WO2021207031A1

WO2021207031A1 - Methods of treating and preventing coronavirus infections using inhibitors of coagulation factor xa

Info

Publication number: WO2021207031A1
Application number: PCT/US2021/025606
Authority: WO
Inventors: Galit H. FRYDMAN
Original assignee: Coagulo Medical Technologies Inc
Current assignee: Coagulo Medical Technologies Inc
Priority date: 2020-04-05
Filing date: 2021-04-02
Publication date: 2021-10-14
Anticipated expiration: 2022-10-05

Abstract

Inhibitors of coagulation Factor Xa such as rivaroxaban, apixaban, edoxaban, and betrixaban are used to treat and/or to prevent coronavirus infections such as Covid-19. Methods employing inhibitors of coagulation Factor Xa can prevent and/or inhibit viral infections of cell and/or proteolytic cleavage of viruses, thereby reducing viral infectivity and its subsequent activation of inflammation and coagulation.

Description

METHODS OF TREATING AND PREVENTING CORONAVIRUS INFECTIONS USING INHIBITORS OF COAGULATION FACTOR XA

FIELD OF THE INVENTION

The present invention relates to methods of treating and/or preventing coronavirus infections using one or more inhibitors of coagulation Factor Xa and, more particularly, to such methods using rivaroxaban, apixaban, edoxaban, and/or betrixaban. The present invention also relates to methods of preventing and/or inhibiting viral infection of a cell and/or the proteolytic cleavage of a virus, thereby reducing viral infectivity and its subsequent activation of inflammation and coagulation.

BACKGROUND OF THE INVENTION

Coronaviruses are small, enveloped, positive-sense single-stranded RNA viruses that can infect multiple species. A unique subgroup, called Betacoronavimses, has caused severe disease in humans over the past few decades and includes Severe Acute Respiratory Syndrome or SARS-CoV-1 (SARS), Middle East Respiratory Syndrome (MERS), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 or SARS-CoV-2 which causes the illness COVID-19. (Chan JF, et al., Interspecies transmission and emergence of novel viruses: lessons from bats and birds, Trends in Microbiology, 2013;21(10):544-555.) Coronavirus infections are very common and clinical symptoms can range from asymptomatic and mild (e.g., “cold” symptoms) to moderate and severe, including fever, upper and lower respiratory tract infection, gastrointestinal symptoms, and even a progression into systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), and multi-organ dysfunction syndrome (MODS). (Arabi YM, et al., Middle East Respiratory Syndrome, NEJM, 2017;376:584-594; Lee N, et al., A major outbreak of severe acute respiratory syndrome in Hong Kong, NEJM, 2003;348: 1986-1994; Guan E, et al., Clinical characteristics of coronavirus disease 2019 in China, NEJM, 2020: doi : 10.1056/NEJMoa2002032.)

There is a need in the art for methods of treating and/or preventing a coronavirus infection in a subject, especially when the subject has one or more co-morbidities like heart disease, pulmonary disease, and diabetes and/or is otherwise at an increased risk of infection and morbidity. There is a particular and urgent need around the world for methods of treating and/or preventing COVID-19 in a subject, especially when the subject has one or more such co-morbidities.

SUMMARY OF THE INVENTION

The present invention provides methods of treating and/or preventing a coronavirus infection in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

The present invention also provides for the use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for treating and/or preventing a coronavirus infection in a subject.

Further provided are methods of preventing and/or inhibiting coronavirus infection of a cell and/or the proteolytic cleavage of a coronavirus in a subject, comprising administering one or more inhibitors of coagulation Factor Xato the subject.

In embodiments of the methods and uses of the present invention, the coronavirus infection may be COVID-19.

In embodiments of the methods and uses of the present invention, the subject may be a human or an animal.

In embodiments of the methods and uses of the present invention, the administering may be by an oral route or by inhalation.

In embodiments of the methods and uses of the present invention, the inhibitor of coagulation Factor Xa may be rivaroxaban, apixaban, edoxaban, and/or betrixaban.

The methods of the present invention reduce viral infectivity and its subsequent activation of inflammation and coagulation in a subject.

Coronaviruses, like SARS-CoV-1 (which causes SARS) and SARS-CoV-2 (which causes COVID-19), contain spike proteins, which determine a virus’s cellular tropism and are a key to successful infection of a host cell by the vims. The SARS and COVID-19 spike protein (SP) recognizes and binds to cells with the Angiotensin-Converting Enzyme-2 (ACE2) receptor. Once bound to this receptor, SP is then subject to one or more protease- mediated cleavages, which converts the spike protein into two subunits: spike protein 1 (SI) and spike protein 2 (S2). SI is then released along with the ACE2 receptor, and S2 plays a role in the fusion of the viral and host cell membrane, allowing for cellular infection to take hold.

Coagulation Factor Xa is a serine protease, which is capable of proteolytically cleaving coronavirus SP into SI and S2, allowing for the virus to fuse with the cell membrane and complete successful infection. Subsequently, the virus inserts its genomic material into the cell, where it then reproduces, resulting in release of increased copies of the virus being released from the cell.

COVID-19 patients who are critically ill have increased inflammatory cytokines and coagulopathies. In accordance with the present invention, because FX/FXa appears to play a role in the infectivity and pathophysiology of COVID-19 infection, the administration of a Factor Xa inhibitor to a subject can prevent or reduce the cleavage of coronavirus SP and reduce the infectivity of the virus to the host cell. Inhibition of Factor Xa should aid in the reduction of cell infectivity by reducing Factor Xa-dependent proteolytic cleavage of SP, whether the Factor Xa is localized on the host cell, released by nearby cells, or in the circulation. Administration of Factor Xa inhibitors will, further, aid in controlling the inflammation and coagulation-related stimulation caused by COVID-19 infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the role of Factor Xa in coronavirus spike protein proteolysis.

FIGS. 2A-2C schematically depict the role of Factor Xa in coronavirus infection.

DETAILED DESCRIPTION OF THE INVENTION

Coronavirus Mechanism of Infection and Pathophysiology

Coronaviruses are internally composed of an RNA (ribonucleic acid) genome and proteins holding the RNA genome, surrounded by a nuclear envelope with spike proteins (glycoproteins). The spike proteins (SP) are peplomers that determine the cellular host tropism. SP contains a type II fusion machine, spike protein 2 (S2), and a receptor binding domain (RBD) on spike protein 1 (SI). (Li F, Structure, function, and evolution of coronavirus spike proteins, Annu. Rev. Virol ., 2016;2:237-261; Hulswit RJ, et al., Coronavirus spike protein and tropism changes, Adv Virus Res, 2016;96:29-57.) For both SARS and COVID-19, one of the primary host cell receptors to which the SP binds is the Angiotensin-Converting Enzyme-2 (ACE2) receptor. (Haga S. et al., Modulation of TNF-a-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-a production and facilitates viral entry, PNAS , 2008;105(22):7809-7814; Imai Y, et al., The discovery of angiotensin-converting enzy me 2 and its role in acute lung injury in mice, Exp Physiol , 2008;93(5):543-548; Wang D, et al., Renin-angiotensin-system, a potential pharmacological candidate, in acute respiratory distress syndrome during mechanical ventilation, Pulmonary Pharm & Ther, 2019;58:101833; Glowacka I, et al., Differential downregulation of ACE2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus NL63, J. of Virol, 2009; doi.org/10.1128/JVI.01248-09.) ACE2 receptors (ACE2Rs) play a primary role in the rennin-angiotensin system (RAS), which is responsible for regulation of blood pressure and fluid volume. More specifically, ACE2 reduces Angiotensin II levels. Angiotensin II is associated with increased inflammation, apoptosis, fibrosis and oxidative stress. (Khan A, et al., A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome, Crit Care , 2017;21(1):234; Batlle D, et al. ACE2 and diabetes: ACE or ACEs? Diabetes , 2010; doi.org/10.2337/dbl0-1205.) Binding of the SARS and COVID-19 SP to the ACE2R has been shown to reduce ACE2 expression via the induction of viral shedding and result in an increase in Tumor Necrosis Factor alpha (TNF- alpha) production. Increased levels of Angiotensin II levels, such as with the blocking or reduction of ACE2, has been shown to play a role in the pathophysiology of ARDS as well as diabetes. (Flaga et al. 2008; Imai et al. 2008; Wang et al. 2019; Glowacka et al. 2009; Khan et al. 2017; Batlle et al. 2010.)

Once the SP is bound to the ACE2R, the SP is subject to one or more proteolytic cleavages into SI and S2. One of the most well-known proteolytic enzymes that performs SP cleavage is Transmembrane Protease Serine 2 (TMPRSS2). (Esuma M, et al., Transmembrane serine protease TMPRSS2 activates hepatitis C virus infection, Hepatology, 2014; 16(2): doi.org/10.1002/hep.27426; Shen LW, et al., TMPRSS2: a potential target for treatment of influenza virus and coronavirus infections, Biochimie, 2017;142: 1-10; Hofmann H, et al., Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor, Biochemical and Biophysical Research Communications , 2004;319(4): 1216-21.) TMPRSS2 is also known to co-localize with ACE2 on Type II pneumocytes and cardiac myocytes and is thought to play a key role in the infection of the pulmonary and cardiovascular system. (Zhen YY, et al.. COVID-19 and the cardiovascular system, Nature Reviews Cardiology’ , 2020, https://www.nature.com/ articles/s41569-020-0360-5; Bertram S, et al., Cleavage and activation of the severe acute respirator syndrome coronavirus spike protein by human airway trypsin-like protease, J Virol 2011;85(24): 13363-13372; Heurich A et al., TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein, J Virol , 2014;88(2): 1293-1307; Bertram S, et al., Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts, PLoS One, 2012;7(4):e35876; Gurwitz D, Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics, Drug Dev Res, 2020;l-4; Xu H, et al., High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa, IJOS, 2020; 12.)

Once the SP is cleaved, SI is released and S2 remains attached to the virus and the host cell, playing a role in cellular-viral cellular membrane fusion. Membrane fusion is the first step to the active infection of the cell, where the genetic material and proteins from the virus are then inserted into the host cell, where it replicates. The new virions are then released, commonly causing cellular stress and cell death, thus further promoting inflammation while increasing the body’s viral load. (Li F 2016; Hulswit et al. 2016; Belouzard et al. 2009.)

Given the mechanism of infection of coronaviruses, patients with comorbidities, such as heart disease, pulmonary disease and diabetes, are at increased risk of infection and morbidity. (Lee et al. 2003; Zhen et al. 2020; Fang L, et al., Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection? The Lancet,

2020;8(4):PE21.) These are individuals who likely already have changes in their ACE2R expression, changes in their RAS system, and pre-existing pulmonary or cardiac injury and fibrosis and/or inflammation. In these cases, it is thought that the virus localizes within these systems and, through viral infection, increases the local inflammation and stress resulting in severe pulmonary and cardiovascular damage. (Zhen et al. 2020; Hofmann et al. 2004; Fang et al. 2020.) Role of Anticoagulants in Systemic Inflammation and Related Sequelae

The coagulation system plays multiple roles in disease, including coagulation, inflammation, and infection. The coagulation system is made up of multiple parts, including: (a) the coagulation cascade, which is a series of zymogens that are activated in a controlled, sequential manner to result in a cross-linked fibrin clot, and (b) platelets, which are acellular circulating cells that have been shown to play roles in infectious agent sequestration, promotion of inflammation via the activation of immune cells and coagulation, serving as an additional activator and scaffold for the fibrin clot. The coagulation system also has a close relationship with inflammation via either direct activation, such as is evidenced by the inflammatory nature of thrombin (Factor Ila), or via interaction with other pathways, including the complement and kallikrein system. (Foley JH, et ak, Cross talk pathways between coagulation and inflammation, Circ Research, 2016;118(9); Engelmann B, et al., Thrombosis as an intravascular effector of innate immunity, Nat Rev Immunol, 2013;13(l):34-45; Schoenmakers et al., Blood coagulation factors as inflammatory mediators, Blood Cells Mol Dis, 2005;34(l):30-7.)

In patients who suffer from systemic inflammation secondary to infection, it is very common to develop coagulopathies. While the initial phase of a severe inflammatory response is typically hypercoagulable (making it more likely for patients to develop pathologic blood clots), after prolonged inflammation, if the hypercoagulable phase is severe enough, the patient can then enter a hypocoagulable phase, usually secondary to consumption of platelets and coagulation factors (i.e., consumptive coagulopathy). During the inflammatory and hypercoagulable phase, there can be an increase in fibrin deposition (such as in the alveolar spaces, resulting in obstructed airways), there can be an increase in immunothrombotic complex formation (such as platelet-leukocyte aggregates), and there can be an increase in extracellular trap formation; all of these then combine to create the perfect storm for obstructing blood vessels and airways via multiple pathways simultaneously. It is usually this complex combination that results in the development of MODS and ARDS in patients with severe infections. (Chang JC, Acute respiratory distress syndrome as an organ phenotype of vascular microthrombotic disease: based on hemostatic theory and endothelial molecular pathogenesis, ClinAppl Thromb Hemost, 2019;25:doi: 10.1177/ 1076029619887437; Levi M, et al., Sepsis and thrombosis, Semin Thromb Hemost, 2013;39(5):559-566; Goeijenbier M et al., Review: viral infections and mechanisms of thrombosis and bleeding, Journal of Medical Virology, 2012;84:1680-1696.) Due to this propensity toward a hypercoagulable state, it is common for patients at risk of severe inflammation to receive prophylactic and therapeutic anticoagulant treatment, usually with unfractionated heparin (UFH) or low molecular weight heparin (LMWH) to prevent the development of deep vein thromboses (DVTs) and pulmonary embolisms (PEs). (Branchford BR, et al., The role of inflammation in venous thromboembolism, Front Pediatr, 2018;6: 142; Kaplan D, et al., VTE incidence and risk factors in patients with severe sepsis and septic shock, Chest , 2015;148(5): 1224-1230.) Heparins are indirect Factor Xa and Factor Ila (thrombin) inhibitors that operate via the binding of Antithrombin III (ATIII). In addition to the traditional intravenous and subcutaneous administration of heparin and its analogs, inhaled UFH and LMWH has been proposed and used in the context of pulmonary inflammation and ARDS. (Artigas A, et al., Inhalation therapies in acute respiratory distress syndrome, Ann Transl Med, 2017;5(14)293; Glas GJ, et al, Nebulized heparin for patients under mechanical ventilation: an individual patient data meta-analysis, Ann Intensive Care , 2016;6:33; Juschten J, et al., Nebulized anticoagulants in lung injury in critically ill patients- an updated systematic review of preclinical and clinical studies. Annals of Transl Medicine, 2017;5(22); Gram J, et al., Inhalation/intravenous recombinant tissue plasminogen activator and inhaled heparin in a patient with acute respiratory distress syndrome, Fibrinolysis and Proteolysis, 1999;13(4-5):209-212.) Poor prognosis is associated with COVID-19 patients who display systemic inflammation in conjunction with coagulopathies, such as increased D- dimers, and increased fibrin degradation products (FDPs). (Tang N, et al, Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia, J Thromb Haemost, 2020; 18(4): 844-847.) It has also been demonstrated that critically -ill COVID-19 patients treated with LMWH have a better prognosis and increased survival rates. (Tang N, et al., Anticoagulation treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. ./ Thromb Haemost, 2020; doi: 10.1111/jth.14817.)

Illustration of the Role of Factor Xa in Coronavirus Spike Protein Proteolysis and Infection

FIG. 1 shows the role of Factor Xa in coronavirus spike protein proteolysis.

At the top of FIG. 1, a coronavirus (100) binds to the host cell (110) expressing a receptor (101) that binds to the SARS coronavirus spike protein (102). Factor Xa (FXa) then acts as a proteolytic enzyme (120), cleaving spike protein into spike protein 1 (103) and spike protein 2 (104). The spike protein 1 then parts from the virus-cell complex, with or without the attached receptor, while spike protein 2 serves to aid in the fusion of the virus and cell membranes. The bottom of FIG. 1 illustrates the addition of a Factor Xa inhibitor (FXa-I) blocking FXa from acting as a proteolytic enzyme (130), therefore leaving the spike protein intact and preventing virus and host cell membrane fusion (140).

FIG. 2A shows how the coronavirus (201) binds to multiple cells, which express receptors that bind to the coronavirus spike protein. For example, the spike protein can bind to ACE2 receptors, which are present in the alveolar and bronchiolar epithelium, cardiac myocytes and the brain/central nervous system.

FIG. 2B depicts a coronavirus once the spike protein (202) is bound to the host cell receptor (203). A proteolytic enzyme binds to the spike protein in order to cleave the protein into spike protein 1 and spike protein 2. In example 210, the proteolytic enzyme is serine protease Factor X and/or Xa and can be expressed by the host cell directly, allowing for co localization of the spike protein receptor and the serine protease (204). As shown in example 220, Factor X and Factor Xa can be present, unbound in the circulation. As shown in example 230, Factor X and Factor Xa can be localized to the spike protein by nearby cells expressing Factor X and Factor Xa, such as macrophages (205).

FIG. 2C depicts a coronavirus once the spike protein is successfully cleaved by the proteolytic enzyme. Spike protein 1 is released with or without the bound host cell receptor (202), while spike protein 2 aids in the fusion of the viral and host cell membrane (240). The virus and host cell membrane are fused and the viral genetic material is inserted into the host cell. The viral genetic material replicates within the host cell (260). New coronavirus viral particles are released by the host cell (250), resulting in infection of new host cells (260) as well as propagation of inflammation and coagulation.

Coagulation Factor X and Coronavirus Spike Protein

While TMPRSS2 has been shown to be one of the primary proteolytic enzymes that cleaves coronavirus SP, this cleavage can be accomplished via multiple other enzymes that bind to the SP. Studies with SARS virus have demonstrated that Factor Xa is capable of cleaving SARS SP into SI and S2 in a dose-dependent manner. The addition of benzamidine hydrochloride (Ben-HCl), a protease and Factor Xa inhibitor, has been shown to inhibit SP cleavage by Factor Xa, and the addition of Ben-HCl also inhibited in vitro cell infectivity.

(Du L, et al., Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity, Biochem Biophys Res Common , 207;359(1): 174-179.)

These findings are consistent with various studies demonstrating that heparin treatment is anti-viral in in vitro experiments with coronaviruses, influenza, metapneumovirus, human immunodeficiency virus (HIV), respiratory syncytial vims (RSV), and hepatitis. (Kanade GD, et al., Activities of thrombin and Factor Xa are essential for replication of hepatitis E virus and are possibly implicated in ORF1 polyprotein processing, J Virol, 2018;92(6):e01853-17; Le BV, et al, Evaluation of anticoagulant agents for the treatment of human metapneumovims infection in mice, J GOT Virol. 2018 Oct;99(10): 1367-1380;

Howell AL, et al., Inhibition of HIV-1 infectivity by low molecular weight heparin. Results of in vitro studies and a pilot clinical trial in patients with advanced AIDS, Int J Clin Lab Res, 1996;26(2): 124-131; Bourgeois C, et al., Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro, Journal of Virol, 1998:7221-7227; Skidmore MA, et al., Inhibition of influenza invasion by modified heparin derivatives, Med Chem Comm, 2015;6:640.) Similarly, other studies have demonstrated the role of various coagulation factors, including Factor Xa, Factor Ila and plasmin, as proteases, which act upon SARS SP. (Simmons G, et al., Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion, Virology, 2011;413(2):265-274.)

Factor X is a coagulation factor and serine protease, which is vitamin K-dependent and primarily synthesized by the liver. (Venkateswarlu D, et al., Structure and dynamics of zymogen human blood coagulation factor X, Biophys J, 2002;82(3):1190-1206.) Factor X and Factor Xa have been proven to also be expressed in other cell types, including alveolar and bronchiolar epithelium, cardiac myocytes and brain tissue. (Hung HL, et al., Liver- enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X. JBC, 1996; doi: 10.1074/jbc.271.4.2323; Alexander R, The regulation of extra-hepatic coagulation factor X production in alveolar and bronchiolar epithelium. Thesis, University College London, https://discovery.ucl.ac.uk/id/eprint/ 1514446/1/RA%20THESIS%20 WITH%20CORRECTIONS.pdf; Scotton CJ, et al., Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury, J Clin Invest, 2009;119(9):2550-2563; Guo X, et ak, Cardiac expression of factor X mediates cardiac hypertrophy and fibrosis in pressure overload, JACC: Basic to Translational Science, 2020;5(1): DOI: 10.1016/j.jacbts.2019.10.006; Shikamoto Y, et al., Expression of factor X in both the rat brain and cells of the central nervous system. FEBS Letters, 1999;463(3):387-389.)

Moreover, many of the cell types that express Factor X and Factor Xa also express ACE2R. (Hu et al. 2020; Xia H, et al., Brain ACE2 shedding contributes to the development of neurogenic hypertension, CircRes, 2015;113(9): 1087-1096; Hamming I, et al., Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis, J Pathol, 2004;203(2):631-637.)

TMPRSS2 is thought to be the primary protease responsible for the cleavage of COVID-19 SP due to its co-localization with ACE2R on host cells, but Factor X and Factor Xa expression in these cells serves as an additional localized protease serving to cleave SP upon host cell receptor binding.

Factor X and its Role in the Pathophysiology of Coronavirus Infection

Factor X and Xa have been shown to play a role in the pathophysiology and progression of various forms of cardiopulmonar disease. Although Factor X and/or Xa is most commonly present in soluble form within the circulatory system, it is frequently expressed by multiple cell types, including alveolar and bronchiolar epithelium. In the alveolar and bronchiolar epithelium, it has been shown that the presence of reactive oxygen species (ROS) increases the expression of Factor X. (Scotton et al. 2009.) Factor Xa has also been shown to be a potent inducer of lung fibrosis via transforming growth factor beta (TGF- beta), mediated by proteinase-activated receptor- 1 (PARI). (Id.) Factor Xa is locally expressed within the lung and drives the fibrotic response to lung injury. (Id. ; Shi M, et al, Direct factor Xa inhibition attenuates acute lung injury progression via modulation of the PAR-2/NF-kB signaling pathway, Am J Transl Res, 2018;10(8):2335-2349; Schuliga M, et al., Annexin A2 contributes to lung injury and fibrosis by augmenting factor Xa fibrogenic activity, Am J Physiol, 2017;312(5): L772-L782.) Considering that patients with pre-existing pulmonary and cardiac disease appear to be amongst the highest risk group for severe COVID-19 infection, these patients likely have a higher baseline expression of Factor X in these cell populations that lend themselves to increased chance of host cell infectivity.

Factor X is also known to be expressed by cardiac myocytes and fibroblasts and has been found to be expressed in the heart following pressure overload. Rivaroxaban, a Factor Xa inhibitor, has been shown to reduce inflammation, hypertrophy and fibrosis secondary to pressure overload and improve diastolic function, even at subtherapeutic doses (i.e., doses that did not affect thrombin generation). (Guo X, et al. 2020) In light of the fact that cardiac myocytes are known to express ACE2R and Factor X, with Factor X and ACE2R expression being increased in cardiac disease, this provides another mechanism by which COVID-19 directly infects cardiac cells at an increased level in patients with pre-existing disease. (Guo J, et al. Coronavirus Disease 2019 (COVID-19) and cardiovascular disease: a viewpoint on the potential influence of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on onset and severity of acute respiratory syndrome coronavirus 2 infection, JAHA, 2020;9(7): doi.org/10.1161/JAHA.120.016219; Mohan R, et al., ACE2: A new target for cardiovascular disease therapeutics, Jof Cardiovascular Pharmacology, 2007;50(2): 112-119; Epelman S, et al., Detection of soluble angiotensin-converting enzyme 2 in heart failure, J Am Coll Cardiol, 2008;52(9):750-754.)

COVID-19 infection has recently been suggested to have primary effects in the brain, with patients presenting with confusion, abnormal behavior, seizures and brain swelling. (Baig AM, et al., Evidence of the COVID-19 virus targeting the CNS: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms, ACS Chemical Neuroscience , 2020;11(7): 995-998.) Factor X has been found to be expressed in the rat brain and central nervous system, and it is well-known that ACE2 is present within the brain. (Shikamoto et al. 1999.) It is, therefore, likely that this is another potential site of Factor X/ACE2R co-localization, allowing for COVID-19 binding and infection. COVID- related neurologic symptoms can be caused by clot formation and/or inflammation, which can be a direct result of either systemic inflammation or local inflammation and coagulation activation within the nervous system secondary to the presence of COVID-19.

Factor X and its Role in Coronavirus-Related Inflammation

Coronavirus infections, especially SARS and COVID-19, frequently result in increased plasma inflammatory cytokines, and evaluation of pulmonary pathology has revealed an increase in macrophage and lymphocyte infiltration with fibrin deposition.

(Mehta P, et al., COVID-19: consider cytokine storm syndromes and immunosuppression,

The Lancet, 2020; DOI: https://doi.org/10.1016/S0140-6736(20)30628-0; Tian S, et al., Pulmonary pathology of early-phase 2019 novel coronavirus (COVID-19) pneumonia in two patients with lung cancer, J Thorac Oncol, 2020; doi: 10.1016/j.jtho.2020.02.010.) Patients suffering from severe coronavirus infection have also been show n to suffer from various states of coagulopathy and systemic inflammation. (Zhou F, et al., Clinical course and risk factors for mortality of adult patients with COVID-19 in Wuhan, China: a retrospective cohort study, The Lancet, 2020; doi.org/10.1016/S0140-6736(20)30566-3; Tang et al , J Thromh Haemost, 2020; 18(4): 844-847.) Macrophages are one of the primary inflammatory cells involved in the response to coronavirus-related immune response. (Cheung CY, et al., Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis, J Virol , 2005;79:7819-7826.) Some SARS studies have demonstrated that SARS is capable of replicating in human peripheral monocytes and macrophages as well as in alveolar macrophages. (Yilla M, et al., SARS- coronavirus replication in human peripheral monocytes/macrophages, Virus Research,

2005 ; 107 (1):93-101; Funk CJ, et al., Infection of human alveolar macrophages by human coronavims strain 229E, J Gen Virology, 2012;93(3): doi.org/10.1099/vir.0.038414-0.) Macrophages, and, more specifically, alveolar macrophages, have been demonstrated to express Factor X and Factor Xa (in addition to other coagulation factors). (McGee MP, et al., Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study, Blood, 1989;74(5): 1583-1590; Zuo P, et al., Factor Xa induces pro- inflammatory cytokine expression in RAW 264.7 macrophages via protease-activated receptor-2 activation, Am J Transl Res, 2015;7(11):2326-2334.) In addition to their role in inflammation and coagulation, localized macrophages provide an additional source of Factor Xa to nearby cells expressing the ACE2 receptor and binding to coronavims. This results in enhanced spike protein cleavage and increased cell infection as well as increased inflammation and activation of coagulation.

Factor Xa also plays a major role in Factor Ila-mediated proteinase-activated receptor (PAR) activation. Factor Xa is upstream of prothrombin (Factor II) and is capable of activating Factor II to Factor Ila, which cleaves PARI, PAR3 and PAR4, resulting in cellular activation. PAR2 is primarily activated by Factor Xa. PARs are expressed on platelets, leukocytes and endothelial cells. PARI plays a large role in inflammation and is present on endothelial cells and fibroblasts. When activated, PARI stimulates the production of monocyte chemoattractant protein- 1 (MCP1), TNF-alpha, interleukin 1-beta (IL1B), interleukin 6 (IL6) and TGB-beta. This PARI activation also activates cells, resulting in P- and E-selectin exposure. (Foley et al. 2016; Zuo et al. 2015.) In the case of a patient with pre-existing lung disease (such as idiopathic fibrosis or asthma, where there is already an increase in Factor X expression and activation, along with increased PARI activation and fibroblasts), there is a naturally hospitable environment for the binding of COVID-19, which, in turn, results in increased inflammation and cell activation, thereby increasing cell infection and the development of severe lung pathology, such as ARDS.

Factor Xa Inhibitors as Coronavirus Infection Preventatives and Treatments

COVID-19 can be very dangerous in at-risk patient populations, although the exact pathophysiology of the increased morbidity and mortality remains under study. COVID-19 is thought to bind to ACE2R on host cells with TMPRSS2 cleaving SP into SI and S2, enabling fusion of the viral and host cell membranes for cellular infection to take place. Factor X acts as an additional co-localized protease on cells co-expressing ACE2 and also serves as a protease for SP cleavage. Additionally, Factor X and Xa are expressed in alveolar macrophages, which provides an additional localized source of protease for the cleavage of SP. In this model of infectivity, there is a continuum of pathology, where patients with pre existing cardiopulmonary inflammatory disease may have increased baseline levels of ACE2R and Factor X/X a, which increases COVID-19 binding and infection, which then results in further cellular activation, inflammation and coagulation, which further perpetuates the binding and COVID-19 infection. As this viral load increases, so does activation of the inflammatory and coagulation system.

The inhibition of Factor Xa aids in the reduction of cell infectivity by reducing Factor Xa-dependent proteolytic cleavage of SP, whether the Factor Xa is localized on the host cell, released by nearby cells or in the circulation.

Administration of Factor Xa inhibitors will, further, aid in controlling inflammation and coagulation-related stimulation caused by COVID-19 infection. This may explain the clinical data suggesting the benefit of LMWH administration in critical patients. (Tang N, et ah, Anticoagulation treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy, J Thromb Haemost, 2020; doi: 10.1111/jth.14817.) There is evidence of specific anti-inflammatory benefits to some of the oral Factor Xa inhibitors, resulting in a dose-dependent decrease in the release of inflammatory cytokines by LPS-activated monocytes. (Laurent M, et ah, Comparative study of the effect of rivaroxaban and fondaparinux on monocyte’s coagulant activity and cytokine release, Experimental Hematology & Oncology , 2014;3:2227.) This is especially true in the case of increased oxidative stress secondary to pulmonary comorbidities, such as pulmonary fibrosis or inflammatory conditions such as asthma or acute respiratory distress syndrome (ARDS). The administration of Factor Xa inhibitors, alone or in combination with other drugs, may also provide a successful prophylaxis against coronavirus infection and/or for the reduction of Factor Xa levels in: subjects that have recently been exposed to a coronavirus, subjects at high risk of exposure, and/or subjects in a high-risk category (such as subjects with a prior or existing cardiac or pulmonary condition or subjects with diabetes). Direct oral Factor Xa inhibitors, such as rivaroxaban, apixaban, edoxaban, and betrixaban, may be drug choices that are easily administered in outpatient and inpatient settings. (Bielecki S, et ak,

The market for oral anticoagulants, Nature Reviews Drug Discovery, 2018;17:617-618.) Where Factor Xa inhibitor is to be administered to subjects who do not require active anticoagulation, the dose of the Factor Xa inhibitor should be worked out in order to help prevent adverse bleeding events. For these cases, there is evidence that Factor Xa inhibitors at subtherapeutic doses (doses that do not result in anticoagulation) reduce the level of Factor Xa, and doses under the current label recommendations for anticoagulation may be sufficient. The dose of Factor Xa inhibitor required will be dependent on the reason for administration and requires evaluation of the subject, including factors that may change the pharmacokinetics of the inhibitors, such as changes in kidney function and polypharmacy.

Current coagulation laboratory tests, such as anti-Xa levels, aPTT/PT and viscoelastic testing, may not accurately determine the levels of cell-associated Factor X/Xa and inhibition, as cell-bound Factor XJX a may not directly correlate with circulating levels of Factor Xa. However, traditional testing may still be useful for the management of the level of anticoagulation, as Factor Xa inhibitor drugs are not benign and will require dose management to reduce the chance of adverse bleeding events. (Douxfils J, et al., Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians, J Thromb Haemost, 2018;16(2):209-219.) Administration of Factor Xa inhibitors via direct inhalation for the treatment of respiratory distress and ARDS, also is worthy of further investigation.

The present invention provides methods of treating a coronavirus infection in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

The present invention provides methods of preventing a coronavirus infection in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject. The present invention also provides for the use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for treating a coronavirus infection in a subject.

The present invention further provides for the use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for preventing a coronavirus infection in a subject.

Also provided by the present invention are methods of preventing and/or inhibiting coronavirus infection of a cell and/or the proteolytic cleavage of a coronavirus in a subject, comprising administering one or more inhibitors of coagulation Factor Xato the subject.

The present invention provides methods of treating COVID-19 in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

The present invention provides methods of preventing COVID-19 in a subject, comprising administering one or more inhibitors of coagulation Factor Xato the subject.

The present invention also provides for the use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for treating COVID-19 in a subject.

The present invention further provides for the use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for preventing COVID-19 in a subject.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered via multiple routes. For example, Factor Xa inhibitors may be administered via an oral route, submucosal route, injection (intradermal, transdermal, subcutaneous, intravenous and intramuscular), or inhalation.

The methods of the present invention may be employed to reduce viral infectivity and its subsequent activation of inflammation and coagulation in a subject. In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered to subjects with active coronavirus infection for the reduction of cell infectivity and viral load.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered to subjects with or without clinical symptoms of a coronavirus infection.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered prophylactically to patients who have been exposed to coronavirus, in order to prevent viral infection and/or to reduce infectivity rate and viral load.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered prophylactically to individuals who are at risk of exposure in order to serve as a protectant against potential coronavirus infection. Such individuals include healthcare workers, first responders and other individuals who are at greater risk of coming in contact with infected individuals and/or materials which may harbor the virus.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered for the prophylaxis or treatment of coronavirus infection in combination with other agents administered for the prevention or treatment of coronavirus infection.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered for the prophylaxis or treatment of coronavirus infection for their anti-viral, anticoagulant, immune-modulatory and/or anti-inflammatory effects.

In embodiments of the methods of the present invention, Factor Xa inhibitors may be administered via specific routes of administration in order to target body systems that may have high burden of viral infection. In embodiments, Factor Xa inhibitors may be administered via aerosolization/inhalation for the treatment of respiratory distress or respiratory failure. In these cases, Factor Xa inhibitors may be administered for both their anti -viral effects as well as their for anticoagulant and/or immune-modulatory and/or anti inflammatory effects.

Rivaroxaban, apixaban, edoxaban, and betrixaban are preferred inhibitors of Factor Xa for use in the methods and uses of the present invention.

The dose of Factor Xa inhibitors in the methods and uses of the present invention may vary based on the specific chemical structure, pharmacokinetics and/or route of administration of the inhibitor. In some embodiments, rivaroxaban is administered at a 20 mg dose by mouth once a day to a subject. In other embodiment, apixaban is administered at a 2.5-5 mg dose by mouth twice a day to a subject. Other embodiments of the instant invention provide for other methods of treating and/or preventing coronavirus infection, by different Factor Xa inhibitors, routes of administration, and/or dosages. For example, the drug, route of administration and/or dosage may vary based on desired therapeutic effect, which may be at sub-, supra- or target-therapeutic levels for anticoagulation, interactions with other drugs that may be administered at the same time, such as drugs that effect CYP3A4 and P-gp, and the patient’s co-morbidities, such as renal insufficiency. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood in light of the present disclosure by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

I. A method of treating a coronavirus infection in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

2. A method of preventing a coronavirus infection in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

3. The method according to claim 1 or claim 2, wherein the coronavirus infection is COVID- 19.

4. The method according to claim 1 or claim 2, wherein the coronavirus infection is Severe Acute Respiratory Syndrome (SARS).

5. The method according to claim 1 or claim 2, wherein the subject is a human.

6. The method according to claim 1 or claim 2, wherein the inhibitor of coagulation Factor Xa is a direct Factor Xa inhibitor.

7. The method according to claim 1 or claim 2, wherein the inhibitor of coagulation Factor Xa is selected from rivaroxaban, apixaban, edoxaban, and betrixaban.

8. The method according to claim 1 or claim 2, wherein the administering is via oral route, submucosal route, injection, or inhalation.

9. The method according to claim 1 or claim 2, wherein the administering is via oral route.

10. The method according to claim 1 or claim 2, wherein the inhibitor of coagulation Factor Xa is administered at a dose at or below the level required for anticoagulation.

II. The method according to claim 1 or claim 2, wherein the inhibitor of coagulation Factor Xa is administered in combination with another agent.

12. The method according to claim 11, wherein the other agent is administered concurrently.

13. The method according to claim 11, wherein the other agent is administered before or after the administering of the inhibitor of coagulation Factor Xa.

14. A method of preventing or inhibiting proteolytic cleavage of a coronavirus in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

15. A method of treating COVID-19 in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

16. A method of preventing COVID-19 in a subject, comprising administering one or more inhibitors of coagulation Factor Xa to the subject.

17. A method of treating COVID-19 in a subject, comprising orally administering one or more inhibitors of coagulation Factor Xa to the subject.

18. A method of preventing COVID-19 in a subject, comprising orally administering one or more inhibitors of coagulation Factor Xa to the subject.

19. A method of treating COVID-19 in a human, comprising orally administering to the subject one or more inhibitors of coagulation Factor Xa selected from rivaroxaban, apixaban, edoxaban, and betrixaban.

20. A method of prev enting COVID-19 in a human, comprising orally administering to the subject one or more inhibitors of coagulation Factor Xa selected from rivaroxaban, apixaban, edoxaban, and betrixaban.

21. Use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for treating a coronavirus infection in a subject.

22, Use of one or more inhibitors of coagulation Factor Xa in the preparation of a medicament for preventing a coronavirus infection in a subject.

23. Preparation of a medicament for treating a coronavirus infection in a subject comprising the use of one or more inhibitors of coagulation Factor Xa.

24. Preparation of a medicament for preventing a coronavirus infection in a subject comprising the use of one or more inhibitors of coagulation Factor Xa.