminal domain (C-domain). Depending on the virus, either the NTD or C-domain can serve as the receptor-binding domain (RBD). While RBD of mouse hepatitis virus (MHV) is located at the NTD14, most of other CoVs, including SARS-CoV and MERS-CoV use C-domain to bind their receptors.

Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The spike protein, which has been imaged at the atomic level using cryogenic electron microscopy, is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell.

The receptors for SARS-CoV and MERS-CoV are human angiotensin-converting enzyme 2 (hACE2) and human dipeptidyl peptidase 4 (hDPP4), respectively. CoV S proteins are typical class I viral fusion proteins, and protease cleavage is required for activation of the fusion potential of S protein. A two-step sequential protease cleavage model has been proposed for activation of S proteins of SARS-CoV and MERS-CoV, priming cleavage between S1 and S2 and activating cleavage on S2’ site. Depending on virus strains and cell types, CoV S proteins may be cleaved by one or several host proteases, including furin, trypsin, cathepsins, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4, or human airway trypsin-like protease (HAT). Availability of these proteases on target cells largely determines whether CoVs enter cells through plasma membrane or endocytosis.

Angiotensin-converting enzyme 2 (ACE2) is a cell membrane linked carboxypeptidase presented on the outer surface (cell membranes) of cells in the vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and lung. ACE2 has the function of lowering blood pressure by catalyzing the hydrolysis of angiotensin II into angiotensin. ACE2 counters the activity of the related angiotensin-converting enzyme (ACE) making it a drug target for treating cardiovascular diseases. ACE2 is a which is expressed in. ACE2 also serves as the entry point into cells for some coronaviruses including Severe acute respiratory syndrome coronavirus 2.

ACE2 is a zinc containing metalloenzyme that contains an N-terminal peptidase M2 domain and a C-terminal collectrin renal amino acid transporter domain. ACE2 is a single-pass type I membrane protein, with its enzymatically active domain exposed on the surface of cells. The extracellular domain of ACE2 is cleaved from the transmembrane domain by another enzyme known as sheddase, and the resulting soluble protein is released into the blood stream and ultimately excreted into urine.

Angiotensin converting enzyme 2 receptor (ACE2) is expressed at the surface of epithelial cells. ACE2 is a cell membrane linked carboxypeptidase which is expressed in vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and lung. As one consequence binding of the virus leads to an epithelial and endothelial cell damage along with vascular leakage, which triggers a secretion of pro-inflammatory cytokines and chemokines. The virus also mediates ACE2 downregulation and shedding which further promotes a dysfunctional renin-angiotensin system (RAS). Once the RAS is disturbed it can lead an inflammatory response and to vascular permeability. Focusing on the respiratory system, ACE2 shedding can lead to pulmonary vascular permeability and subsequently to pulmonary edema. pH dependent virus:

As used herein, the term “pH dependent virus” describes a group of viruses that enter the cell by the cellular endosomal route in a pH dependent manner. The endosomal pH, usually an acidic pH, triggers fusion of virus to cell membranes. Changes in endosomal pH reduces viral infection of the target cell.

Details of a fusion process being dependent on pH were initially described for influenza HA, where a low pH value is sufficient to trigger all steps of the fusion cascade. A low pH is also sufficient to trigger fusion proteins of the alpha, arena, bunya, flavi and rhabdovirus. For viruses activated solely by a low pH, fusion in early or late endosomes is generally determined by pH dependence for important conformational changes in the viral fusion protein; those with higher pH thresholds generally fuse in earlier endosomes than those requiring a lower pH. A list of virus families with viral membrane fusion proteins and pH dependence is provided. Further reference may be obtained from Traffic. 2016 Jun; 17(6): 593-614. In some embodiments, the pH dependent virus is selected from Orthomyxoviridae, Paramyxoviridae, Retroviridae,

Coronaviridae, Arenaviridae, Filoviridae, Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae, Herpesviridae, Bornaviridae, Poxviridae, Asfarviridae, Arteriviridae, or Hepadnaviridae.

Some virus types require priming of viral fusion proteins by binding to a receptor, followed by the action of a protease, which may or may not be low pH-dependent. An exemplarily list of virus with pH triggered endosomal viral fusion is provided. Such triggering mechanisms are used by many coronaviruses and the paramyxovirus respiratory syncytial virus (RSV). SARS-CoV has been extensively studied in this respect. After its interaction with the ACE2 receptor, SARS-CoV is activated by proteolytic cleavage; cathepsin L activates the virus in late endosomes pH- dependent, but the virus can also be activated pH-independently by trypsin-like proteases TMPRSS2 on the cell surface. As first identified with SARS-CoV 208, coronaviruses are somewhat unusual in that they have two different cleavage sites within their spike proteins and S2', which are known as S1/S2. In the case of SARS-CoV, either cathepsin L or trypsin-like enzymes cleave at both positions, but probably in a sequential manner (S1/S2 followed by S2'). The use of each protease may be different for each cell type (e.g. vero cells versus respiratory epithelial cells). In some embodiments, the pH dependent virus is selected from Influenza, SFV, Rubella, VSV, Dengue, Andes, UUKV, LCMV, ASLV, JSRV, HCV, LASV, EBOV, SARS, and MERS.

Subject

As used herein, the term "subject" refers to a mammal, such as humans, but can also be another animal, such as a domestic animal (e.g. a dog, cat or the like), a farm animal (e.g. a cow, sheep, pig, horse or the like) or a laboratory animal (e.g. a monkey, rat, mouse, rabbit, guinea pig or the like). The term "patient" refers to a "subject" suffering from or suspected of suffering from a viral infection.

Medical Indications:

In one aspect of the present invention there is provided a pharmaceutical combination according to the invention as herein described for use in the treatment and/or prevention of a viral infection in a subject and/or a medical condition associated with a viral infection. Preferred viral infections to be or associated diseases are those described herein. In some embodiments, without limitation, the virus Amy be pH dependent.

In one aspect of the present invention there is provided a pharmaceutical combination according to the invention as herein described for use in the treatment of a medical condition associated with a SARS Coronavirus, wherein the medical condition associated with a SARS Coronavirus is preferably COVID-19 or a SARS Coronavirus-associated respiratory disease.

As used herein, the “patient” or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms.

In the present invention "treatment" or “therapy” generally means to obtain a desired pharmacological effect and/or physiological effect. The effect may be prophylactic in view of completely or partially preventing a disease and/or a symptom, for example by reducing the risk of a subject having a disease or symptom or may be therapeutic in view of partially or completely curing a disease and/or adverse effect of the disease.

In the present invention, "therapy" includes arbitrary treatments of diseases or conditions in mammals, in particular, humans, for example, the following treatments (a) to (c): (a) Prevention of onset of a disease, condition or symptom in a patient; (b) Inhibition of a symptom of a condition, that is, prevention of progression of the symptom; (c) Amelioration of a symptom of a condition, that is, induction of regression of the disease or symptom.

In particular, the treatment described herein relates to either reducing or inhibiting Coronavirus infection or symptoms thereof via binding the viral Spike protein with the antibodies or fragments thereof of the present invention. The prophylactic therapy as described herein is intended to encompass prevention or reduction of risk of Coronavirus infection, due to a reduced likelihood of Coronavirus infection of cells via interaction with the ACE2 protein after treatment with the antibodies or fragments thereof described herein.

As used herein, a “patient with symptoms of an infectious disease” is a subject who presents with one or more of, without limitation, fever, diarrhea, fatigue, muscle aches, coughing, if have been bitten by an animal, having trouble breathing, severe headache with fever, rash or swelling, unexplained or prolonged fever or vision problems. Other symptoms may be fever and chills, very low body temperature, decreased output of urine (oliguria), rapid pulse, rapid breathing, nausea and vomiting. In preferred embodiments the symptoms of an infectious disease are fever, diarrhea, fatigue, muscle aches, rapid pulse, rapid breathing, nausea and vomiting and/or coughing. As used herein ..infectious disease” comprises all diseases or disorders that are associated with bacterial and/or viral and/or fungal infections. As used herein, a patient with “symptoms of a viral infection of the respiratory tract” is a subject who presents with one or more of, without limitation, cold-like symptoms or flu-like illnesses, such as fever, cough, runny nose, sneezing, sore throat, having trouble breathing, headache, muscle aches, fatigue, rapid pulse, rapid breathing, nausea and vomiting, lack of taste and/or smell and/or malaise (feeling unwell).

In some embodiments, symptoms of infection with a SARS-virus are fever, sore throat, cough, myalgia or fatigue, and in some embodiments, additionally, sputum production, headache, hemoptysis and/or diarrhea. In some embodiments, symptoms of an infection with a SARS- coronavirus, for example SARS-CoV-2, are fever, sore throat, cough, lack of taste and/or smell, shortness of breath and/or fatigue.

As used herein, the term “a patient that is at risk of developing a severe acute respiratory syndrome (SARS)” relates to a subject, preferably distinct from any given person in the general population, who has an increased (e.g. above-average) risk of developing SARS. In some embodiments, the patient has symptoms of SARS or symptoms of a SARS Coronavirus infection. In some embodiments, the patient has no symptoms of SARS or symptoms of a SARS Coronavirus infection. In some embodiments, the subject has been in contact with people with SARS Coronavirus infections or symptoms. In some embodiments, the person at risk of developing SARS has been tested for the presence of a SARS Coronavirus infection. In some embodiments, the person at risk of developing SARS has tested positive for the presence of a SARS Coronavirus infection, preferably a coronavirus infection.

In embodiments, the patient at risk of developing SARS is an asymptomatic patient that shows no specific symptoms of SARS (yet). An asymptomatic patient may be at risk of developing SARS because the patient has been in contact with a person infected with a SARS Coronavirus. For example, the asymptomatic patient may have been identified as being at risk of developing SARS by a software application (app) that is installed on his smart phone or corresponding (portable) device and that indicates physical proximity or short physical distance to an infected patient that uses a corresponding app on its respective mobile device/smart phone. Other methods of determining contact/physical proximity to an infected person are known to the skilled person and equally apply to the method of the invention.

In some embodiments, the patient that has or is at risk of developing a severe acute respiratory syndrome (SARS) has a coronavirus infection.

Coronaviruses are a group of related viruses that cause diseases in mammals and birds. The scientific name for coronavirus is Orthocoronavirinae or Coronavirinae. Coronavirus belongs to the family of Coronaviridae. The family is divided into Coronavirinae and Torovirinae sub-families, which are further divided into six genera: Alphacoronavirus, Betacoronavirus,

Gammacoronavirus, Deltacoronavirus, Torovirus, and Bafinivirus. While viruses in the genera Alphacoronaviruses and Betacoronaviruses infect mostly mammals, the Gammacoronavirus infect avian species and members of the Deltacoronavirus genus have been found in both mammalian and avian hosts.

In humans, coronaviruses cause respiratory tract infections that can be mild, such as some cases of the common cold, and others that can be lethal, such as SARS, MERS, and COVID-19. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases, the largest among known RNA viruses.

Various species of human coronaviruses are known, such as, without limitation, Human coronavirus OC43 (HCoV-OC43), of the genus b-CoV, Human coronavirus HKU1 (HCoV-HKLH), of the genus b-CoV, Human coronavirus 229E (HCoV-229E), a-CoV, Human coronavirus NL63 (HCoV-NL63), a-CoV, Middle East respiratory syndrome-related coronavirus (MERS-CoV), Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Coronaviruses vary significantly in risk factor. Some can kill more than 30% of those infected (such as MERS-CoV), and some are relatively harmless, such as the common cold. Coronaviruses cause colds with major symptoms, such as fever, and a sore throat, e.g. from swollen adenoids, occurring primarily in the winter and early spring seasons. Coronaviruses can cause pneumonia (either direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (either direct viral bronchitis or secondary bacterial bronchitis). Coronaviruses can also cause SARS.

Advances in nucleic acid sequencing technology (commonly termed Next-Generation Sequencing, NGS) are providing large sets of sequence data obtained from a variety of biological samples and allowing the characterization of both known and novel virus strains. Established methods are therefore available for determining a Coronavirus infection.

Viruses encode a collection of proteins required to ensure self-replication and persistence of the encoding virus. Enzymes for genome mRNA production and genome replication, proteases for protein maturation, proteins for genome encapsidation, and proteins for undermining the host anti-viral responses can be identified conserved protein motifs or domains. Likely because of selective pressures, viral genomes are streamlined and the functional protein content encoded by viruses is much higher than for a cellular organisms. Thus, describing a viral genome by the collection of encoded protein domains is a potentially useful classification method. Viral evolution can therefore be followed and novel strains of coronavirus can be determined based on sequence comparison to known coronavirus strains.

In some embodiments, the patient suffers from an infection, preferably with a SARS Coronavirus (SARS-CoV). As used herein, SARS Coronavirus refers to a Coronavirus that leads to severe acute respiratory syndrome (SARS). This syndrome is a viral respiratory disease of zoonotic origin that first surfaced in the early 2000s caused by the first-identified strain of the SARS coronavirus (SARS-CoV or SARS-CoV-1).

SARS is induced via droplet transmission and replication of the virus could be shown in the upper and lower respiratory tract or gastrointestinal mucosa. In parallel the virus is also capable of directly invading cells of different organs such as liver, kidney, heart and brain. Another distinct mechanism appears to be the direct invasion of the virus in T-cells. A significant number of SARS patients, who suffer COVID-19, have a clinically low concentration of lymphocytes in the blood, also known as Lymphocytopenia. Clinically, patients show respiratory symptoms such as dry cough and shortness of breath, fever or diarrhea. But symptoms associated with acute liver injury, heart injury or kidney injury can also occur. In a less severe state of SARS, patients can show mild or even no symptoms.

Clinical and scientific investigations show that SARS-CoVs bind to the epithelial cells via the Angiotensin converting enzyme 2 receptor (ACE2). ACE2 is a cell membrane linked carboxypeptidase which is expressed in vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and lung. As one consequence binding of the virus leads to an epithelial and endothelial cell damage along with vascular leakage, which triggers a secretion of pro- inflammatory cytokines and chemokines. The virus also mediates ACE2 downregulation and shedding which further promotes a dysfunctional renin-angiotensin system (RAS). Once the RAS is disturbed it can lead an inflammatory response and to vascular permeability. Focusing on the respiratory system, ACE2 shedding can lead to pulmonary vascular permeability and subsequently to pulmonary edema.

Older patients (above 60 years), patients with chronic diseases (e.g. cardiovascular diseases, diabetes, cancer, COPD) or immune compromised patients are considered to be at a higher risk to face a severe development of SARS. Smoking and obesity are also considered as risk factors.

Embodiments of a SARS Coronavirus include, without limitation, any coronavirus that induces a SARS or SARS-similar pathology. Particular embodiments include, without limitation, the SARS Coronavirus (SARS-CoV-1) first discovered in 2003 (as described above), the Middle East respiratory syndrome (MERS-CoV) first discovered in 2012, and the SARS-CoV-2, which causes COVID-19, a disease which brought about the 2019-2020 coronavirus pandemic.

The strain SARS-CoV-2 causes COVID-19, a disease which brought about the ongoing 2019- 2020 coronavirus pandemic. The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and spread globally. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include muscle pain, diarrhea, sore throat, loss of taste and/or smell, and abdominal pain. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure.

Coronaviruses can be determined by molecular techniques, for example sequence-based analysis, for example by PCR-based amplification of viral genetic material. Genome-wide phylogenetic analysis indicates that SARS-CoV-2 shares 79.5% and 50% sequence identity to SARS-CoV and MERS-CoV, respectively. However, there is 94.6% sequence identity between the seven conserved replicase domains in ORFlab of SARS-CoV-2 and SARS-CoV, and less than 90% sequence identity between those of SARS-CoV-2 and other -CoVs, implying that SARS-CoV-2 belongs to the lineage of Beta-CoVs.

Similar to other CoVs, the SARS-CoV-2 virion with a genome size of 29.9 kb possesses a nucleocapsid composed of genomic RNA and phosphorylated nucleocapsid (N) protein. The nucleocapsid is buried inside phospholipid bilayers and covered by two di erent types of spike proteins: the spike glycoprotein trimmer (S) that exists in all CoVs, and the hemagglutinin- esterase (HE) only shared among some CoVs. The membrane (M) protein and the envelope (E) protein are located among the S proteins in the viral envelope. The SARS-CoV-2 genome has 5’ and 3’ terminal sequences (265 nt at the 5’ terminal and 229 nt at the 3’ terminal region), which is typical of -CoVs, with a gene order 5’-replicase open reading frame (ORF) lab-S-envelope(E)- membrane(M)-N-30. The predicted S, 0RF3a, E, M, and N genes of SARS-CoV-2 are 3822, 828, 228, 669, and 1260 nt in length, respectively. Similar to SARS-CoV, SARS-CoV-2 carries a predicted ORF8 gene (366 nt in length) located between the M and N ORF genes.

According to Jin et al (Jin et al. 2020), in the initial 41 patients, fever (98%), cough (76%), and myalgia or fatigue (44%) were the most common symptoms. Less common symptoms were sputum production (28%), headache (8%), hemoptysis (5%), and diarrhea (3%). More than half of patients developed dyspnea. The average incubation period and basic reproduction number (R0) were estimated to be 5.2 d (95% Cl: 4.1-7.0) and 2.2 (95% Cl, 1.4-3.9), respectively.

Treatment:

In the present invention "treatment" or “therapy” generally means to obtain a desired pharmacological effect and/or physiological effect. The effect may be prophylactic in view of completely or partially preventing a disease and/or a symptom, for example by reducing the risk of a subject having a particular disease or symptom, or may be therapeutic in view of partially or completely curing a disease and/or adverse effect of the disease.

In the present invention, "therapy" includes arbitrary treatments of diseases or conditions in mammals, in particular, humans, for example, the following treatments (a) to (c): (a) Prevention of onset of a disease, condition or symptom in a patient; (b) Inhibition of a symptom of a condition, that is, prevention of progression of the symptom; (c) Amelioration of a symptom of a condition, that is, induction of regression of the disease or symptom.

In particular, the treatment described herein relates to viral clearance either by autophagy or by blockade of viral cell entry. Therefore, the surrogate parameter viral load will be assessed on a daily basis. The prophylactic therapy as described herein is intended to encompass prevention or reduction of risk of infection with SARS-CoV, due to a blockade of viral cell entry after treatment with the compounds described herein.

Pharmaceutical Compositions and Methods of administration:

The present invention also relates to a pharmaceutical composition comprising the compounds described herein. The invention also relates to pharmaceutically acceptable salts of the compounds described herein, in addition to enantiomers and/or tautomers of the compounds described.

The term “pharmaceutical composition” refers to a combination of the agent as described herein with a pharmaceutically acceptable carrier. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce a severe allergic or similar untoward reaction when administered to a human. As used herein, "carrier" or “carrier substance” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, chewing tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

Dosage levels of the order of from about 0.01 mg to about 500 mg per kilogram of body weight per day are useful in the treatment of the indicated conditions. For example, a viral infection may be effectively treated by the administration of from about 0.01 to 50 mg of the inventive molecule per kilogram of body weight per day (about 0.5 mg to about 5 g per patient per day). The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may vary from about 5 to about 95% of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 5000 mg of active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The dosage effective amount of compounds according to the invention will vary depending upon factors including the particular compound, toxicity, and inhibitory activity, the condition treated, and whether the compound is administered alone or with other therapies.

The invention relates also to a process or a method for the treatment of the mentioned pathological conditions. The compounds of the present invention can be administered prophylactically or therapeutically, preferably in an amount that is effective against the mentioned disorders, to a warm-blooded animal, for example a human, requiring such treatment, the compounds preferably being used in the form of pharmaceutical compositions.

In some embodiments, a patient may receive therapy for the treatment and/or management of the viral infection before, during or after the administration of the therapeutically effective regimen of the compound of the invention, or a pharmaceutically acceptable salt thereof. Non-limiting examples of such a therapy include pain management, anti-inflammatory drugs, antibody therapy, immunotherapy, targeted therapy (i.e. therapy directed toward a specific target or pathway, e.g. virus replication, etc.), and any combination thereof. In some embodiments, the patient has not previously received a therapy for the treatment and/or management of virus infection.

FIGURES

The invention is further described by the figures. These are not intended to limit the scope of the invention.

Short description of the figures: Figure 1 : SARS-CoV-2 growth inhibition by camostat mesylate and niclosamide (including co-treatments) in human lung cells.

Figure 2: Camostat mesylate, its metabolite (GBPA), and niclosamide show synergistic effects and are able to abrogate SARS-CoV-2 infection in human lung cells.

Figure 3: Camostat mesylate and niclosamide show synergistic effects and are able to abrogate SARS-CoV-2 infection in human lung cells.

Detailed description of the figures:

Figure 1 : SARS-CoV-2 growth inhibition by camostat mesylate and niclosamide (including cotreatments) in human lung cells.

3x10e5 Calu-3 human lung cells were pretreated with 0.1 mM or 1 mM camostat mesylate (Sigma- Aldrich, SML0057) for 2 hours and infected with SARS-CoV-2 (strain Munich) using a multiplicity of infection of 0.001 . One hour post infection, supernatants were removed and cells were washed three times with PBS. Subsequently, DMEM cell culture medium with indicated concentrations of camostat mesylate and/or niclosamide (Sigma-Aldrich, N3510) was added and cells were incubated for 24 hours at 37°C. Samples from supernatants were used for viral RNA extraction (MagNApure Roche) and SARS-CoV-2 specific real time RT-PCR to determine viral genome equivalents.

Figure 2: Camostat mesylate, its metabolite (GBPA), and niclosamide show synergistic effects and are able to abrogate SARS-CoV-2 infection in human lung cells. (A,B) The human bronchial epithelial cell line Calu-3 was infected with SARS-CoV-2 (strain Munich/2020/984; BetaCoV/Munich/BavPat1/2020|EPI_ISL_406862) in a multicycle infection setting (MOI = 0.0005) under the influence of camostat mesylate, its metabolite GBPA and niclosamide, or a combination of camostat mesylate or GBPA with niclosamide using indicated concentrations in a range of 0-6,400 nmol/l (or 3,200 nmol/l per compound in combined applications). Control infections (“0”) were performed with DMSO treatment. At 24 h post infection, genomic viral RNA was quantified by qRT-PCR. The average of n = 4 biological replicates is displayed. (A) Relative values for infectious viral titers for each treatment. The results were calculated relatively to the DMSO control (“0”). (B) Absolute SARS-Cov-2 RNA copy numbers per ml culture supernatant.

Figure 3: Camostat mesylate and niclosamide show synergistic effects and are able to abrogate SARS-CoV-2 infection in human lung cells. The human bronchial epithelial cell line Calu-3 was infected with SARS-CoV-2 (strain Munich/2020/984;

BetaCoV/Munich/BavPat1/2020|EPI_ISL_406862) in a multicycle infection setting (MOI = 0.001) under the influence of camostat mesylate, niclosamide, or a combination of both. Control infections were performed with and without DMSO treatment. At 24 h post infection, infectious viral titers in the culture supernatant were quantified by plaque titration on VeroE6 cells (A) and genomic RNA was by quantitative real-time PCR (3) (B). The average of n = 2 experiments is displayed, with each treatment condition conducted in triplicates. PFU, plaque forming units; GE, genome equivalents EXAMPLES

The invention is further described by the following examples. These are not intended to limit the scope of the invention.

Example 1: Camostat mesylate and/or niclosamide treatment results in SARS-CoV-2 viral clearance

Camostat mesylate and/or niclosamide treatment significantly reduced Calu-3 lung cell line infection with authentic SARS-CoV-2. In these experiments, SARS-CoV-2 entry into human lung cells was inhibited by 50% (EC50) using 5 mM niclosamide (genomic titer) or with 1 pM camostat alone. At concentrations of 5 pM camostat mesylate viral entry was inhibited by > 90% (EC90). In combination, 5 pM niclosamide + 0.1 pM camostat showed viral clearance of 95 % (genomic titer).

The inventors show that niclosamide and camostat inhibit SARS-CoV-2 in cell culture when applied separately. These findings prompted the inventors to exploit these targets by combined intervention of SARS-CoV2 infection using repositioned small molecule inhibitors such as niclosamide and camostat mesylate. This intervention strategy using drug combinations showed an unexpected synergistic efficacy for viral clearance. This is a strong indication, that targeting of these key pathways and molecules causally involved in SARS-CoV2 viral entry can lead to efficient intervention of SARS-CoV2 viral infection. Thus, treatment of those patients infected with SARS-CoV2 virus can be beneficial for these patients and holds enormous promise for clinical use.

Example 2: Cotreatment of Camostatmesylate, its metabolite (GBPA) and niclosamide efficiently inhibits SARS-CoV-2 infection of human lung cells

Materials and Methods:

Calu-3 cells (ATCC HTB-55) and Vero E6 cells (ATCC CRL-1586) were maintained at 37°C, 5% C02 in Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 10% FCS, 1%

Penicillin, 1% non-essential amino acids, 1% sodium pyruvate and 1% L-Glutamine.

Virus Infections; camostat mesylate and niclosamide treatment camostat mesylate was diluted in culture medium and treatment was started 2 h prior to infection. Calu-3 seeded at 3x10e5 cells in 24-well format were infected with concentrated passage two SARS-CoV-2 stocks, strain Munich/2020/984 (BetaCoV/Munich/BavPat1/2020|EPI_ISL_406862), for one hour at 37°C. Cells were washed two times with PBS and further incubated in culture medium supplemented with either camostat mesylate (suspended in water), GBPA, niclosamide (suspended in DMSO), a combination of niclosamide and camostat mesylate or a combination of niclosamide and GBPA, DMSO alone, or no further supplements.

Results and Discussion

In another experiment, the inventors tested if the combination of camostat, its metabolite (GBPA), and niclosamide treatment has synergistic effects compared to single treatments using human lung cells (Calu-3). Infection of human lung cells Calu-3 with SARS-CoV-2 was inhibited under treatment with camostat mesylate, its metabolite (GBPA) and niclosamide and affect synergistically if camostat mesylate and niclosamide or GBPA and niclosamide are combinatorically applied. (Figure 2 a and b). The combination of niclosamide and camostat mesylate showed approximately 10,000- fold and the combination of niclosamide and GBPA showed 100-fold reduction in SARS-Cov-2 viral RNA compared to an untreated control infected with SARS-Cov-2 (Figure 3b).

Calu-3 cells treated with a combination of camostat and niclosamide showed a stronger reduction in virus infectious particle production (PFU/ml) and genome replication (gene equivalents, GE/ml) over samples treated with either drug alone (Figure 3 a and b). In Calu-3 cells infected at a low multiplicity of infection (MOI = 0.001) with SARS-CoV-2 (Munich strain,

BetaCoV/Munich/BavPat1/2020|EPI_ISL_406862) 0.1 mM camostat treatment alone reduced averaged virus titers 2.5-fold over control samples, while co-treatment with 5 pM and 10 pM niclosamide reduced virus titer to 43.3-fold and 221 -fold, respectively. The same trend was observed for 1 pM camostat treatment. Here, virus titers were reduced from 26.8-fold by 1 pM camostat treatment alone to 231.7-fold and 708.1-fold, respectively, in 5 pM and 10 pM niclosamide co-treated samples. These data demonstrate that combinational treatment with camostat mesylate and niclosamide have synergistic properties in impeding SARS-CoV-2 infection in vitro. These data encourage a combinational use in clinical trials.

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Claims

1. A pharmaceutical combination, comprising:

(a.) a therapeutically effective amount of an anti-viral protonophore, and (b.) a therapeutically effective amount of a serine protease inhibitor.

2. The pharmaceutical combination according to the preceding claim, wherein the anti-viral protonophore increases the pH in acidic endosomes of a target cell.

3. The pharmaceutical combination according to any one of the preceding claims, wherein the anti-viral protonophore is an autophagy inducer.

4. The pharmaceutical combination according to any one of the preceding claims, wherein the anti-viral protonophore is a salicylanilide or derivative thereof.

5. The pharmaceutical combination according to any one of the preceding claims, wherein the anti-viral protonophore is niclosamide, or a derivative thereof.

6. The pharmaceutical combination according to any one of the preceding claims, wherein the serine protease inhibitor is a trypsin-like serine protease inhibitor.

7. The pharmaceutical combination according to any one of the preceding claims, wherein the serine protease inhibitor inhibits transmembrane serine proteases.

8. The pharmaceutical combination according to any one of the preceding claims, wherein the serine protease inhibitor inhibits the TMPRSS2 serine protease.

9. The pharmaceutical combination according to any one of the preceding claims, wherein the serine protease inhibitor is camostat or a salt thereof.

10. The pharmaceutical combination according to any one of the preceding claims, wherein the serine protease inhibitor is camostat mesylate.

11 . The pharmaceutical combination according to any one of the preceding claims, wherein the serine protease inhibitor is a camostat metabolite.

12. The pharmaceutical combination according to any one of the preceding claims, wherein said camostat metabolite is 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA).

13. The pharmaceutical combination according to any one of the preceding claims, comprising:

(a.) niclosamide, and

(b.) camostat mesylate.

14. The pharmaceutical combination according to any one of the preceding claims, comprising

(a.) niclosamide, and (b.) GBPA.

15. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore and (b.) the serine protease inhibitor act synergistically to inhibit SARS-CoV infection of a target cell and/or replication of a SARS-CoV.

16. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore and (b.) the serine protease inhibitor act synergistically to inhibit SARS-CoV-2 infection of a target cell and/or replication of a SARS-CoV-2.

17. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor camostat mesylate act synergistically to inhibit SARS-CoV infection of a target cell and/or replication of a SARS-CoV.

18. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor camostat mesylate act synergistically to inhibit SARS-CoV-2 infection of a target cell and/or replication of a SARS-CoV-2.

19. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor GBPA act synergistically to inhibit SARS-CoV infection of a target cell and/or replication of a SARS-CoV.

20. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor GBPA act synergistically to inhibit SARS-CoV-2 infection of a target cell and/or replication of a SARS-CoV-2.

21 . The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore and (b.) the serine protease inhibitor have relative amounts of 10000:1 to 1 :10000 by weight.

22. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore and (b.) the serine protease inhibitor have relative amounts of 10:1 to 1 :1 by weight,.

23. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor camostat mesylate, have relative amounts of 1000:1 to 1 :1.

24. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor camostat mesylate, have relative amounts of 100:1 to 1 :1.

25. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor camostat mesylate, have relative amounts of 10:1 to 1 :1.

26. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor camostat mesylate, have relative amounts of about 3.3:1.

27. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor GBPA, have relative amounts of 1000:1 to 1 :1.

28. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor GBPA, have relative amounts of 100:1 to 1 :1.

29. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor GBPA, have relative amounts of 10:1 to 1 :1.

30. The pharmaceutical combination according to any one of the preceding claims, wherein (a.) the anti-viral protonophore niclosamide and (b.) the serine protease inhibitor GBPA, have relative amounts of about 3.3:1.

31 . The pharmaceutical combination according to any one of the preceding claims, wherein the camostat mesylate metabolite GBPA can be administered in an equivalent amount to camostat mesylate.

32. The pharmaceutical combination according to any one of the preceding claims, wherein the equivalent amounts of GBPA and camostat mesylate are determined by the weight ratio of the GBPA and camostat mesylate.

33. The pharmaceutical combination according to any one of the preceding claims, wherein

(a.) the anti-viral protonophore is in a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, and (b.) the serine protease inhibitor is in a separate pharmaceutical composition in admixture with a pharmaceutically acceptable carrier, or

(a.) the anti-viral protonophore and (b.) the serine protease inhibitor according to any one of the preceding claims are present in a kit, in spatial proximity but in separate containers and/or compositions, or

(a.) the anti-viral protonophore and (b.) the serine protease inhibitor according to any one of the preceding claims are combined in a single pharmaceutical composition in admixture with a pharmaceutically acceptable carrier.

34. The pharmaceutical combination according to any one of the preceding claims, for use in the treatment and/or prevention of a viral infection in a subject and/or a medical condition associated with a viral infection.

35. The pharmaceutical combination for use according to the preceding claim, wherein the virus is a pH-dependent virus, wherein infection of a target cell with the virus is dependent on pH.

36. The pharmaceutical combination for use according to any one of claims 13 or 14, wherein the virus is a SARS-CoV virus.

37. The pharmaceutical combination for use according to any one of claims 13 or 14, wherein the virus is a SARS-CoV-2 virus.