WO2023084489A1 - Methods of treating coronavirus disease 2019 - Google Patents

Abstract

The present invention is related to the discovery of new methods for treating the patients infected with SARS-CoV-2 comprising administering orally to the patient in need of such treatment a therapeutically effective amount of a MEK inhibitor.

Description

METHODS OF TREATING CORONAVIRUS DISEASE 2019

FIELD OF THE INVENTION

The present invention relates to the discovery of new methods for treating patients that are afflicted with coronavirus disease 2019 (COVID-19).

BACKGROUND OF THE INVENTION

Coronavirus disease 2019 (COVID-19) is a viral disease caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that can cause acute respiratory distress syndrome (ARDS). ARDS is an acute lung disease resulting from destruction of the alveolar epithelium (diffuse alveolar damage) that is a response to a variety of injurious stimuli including viral pathogens such as SARS-CoV-2. The destruction of the alveolar epithelial barrier leads to an exudation of interstitial fluid and inflammatory cells (neutrophils and macrophages) that ultimately compromises lung dynamics, ventilation, and oxygenation. Clinically, development of ARDS is characterized by bilateral pulmonary infiltrates, decreased pulmonary compliance, and progressive hypoxemia. The severity of COVID-19 can vary from asymptomatic illness to severe or fatal disease. Many patients may rapidly (within 1-2 weeks of infection) develop dyspnea and pneumonia and require hospitalization for respiratory support. Of these hospitalized patients, 20 - 30% have required admission to intensive care units (ICUs) for ventilatory support due to development of ARDS, with ventilatory failure being a major cause of overall mortality due to COVID-19.

The genome sequence of SARS-CoV-2 was sequenced from isolates from nine patients in Wuhan, China and found to be of the genus betacoronavirus sharing about 79% homology with severe acute respiratory syndrome coronavirus (SARS-CoV), the causative agent of the SARS outbreak in 2002-2003. Preclinical data from betacoronaviruses that are similar to SARS-CoV-2 suggest that the pathogenic characteristics of progressive disease are dominated by an intense inflammatory response. The ultimate result is progressive destruction of the alveolar epithelium leading to ARDS. Moreover, the exudative phase of ARDS is due, at least in part, to a pro- inflammatory response involving influx of innate immune cells (neutrophils and macrophages) and elevations of inflammatory cytokines such as interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-a, with higher levels of both IL-6 and IL-8 levels being correlated with increased mortality. While innate immune signaling is likely important for the initial response to SARS-CoV- 2 infection, once pneumonia has developed, immunomodulatory therapy may be beneficial in reducing the deleterious effects of lung inflammation and mitigating progressive lung injury. The Ras/Raf/MEK/ERK pathway may play a role in the progression of viral disease. Inhibition of mitogen-activated protein kinase kinase (MEK) via a pharmacologic MEK inhibitor may lead to two beneficial effects in COVID-19 patients: 1) by inhibiting RNA replication of the coronavirus; and 2) by also reducing the levels of deleterious inflammatory cytokines (e.g.,TNF, IL-1 , and IL-6).

The Ras/Raf/MEK/ERK signaling pathway is one of the three mitogen-activated protein kinase (MARK) cascades that play important roles in the regulation of cell growth, differentiation, and survival. The functions of this pathway can also be important to viral propagation since viruses are obligate intracellular parasites that require host cellular machinery for survival. MEK inhibition is an interesting molecular target for anti-viral therapy against a diverse range of viruses (Baturcam et al., 2019, Cell Communication and Signaling 17: 78; Haasbach et al., 2017, Antiviral Research 142: 178-184; Bonjardim, 2017, Virology 507: 267-275). Research studying an antiviral effect in coronavirus (murine coronavirus mouse hepatitis virus (MHV)) demonstrated that inhibition of MEK signaling reduced MHV propagation which was mediated by decreased viral RNA synthesis (Cai et al., 2007, Journal of Virology 81 : 446-456).

The airway epithelium is a major target tissue for respiratory infections. The epithelial anti-viral response is orchestrated by the interferon regulatory factor-3 (IRF3) which induces type I and type III interferon (IFN) signaling. The MEK pathway is involved in regulating the IFN response to viral infection and pharmacological MEK inhibition has been shown to enhance type I IFN response as well as reduce IL-8 production (Baturcam et al., supra).

Coronavirus is an enveloped, single-stranded positive sense RNA virus. Approximately two-thirds of the 5' genome encodes two overlapping polyproteins, pp1 a and ppl ab, which are essential for viral replication and transcription. The 3' terminus encodes a set of four structural proteins for coronavirus: nucleocapsid (N), spike protein (S), membrane protein (M), and envelope protein (E), which are responsible for virion assembly and suppression of host immune response. In the life cycle of coronavirus infection, it mainly uses spike proteins to bind to their receptors for attachment onto the host cell membrane. Then, the coronavirus fuses with host cellular membrane and releases its genomic RNA. Subsequently, the two polyproteins are expressed through hijacking host ribosomes, which are further processed by two viral proteases, papain-like protease and main protease (Mpro), also termed 3CL protease, into 16 mature non- structural proteins (nsps). These nsps, including helicase, RNA-dependent RNA polymerase (RdRp), and methyltransferase, can then assemble into the replication:transcription complex and initiate viral RNA replication and translation (Thiel et al., 2003, J. Gen. Virol. 84(Pt.9): 2305-2315). The newly produced viral RNA and proteins are then packaged into mature progeny virions, which are subsequently released through exocytosis to infect other healthy cells.

Mpro, or 3CL protease, is a 33.8-kDa cysteine protease which mediates the maturation of functional polypeptides involved in the assembly of replication-transcription machinery (Wang et al., 2016, Virol. Sin. 31 :24-30). Mpro digests the polyprotein at no less than 11 conserved sites, starting with the autolytic cleavage of this enzyme itself from pp1 a and ppl ab. In addition, 3CL has no human homolog and is highly conserved among coronaviruses (Yang et al., 2006, Curr. Pharm. Des. 12: 4573-4590). These above features make it an attractive drug target.

MEK activity is thought to play a role in viral infection and replication beyond coronavirus infection; MEK activity has been suggested as a potential antiviral therapeutic strategy. For example, activation of the Raf/MEK/ERK pathway is required for translation of respiratory syncytial virus (RSV) (Preugschas et al, 2019, Cell Micriobiol, 21 :e12955). MEK inhibition has also provided broad anti-influenza virus activity and improved duration of treatment. Haasback et al, 2017, Antiviral Research (142: 178-184).

There are several animal models which recapitulate disease and virus replication of SARS-CoV-2. For example, transgenic mice expressing the SARS-CoV-2 receptor, human ACE2 have been produced that support viral replication of SARS-CoV-2 (Bao et al, 2020, Nature-. 583, 830-833; Sun et aL 2020, Cell Host & Microbe-. 28, 124-133). However, with ectopic expression of ACE2 in these models, the virus appears to cause the death of animals due to inappropriately high-level expression of ACE2 in the brain, resulting in viral encephalitis (Jiang et al, 2020). Subsequently, a robust MA-SARS-CoV-2 model was developed by site directed mutations in spike gene and multiple passages; thus, allowing for evaluation of antiviral agents in vivo against SARS-CoV-2 (Dinnon et al, 2020, Nature’. 586(7830), 560-566; Leist et al., 2020, Cell: 183, 1070-1085).

To date there are few treatments for CVOID-19. Paxlovid (nirmatrelvir, ritonavir) has been authorized in United States for emergency use under an Emergency Use Authorization for the treatment of mild-to-moderate COVD-19 in adults and pediatric patients with positive results of direct SARS-CoV-2 viral testing, and who are at high risk for progression to severe COVID-19, including hospitalization or death. Lagevrio (molnupiravir) has been authorized in United States for emergency use under an Emergency Use Authorization, for the treatment of mild-to-moderate COVID-19 in adults who are at risk for progression to severe COVID-19, including hospitalization or death, and for whom alternative COVID-19 treatment options authorize d by FDA are no accessible or clinically appropriate. Veklury (remdesivir) is approved in United States for the treatment of adults and pediatric patients for the treatment of COVID-19 requiring hospitalization. There remains a need for therapies for COVID-19, for example that are effective treatments for COVID-19 beyond the initial days of invention, or that prevent the progression of infection to severe disease and death, in particular among hospitalized patients with active pneumonia. Further there remains a need for therapies that have a direct effect on reducing viral replication, for example by inhibiting post-cell entry viral RNA synthesis. The present invention provides methods for treating SARS-CoV-2 patients for example by inhibiting the inflammatory pathways activated by COVID-19 SARS-CoV-2 infection. There also remains a need for additional therapies that treat other viral infections, including respiratory viral infections.

SUMMARY OF THE INVENTION

The present invention provides a method of treating a patient infected with SARS-CoV-2 comprising administering to the patient in need of such treatment a therapeutically effective amount of a MEK inhibitor, preferably binimetinib (MEKTOVI®) or a pharmaceutically acceptable salt thereof.

In preferred embodiments, the method comprises administering 45 mg, or 30 mg, or 60mg of binimetinib orally, twice daily, or an equivalent amount of binimetinib in the form of a pharmaceutically acceptable salt thereof.

In another embodiment the invention provides for administering binimetinib in combination with a protease inhibitor.

In another embodiment, the method provides for any of the above embodiments, wherein one or more cytokine selected from the list of consisting of tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8) levels, is reduced for at least 4 hours after administering binimetinib. Preferably, the cytokine is reduced by at least 40%. Alternatively, the cytokine is reduced by at least 80%.

The invention provides a method of treating a patient infected with SARS-CoV-2 comprising administering to the patient in need of such treatment a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a 3CL protease inhibitor. Preferably, the 3CL protease inhibitor is PF-07321332 or a pharmaceutically acceptable salt thereof. In other preferred embodiments, the 3CL protease inhibitor is PF-07304814 or PF-00835231 or a pharmaceutically acceptable salt thereof.

In some embodiments, the patient is asymptomatic from a coronavirus infection. In some embodiments, the patient is symptomatic from the coronavirus infection. In some embodiments, the patient is symptomatic from the coronavirus infection with one or more symptom selected from the group consisting of fever, dry cough, diarrhea, headache, lymphopenia, hypoxia, shortness of breath, transaminitis, pulmonary edema, acute hypoxemic respiratory failure, kidney failure, respiratory failure, pneumonia, macrophage activation syndrome (MAS), cytokine storm, severe acute respiratory syndrome (SARS), acute respiratory distress syndrome (ARDS), and cardiac failure.

The invention also provides a method for treating severe acute respiratory syndrome (SARS) or acute respiratory distress syndrome (ARDS) comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof. In another embodiment, the method also provides for reducing a risk that the patient will develop severe acute respiratory syndrome (SARS) or acute respiratory distress syndrome (ARDS) comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof. The invention also provides a method for preventing a patient from developing severe acute respiratory syndrome (SARS) or acute respiratory distress syndrome (ARDS) comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the patient has tested positive for infection by SARS-CoV-2. In other preferred embodiments, the patient has one or more symptoms selected from the group consisting of fever, dry cough, diarrhea, headache, lymphopenia, hypoxia, shortness of breath, transaminitis, pulmonary edema, acute hypoxemic respiratory failure, kidney failure, respiratory failure, pneumonia, MAS or cytokine storm. In another preferred embodiment, the method further comprises administering a therapeutically effective amount of additional therapeutic agent that is an anti-viral agent, an anti-cytokine agent, a steroid, or an anti-inflammatory agent. Preferably, the additional therapeutic agent is a 3CL protease inhibitor. More preferably, the 3CL protease inhibitor is PF-07321332 or a pharmaceutically acceptable salt thereof. In other preferred embodiments, the 3CL protease inhibitor is PF-07304814 or PF-00835231 or a pharmaceutically acceptable salt thereof.

In some embodiments the anti-cytokine agent is selected from an anti-IL-6 agent, anti-IL- 1 agent, and an anti-TNF agent.

The invention also provides a method of reducing viral load in a patient infected with SARS-CoV-2 comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

The invention also provides a method of reducing morbidity and mortality in a patient infected with SARS-CoV-2, wherein the patient has active pneumonia, comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

The invention also provides a method of mitigating lung injury leading to ARDS in a patient infected with SARS-CoV-2, comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating a lung inflammation in a patient infected with SARS-CoV-2 comprising administering to the patient in need of such treatment a therapeutically effective amount of a MEK inhibitor, preferably or a pharmaceutically acceptable salt thereof and whereby the lung inflammation is reduced in the subject.

In some embodiments, the lung inflammation comprises bronchial epithelial cells infected with SARS-CoV-2. Definitions

As used herein, the singular form "a", "an", and "the" include plural references unless indicated otherwise. For example, "a" substituent includes one or more substituents.

The term “about” when used to modify a numerically defined parameter (e.g., the dose of an inhibitor, the dose of a drug and the like) means that the parameter may vary by as much as 10% above or below the stated numerical value for that parameter. For example a dose of about 5 mg/kg should be understood to mean that the dose may vary between 4.5 mg/kg and 5.5 mg. kg.

The term “BID,” as used herein means administration of drug twice a day to patients.

The term “QD,” as used herein means administration of drug once a day to patients.

The term “immune” or “immune system,” as used herein means the innate and adaptive immune systems.

The term “patient” or “subject,” as used herein, means a human being in need of the treatments or therapies as described herein.

The term “treating” or “treatment” means an alleviation of symptoms associated with the relevant virus, for example with COVID-19 disease, or halt of further progression or worsening of those symptoms, including where applicable, syndrome coronavirus 2, severe acute respiratory syndrome (SARS) and/or acute respiratory distress syndrome (ARDS). Depending on the condition of the patient, the term “treatment” as used herein may include one or more of curative, palliative and prophylactic treatment. Treatment can also include administering a pharmaceutical formulation of the present invention in combination with other therapies.

The term "therapeutically-effective" indicates the capability of an agent to prevent or improve the severity of the underlying viral disease, for example COVID-19 disease, or halt of further progression or worsening of those symptoms, including, where applicable, syndrome coronavirus 2, severe acute respiratory syndrome (SARS) and acute respiratory distress syndrome (ARDS), while avoiding adverse side effects typically associated with alternative therapies.

“Pharmaceutically acceptable” means suitable for use in a “patient” or “subject.”

“Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a therapeutic agent of a method or regimen of the invention. “Ameliorating” also includes shortening or reduction in duration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug, compound or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired, including biochemical, histological and I or behavioral symptoms, of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, a “therapeutically effective amount” refers to that amount of a compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. An effective dosage can be administered in one or more administrations. For the purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition.

Each ofthe embodiments of the present invention described below may be combined with one or more other embodiments of the present invention described herein which is not inconsistent with the embodiment(s) with which it is combined. In addition, each of the embodiments below describing the invention envisions within its scope the pharmaceutically acceptable salts, solvates, hydrates and complexes thereof, and to solvates, hydrates and complexes of salts thereof, including polymorphs, stereoisomers, and isotopically labelled versions thereof of the compounds of the invention. Accordingly, the phrase “or a pharmaceutically acceptable salt thereof’ is implicit in the description of all compounds described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A provides a graph showing the dose-dependent effect of binimetinib (ARRY-162) reducing viral titer in SARS-CoV-2 infected human airway epithelial cells. Results are shown as percent (%) virus inhibition as compared to uninfected cells at day 3 and day 5 post-infection.

Figure 1 B provides a graph showing the dose-dependent effect of remdesivir reducing viral titer in SARS-CoV-2 infected human airway epithelial cells. Results are shown as percent (%) virus inhibition as compared to uninfected cells at day 3 and day 5 post-infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.

There are three clinical phases associated with patients infected with SARS-CoV-2.

The first phase is characterized by robust virus replication that initiates the patient’s antiviral defense that includes: early IFN response; inflammatory monocyte-macrophage and neutrophil infiltration; and pro-inflammatory cytokines and chemokines. An effective endogenous response at this stage leads to: minimal epithelial and endothelial cell apoptosis; reduced vascular leakage; optimal T cell and antibody responses; reduced virus replication and effective virus clearance. Reducing MEK dependent cytokines at this stage may not be desired, but inhibiting MEKto reduce viral replication and viral titers as well as to enhance IFN response would be desired.

The second phase is associated with high fever, hypoxemia, and progression to pneumonia-like symptoms despite a progressive decline in virus titers towards the end of this phase. Anti-viral cytokines and chemokines at this stage lead to an overexuberant response including monocyte/macrophage and polymorphonuclear leukocytes (neutrophils, eosinophils, and basophils). Reducing pathologic levels of MEK dependent cytokines/chemokines may have benefit at this stage of disease.

The third phase, of which ~20% of patients progress, is characterized by ARDS and often results in death. Due to the progressive decline in virus titers, this phase may result from overexpression of pro-inflammatory cytokines/chemokines. Reducing pathologic levels of MEK dependent cytokines/chemokines may have benefit at this stage of disease.

The present invention provides a method of treating a patient infected with SARS-CoV-2 comprising administering to the patient in need of such treatment a therapeutically effective amount of MEK inhibitor, preferably, binimetinib or a pharmaceutically acceptable salt thereof.

Binimetinib is a potent inhibitor of mitogen-activated extracellular signal regulated kinase 1 (MEK1) and MEK2 activity, having the structure.

Binimetinib is also known as 5-[(4-bromo-2-fluorophenyl)amino]-4-fluoro-N-(2- hydroxyethoxy)-1-methyl-1 H-benzimidazole-6-carboxamide and 6-(4-bromo-2- fluorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)- amide (and also known as ARRY-162). Binimetinib is described in WHO Drug information 2014, 28(1), 78-79. Binimetinib and pharmaceutically acceptable salts thereof are disclosed in International patent application PCT/US2003/007864, which published on March 13, 2003 as Publication No. WO 03/077914. Crystallized binimetinib is disclosed in International patent application PCT/US2013/065633 which published on October 18, 2013 as Publication No. WO 2014/063024. The contents of each of the foregoing references are incorporated herein by reference in their entirety.

Binimetinib is an orally bioavailable, potent, selective, allosteric small-molecule inhibitor of MEK1/2 that is uncompetitive with adenosine triphosphate. MEK1/2 are key intracellular enzymes in the RAS/RAF/MEK/ERK signal transduction pathway. Specifically, inhibition of the RAS/RAF/MEK/ERK pathway has the potential to block cytokine signaling. MEK inhibition selectively blocks biosynthesis of several inflammatory cytokines, including IL-1 p, IL-6, and TNFa. Binimetinib has also demonstrated anti-tumor activity via direct effects on tumor cell proliferation as a single agent and in combination with a BRAF inhibitor, encorafenib.

Binimetinib has been shown to reduce inflammatory cytokines (TNF, IL-1 , IL-6) in healthy subjects and in patients with rheumatoid arthritis, and, therefore, could ameliorate the cytokine storm that is the predecessor to ventilator dependence and MOF in patients infected with SARS- CoV-2. Published studies have shown the RAS/RAF/MEK/ERK pathway also plays a role in the progression of infectious diseases, and, particularly, viral disease. The antiviral activity of MEK inhibitors, and the fact that inhibition of the cascade does not severely affect cell viability, suggest that the signaling pathway serves as a new target for an antiviral approach. MEK inhibition promotes anti-viral defense in rhinovirus and has been shown to inhibit replication (direct interference with viral RNA synthesis) of mouse coronaviruses and H1 N1 influenza virus in mouse. Inhibition of MEK with a MEK inhibitor, and specifically, binimetinib, will be clinically beneficial in producing an anti-viral effect and in down-regulating the cytokine-driven inflammation, in particular, that driven by IL-6, TNF and IL-1 , in patients. Binimetinib has been demonstrated to reduce IL-1 , TNFa, and IL-6 levels in human whole blood (IC50s of 23, 21 , and 21 nM, respectively) (Koch et al., EULAR Barcelona, Spain, 2007, Ann Rheum Dis 66 (suppl II): Abstract SAT0051 , 444).

Binimetinib has been shown to reduce IL-6 levels in treated rats. The efficacy of repeated doses of binimetinib with and without methotrexate was evaluated in rats with adjuvant-induced arthritis (AIA). Female Lewis rates (7-9 weeks of age; 8 per group for arthritis, 4 per group for control) were acclimated for 4-8 days after arrival. Arthritis was induced in animals by injections of lipoidal amine (LA), 7 mg in Freund’s Complete Adjuvant on pre-Study Day 0. For the LA injections, the rats were anesthetized with isoflurane and given 100 pL of LA, intradermal at the base of the tail. Animals randomized to the binimetinib treatment groups received 1 , 3, or 10 mg/kg QD, PO, or vehicle for 7 days. The binimetinib combination treatment group received once daily doses of binimetinib or vehicle with methotrexate (0.05 mg/kg, QD) on Study Days 1-14. Serum samples were obtained at necropsy to evaluate IL-6 concentrations as a measure of systemic anti-inflammatory activity. Methotrexate alone had no effect on serum IL-6 concentrations. Binimetinib significantly reduced serum IL-6 concentrations (33% at 1 mg/kg; 61 % at 30 mg/kg). Greater inhibition of serum IL-6 was observed when binimetinib was dosed in combination with methotrexate (74% at 1 mg/kg; 88% at 10 mg/kg).

In one embodiment, the present invention also provides a method of treating a patient infected with SARS-CoV-2 by administering a MEK inhibitor such as binimetinib such that one or more cytokines selected from the group consisting of interleukin-1 p (IL-1 p), tumor necrosis factor a (TNFa), interleukin-6 (IL-6) or prostaglandin E2 (PGE2) is reduced in said patient, preferably, by at least 25%, 50%, or 70-80% for a sustained period following multiple doses (e.g., 5, 7, 10, or 14 daily doses) or reduced for at least 2, 4 or 12 hours following a single dose. Preferably one or more cytokines selected from the group consisting of interleukin-1 p (IL-1 p), tumor necrosis factor a (TNFa), interleukin-6 (IL-6) or prostaglandin E2 (PGE2) is reduced in said patient following at least 14 daily doses of a MEK inhibitor. Preferably, the one or more cytokines are reduced by at least 25%.

In phase 1 clinical studies, TNFa levels were reduced following TPA stimulation for at least 4 hours (60% inhibition post treatment at 1 hour, 54% inhibition at 2 hours, 42% inhibition at 4 hours). Inhibition was also seen in IL-1 p levels for at least 4 hours following TPA stimulation in these cohorts. In another study with a single ascending dose study of binimetinib, healthy volunteers were administered an oral dose of binimetinib (5, 10, 20, 30, 40 or 80 mg), blood was drawn at various times after dosing and stimulated ex vivo with TPA. Binimetinib was well- tolerated and drug exposure was dose-proportional. In ex vivo samples, there was both a time- and concentration-dependent inhibition of TPA-induced IL-1 p and TNFa, with more than 80% inhibition observed at plasma concentrations of binimetinib of greater than 50 ng/ml and greater than 150 ng/ml, respectively (Koch et al, 2007, supra).

The present invention also provides a method of treating respiratory syncytial virus (RSV) infection in a patient, comprising administering to the patient in need of such treatment a therapeutically effective among of a MEK inhibitor, preferably binimetinib, a crystalline form, or a pharmaceutically acceptable salt thereof.

As used herein, the term “respiratory syncytial virus infection” refers to a subject who is infected with the RSV virus and, therefore, may exhibit RSV-associated disorders or symptoms including, but not limited to, nasal congestion, nasal flaring, coughing, rapid breathing, breathing difficulty, fever, shortness of breath, wheezing and hypoxia. RSV infection may also result in respiratory complications such as pneumonia, bronchiolitis, bronchitis and croup. Methods of treatment of RSV infection include acute management and chronic management of the disease.

In some embodiments of the method of treating respiratory syncytial virus (RSV) infection, the method further comprises administering a therapeutically effective amount of additional therapeutic agent that is an anti-viral agent, an anti-cytokine agent such as an anti-TNFalpha antibody, a bronchodilator drug, supplemental oxygen or a corticosteroid.

The present invention also provides a method of treating herpes simplex 1 infection in a patient, comprising administering to the patient in need of such treatment a therapeutically effective among of a MEK inhibitor, preferably binimetinib or a pharmaceutically acceptable salt, thereof.

As used herein, the term “herpes simplex 1 infection” refers to a subject who is infected with the herpes simplex 1 virus and, therefore, may exhibit herpes simplex 1 associated disorders or symptoms including, but not limited to, skin lesions such as sores or blisters and associated symptoms such as skin tingling, itching or burning sensation.

In some embodiments of the method of treating herpes simplex 1 infection, the method further comprises administering a therapeutically effective amount of additional therapeutic agent that is gamma interferon antibody, anti-TNF-alpha agent, an antibody to IL-1 , or topical retinoid.

The present invention also provides a method of treating hepatitis C virus infection in a patient, comprising administering to the patient in need of such treatment a therapeutically effective among of a MEK inhibitor, preferably binimetinib or a pharmaceutically acceptable salt thereof.

As used herein, the term “hepatitis C virus infection” refers to a subject who is infected with hepatitis C virus infected with HCV genotype 1 , 1 a, 1 b, 2, 3, 4, 5, or 6. In addition the subject may optionally be renal impaired, for example the subject may optionally have chronic kidney disease. Furthermore, the subject may optionally be without cirrhosis. Alternatively, the subject may optionally be with compensated cirrhosis.

In some embodiments of the method of treating hepatitis C virus infection, the method further comprises administering a therapeutically effective amount of additional therapeutic agent that is a protease inhibitor, a nucleoside or nucleotide polymerase inhibitor, a non-nucleoside polymerase inhibitor, a NS3B inhibitor, a NS4A inhibitor, a NS5A inhibitor, a NS5B inhibitor, or a cyclophilin inhibitor.

The present invention also provides a method of treating influenza A virus infection in a patient, comprising administering to the patient in need of such treatment a therapeutically effective among of a MEK inhibitor, preferably binimetinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the method of treating influenza A virus infection, the method further comprises administering a therapeutically effective amount of additional therapeutic agent that is a additional antiviral agents, such as oseltamivir or zanamivir or an adamantane such as amantadine and rimantadine.

The present invention also provides a method of treating a human coronavirus infection in a patient, comprising administering to the patient in need of such treatment a therapeutically effective among of a MEK inhibitor, preferably binimetinib or a pharmaceutically acceptable salt, thereof.

In one embodiment of the method of treating a human coronavirus infection, the human coronavirus infection is 229E (alpha coronavirus). In another embodiment of the method of treating a human coronavirus infection, the human coronavirus infection is NL63 (alpha coronavirus). In another embodiment of the method of treating a human coronavirus infection, the human coronavirus infection is OC43 (beta coronavirus). In another embodiment of the method of treating a human coronavirus infection, the human coronavirus infection is HKU1 (beta coronavirus). In another embodiment of the method of treating a human coronavirus infection, the human coronavirus infection is MERS-CoV (beta coronavirus that causes Middle East Respiratory Syndrome or MERS). In another embodiment of the method of treating a human coronavirus infection, the human coronavirus infection is SARS-CoV (beta coronavirus that causes severe acute respiratory syndrome or SARS).

MEK Inhibitors

As an alternative to binimetinib, other MEK inhibitors that can be employed in the present invention, include, for example, trametinib, cobimetinib, selumetinib, pimasertib, refametinib, N- [2(R),3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide (PD-325901),

2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (CI-1040), and

3-[2(R),3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3- d]pyrimidine-4,7(3H,8H)-dione (TAK-733), or 8-((2-fluoro-4-(methylthio)phenyl)amino)-2-(2- hydroxyethoxy)-7-methyl-3,4-dihydro-2,7-naphthyridine-1 ,6(2H,7H)-dione (PF-07799544 or ARRY-134) as disclosed in PCT /IB2022/052952, the contents of which are incorporated in their entirety herein.

In one embodiment, the present invention provides a method of treating a patient infected with a viral disease such as SARS-CoV-2 by administering a MEK inhibitor such as binimetinib such that one or more cytokines selected from the group consisting of interleukin-1 p (IL-1 p), tumor necrosis factor a (TNFa), interleukin-6 (IL-6) or prostaglandin E2 (PGE2) is reduced in said patient, preferably, by at least 25%, more preferably 50%, or even more preferably 70-80% for a sustained period following multiple doses (e.g., 5, 7, 10, or 14 daily doses) or reduced for at least 2, 4 or 12 hours following a single dose. Preferably one or more cytokines selected from the group consisting of interleukin-1 p (IL-1 p), tumor necrosis factor a (TNFa), interleukin-6 (IL-6) or prostaglandin E2 (PGE2) is reduced in said patient following at least 14 daily doses of binimetinib. Preferably, the one or more cytokines are reduced by at least 25%.

In one embodiment, the MEK inhibitor is binimetinib, or a pharmaceutically acceptable salt thereof.

In another embodiment, the MEK inhibitor is 8-((2-fluoro-4-(methylthio)phenyl)amino)-2- (2-hydroxyethoxy)-7-methyl-3,4-dihydro-2,7-naphthyridine-1 ,6(2H,7H)-dione (PF- 07799544 or ARRY-134), or a pharmaceutically acceptable salt thereof.

Further Therapeutic Agents

As used herein, the term “combination therapy” refers to the administration of each therapeutic agent of the combination therapy of the invention, either alone or in a medicament, either sequentially, concurrently or simultaneously. As used herein, the term “sequential” or “sequentially” refers to the administration of each therapeutic agent of the combination therapy of the invention, either alone or in a medicament, one after the other, wherein each therapeutic agent can be administered in any order. Sequential administration is particularly useful when the therapeutic agents in the combination therapy are in different dosage forms, for example, one agent is a tablet and another agent is a sterile liquid, and I or are administered according to different dosing schedules, for example, one agent is administered daily, and the second agent is administered less frequently such as weekly.

As used herein, the term “concurrently” refers to the administration of each therapeutic agent in the combination therapy of the invention, either alone or in separate medicaments, wherein the second therapeutic agent is administered immediately after the first therapeutic agent, but that the therapeutic agents can be administered in any order. In a preferred embodiment the therapeutic agents are administered concurrently.

As used herein, the term “simultaneous” refers to the administration of each therapeutic agent of the combination therapy of the invention in the same medicament.

The MEK inhibitor, preferably binimetinib, can be administered in combination with another or other agents, for example, another anti-viral therapeutic that targets the viral disease, including SARS-CoV-2.

For example, numerous therapeutics have been reported to effectively inhibit SARS-CoV- 2 replication since the outbreak of the pandemic in late 2019 (Tu et al., 2020, Int. J. Mol. Sci. 12: 2657). They mainly target the essential proteins in the life cycle of the virus. Remdesivir is a promising drug which interferes the viral genome replication by targeting RdRp (Warren et al., 2016, Nature 531 : 381-385). Remdesivir resembles the structure of adenosine, enabling it to incorporate into nascent viral RNA and result in premature termination of the viral RNA chain. Another recently reported potential drug is APN01 , which could inhibit SARS-CoV-2 replication in cellular and embryonic stem cell-derived organoids. It is a soluble recombinant human angiotensin-converting enzyme 2 (ACE2), and could prevent the activation of cellular ACE2, which is the host receptor for viral S protein (Monteil et al., 2020, Cell 181 : 905-913).

Numerous drug candidates which inhibit the 3CL protease activity and the maturation of nsps have been discovered, such as ebselen, disulfiram, carmofur, a-ketoamides, and peptidomimetic aldehydes 11 a/11 b (Dai et al., 2020, Science 368: 1331 -1335; Jin et al., 2020a, Nature 582: 289-293; Jin et al., 2020b, Nat. Struct. Mol. Biol. 27: 529-532; Zhang et al., 2020, Science 368: 409-412). Although several existing antiviral drugs have shown good results in clinical trials, continued efforts to discover new drugs that efficiently treat COVID-19 are ongoing.

Lopinavir and ritonavir were among the first drugs used in clinical trials to treat COVID-19 targeting 3CL protease (Cao et al., 2020, N. Engl. J. Med. 382: 1787-1799). They are inhibitors to human immunodeficiency virus (HIV) aspartyl protease, which is encoded by the pol gene of HIV and cleaves the precursor polypeptides in HIV (Walmsley et al., 2002, N. Engl. J. Med. 346: 2039-2046). The combination of lopinavir and ritonavir are commonly used as a therapeutic regimen for patients with HIV infection (Cvetkovic and Goa, 2003, Drugs 63: 769-802). Lopinavir was previously shown to inhibit 3CL protease of SARS-CoV in vitro (Wu et al., 2004, Proc. Natl. Acad. Sci. USA 101 : 10012-10017), and further studies demonstrated promising antiviral capacity of lopinavir/ritonavir against SARS-CoV and MERS-CoV (Chan et al., 2003, Hong Kong Med. J. 9: 399-406; Chan et al., 2015, J. Infect. Dis. 212: 1904-1913).

N3 is a Michael acceptor-based peptidomimetic inhibitor (Yang et al., 2005, PLoS Biol. 3: e324) which exhibits inhibition of SARS-CoV-2 3CL protease (Jin et al., 2020a, supra). Also identified as potent inhibitors are disulfiram, carmofur, Ebselen, shikonin, tideglusib, PX-12, and TDZD-8 (Jin et al., 2020a, supra) as well as bocepravir, GC-376, and calpain inhibitors II and XII (Ma et al., 2020, Cell Res. 31 : 678-692). Other molecules are disclosed in Rathnayake et al., 2020, Sci. Transl. Med. 12:eabc5332) and the review by Cui et al. (2020, Frontiers in Molecular Biosciences 7:Article 616341).

A preferred 3CL protease inhibitor for use in combination in the present invention is Paxlovid™, also referred herein as PF 07321332, (1R,2S,5S)-/V-{(1S)-1-Cyano-2-[(3S)-2- oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-A/-(trifluoroacetyl)-L-valyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide, or as nirmatrelvir and which is of the formula:.

PF-07321332

Another preferred 3CL protease inhibitor for use in combination in the present invention is ((S)-3-((S)-2-(4-methoxy-1 H-indole-2-carboxamido)-4-methylpentanamido)-2-oxo-4-((S)-2- oxopyrrolidin-3-yl)butyl dihydrogen phosphate) also referred herein as PF-07304814, which is cleaved by alkaline phosphatase enzymes in tissue, releasing the active antiviral (N-((S)-1-(((S)- 4-hydroxy-3-oxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-4- methoxy-1 H-indole-2-carboxamide) also referred herein as PF-00835231 (Boras et al., 2021 , BioRxiv, doi: 0.1101/2020,09.12.293498), and which are of formula:

Additional 3CL protease inhibitors that can be used in combination in the present invention are disclosed, for example, in International Patent Applications PCT/IB2021/051768, PCT/IB2021/052738, and PCT/IB2021/052689, in US Patent Application Ser. Nos. 17/221 ,676 and 17/395,139, and in US Provisional Patent Application Ser. Nos. 63/073,982, 63/143,435, 63/170,158, 63/050,766, 63/167,714, and 63/170,801 , US 63/194,241. All patent applications and provisional patent applications cited above are herein incorporated by reference.

Dosage Forms and Regimens

Each therapeutic agent of the methods of the present invention may be administered either alone, or in a medicament (also referred to herein as a pharmaceutical composition) which comprises the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients, or diluents, according to pharmaceutical practice.

When using binimetinib in each of the present invention described and claimed herein, the invention provides the method wherein said therapeutically effective amount for binimetinib is about 30 mg, 45 mg, 60 mg, or 75 mg, administered orally BID, as well as lower dosage amounts, 3 mg, 10 mg, 15 mg, 20 mg and 25 mg or eguivalents. Preferably, the therapeutically effective amount is about 30 mg or 45 mg administered BID.

When using in combination with the 3CL protease inhibitor PF-07321332, or a pharmaceutically acceptable salt thereof, in the present invention, the invention provides a method wherein said effective amount of PF-07321332 is about 300 mg of PF-07321332 twice per day, or a lower dosage amount of 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or an equivalent amount of PF-07321332 (nirmatrelvir) in the form of a pharmaceutically acceptable salt thereof.

In some embodiments the present invention provides a method comprising administering a therapeutically effective amount of PF-07321332 (nirmatrelvir), or a pharmaceutically acceptable salt thereof, wherein said effective amount of PF-07321332 (nirmatrelvir) is about 300 mg of PF-07321332 twice per day, or a lower dosage amount of 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, twice per day or the free base equivalent amount of PF-07321332 (nirmaltrelvir) in the form of a pharmaceutically acceptable salt thereof.

In some embodiments the present invention provides a method comprising administering a therapeutically effective among of the 3CL protease inhibitor is PF-07321332 (nirmatrelvir), or a pharmaceutically acceptable salt thereof, and further comprises administering to the patient a therapeutically effective amount of ritonavir, or a pharmaceutically acceptable salt thereof, wherein said effective amount of ritonavir, or a pharmaceutically acceptable salt thereof, is about 150 mg of ritonavir twice per day, or a lower dosage amount of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, twice per day, or the free base equivalent amount of ritonavir in the form of a pharmaceutically acceptable salt thereof. When the present invention comprises administering a therapeutically effective amount of ritonavir, or a pharmaceutically acceptable salt thereof, said ritonavir, or a pharmaceutically acceptable salt, thereof, is administered in combination with PF- 07321332 (nirmatrelvir), or a pharmaceutically acceptable salt, thereof. It is to be understood that the dosages may vary depending upon the requirements of each subject and the severity of the disorders or diseases being treated.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.

Also, it is to be understood that the initial dosage administered may be increased above highest dosage level in order to rapidly achieve the desired plasma concentration. On the other hand, the initial dosage may be smaller than the optimal dosage level and the daily dosage may be progressively increased during the course of treatment depending on the particular situation. If desired, the daily dose may also be divided into multiple doses for administration, e.g., two to four times per day.

In therapeutic use for treating disorders in a patient or subject, a compound of the present invention or its pharmaceutical compositions can be administered orally, parenterally, topically, rectally, transmucosally, or intestinally. Parenteral administrations include indirect injections to generate a systemic effect or direct injections to the afflicted area. It also includes transdermal delivery to generate a systemic effect. The rectal administration includes the form of suppositories. The preferred routes of administration are oral and parenteral.

Dosage regimens may be adjusted to provide the optimum desired response. For example, a therapeutic agent of the combination therapy of the present invention may be administered as a single bolus, as several divided doses administered overtime, orthe dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be particularly advantageous to formulate a therapeutic agent in a dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages forthe mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention may be dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose may be readily established, and the effective amount providing a detectable therapeutic benefit to a subject may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the subject. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a subject in practicing the present invention.

It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, taking into consideration factors such as the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. The dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well- known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

Pharmaceutical Compositions and Routes of Administration

A "pharmaceutical composition" refers to a mixture of one or more of the therapeutic agents described herein, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof as an active ingredient, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises two or more pharmaceutically acceptable carriers and/or excipients. As used herein, a "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active compound or therapeutic agent.

Pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, e.g., by means of conventional mixing, dissolving, granulation, levigating, emulsifying, encapsulating, entrapping, lyophilizing processes or spray drying.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients, diluents, and auxiliaries, which facilitate processing of the active compound into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant invention. Such excipients and carriers are described, for example, in Remington’s Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991). The formulations of the invention can be designed to be short-acting, fast-releasing, long-acting, and sustained-releasing. Thus, the pharmaceutical formulations can also be formulated for controlled release or for slow release.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose, i.e., treatment of a patient infected with SARS-CoV-2. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms/signs of the disease or prolong the survival of the subject being treated. The quantity of active component, which is the compound of this invention, in the pharmaceutical composition and unit dosage form thereof, may be varied or adjusted widely depending upon the manner of administration, the potency of the particular compound and the desired concentration. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, the quantity of active component will range between 0.01 % to 99% by weight of the composition. The dosage regimen for the compounds and/or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary widely. Dosage levels of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions. In one embodiment, the total daily dose of a compound of the invention (administered in single or divided doses) is typically from about 0.01 to about 100 mg/kg. In another embodiment, total daily dose of the compound of the invention is from about 0.1 to about 50 mg/kg, and in another embodiment, from about 0.5 to about 30 mg/kg (i.e., mg compound of the invention per kg body weight). In one embodiment, dosing is from 0.01 to 10 mg/kg/day. In another embodiment, dosing is from 0.1 to 1 .0 mg/kg/day. Dosage unit compositions may contain such amounts or submultiples thereof to make up the daily dose. In many instances, the administration of the compound will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired.

For oral administration, the compositions may be provided in the form of tablets containing from about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of active ingredient. Intravenously, doses may range from about 0.1 to about 10 mg/kg/minute during a constant rate infusion.

Kits

The therapeutic agents of the combination therapies of the present invention may conveniently be combined in the form of a kit suitable for coadministration of the compositions.

In one aspect, the present invention relates to a kit which comprises a first container, a second container and a package insert, wherein the first container comprises at least one dose of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, the second container comprises at least one dose of a further therapeutic agent, and the package insert comprises instructions for treating a subject.

In one embodiment, the kit of the present invention may comprise one or both of the active agents in the form of a pharmaceutical composition, which pharmaceutical composition comprises an active agent, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The kit may contain means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

The kit may be particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically includes directions for administration and may be provided with a memory aid. The kit may further comprise other materials that may be useful in administering the medicaments, such as diluents, filters, IV bags and lines, needles and syringes, and the like.

It is to be understood that all references, publications and patent applications cited herein are incorporated by reference in their entireties.

EXAMPLES

In orderthat this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. Example 1 - Effect of binimetinib to inhibit SARS-CoV-2 virus in a cell-based assay

Binimetinib effects on SARS-CoV-2 virus inhibition was assessed in human airway epithelial cells and compared to remdesivir.

Test Compound: Prior to the assay, the compounds were dissolved in 100% DMSO at a concentration of 20 mg/ml and further diluted to the test dilutions in the MatTek (MatTek Corporation, Ashland, MD) culture medium (AIR-100-MM). Uninfected cells were used as controls.

Cell Culture: Antiviral activity was evaluated in differentiated normal human bronchial epithelial (dNHBE) cells in a BSL-3 facility. The dNHBE cells (EpiAirway) were obtained from MatTek and were grown on trans-well inserts consisting of approximately 1 .2 x 10⁶ cells in AIR-100-MM added to the basolateral side, with the apical side exposed to a humidified 5% CO₂ environment at 37 °C. On day 1 , dNHBE cells were infected with SARS-CoV-2 strain USA- WA1/2020 at a MOI of approximately 0.0015 50% of the cell culture infectious dose (CCID50) per cell, and treatment was carried out by inclusion of drug dilutions in basolateral culture media.

Experimental design: Each compound treatment (120 pl) and virus treatment (120 pl) was applied to the apical side. At the same time, the compound treatment (1 ml) was applied to the basal side for a 2-hour incubation. As a virus control, some of the cells were treated with placebo (cell culture medium only). Following the 2-hour infection, the apical medium was removed, and the basal side was replaced with fresh compound or medium (1 ml). The cells were maintained at the air-liquid interface. On day 5, the medium was removed and discarded from the basal side. Virus released into the apical compartment of the dNHBE cells was harvested by the addition of 400 pl of culture medium that was prewarmed at 37°C. The contents were incubated for 30 minutes, mixed well, collected, thoroughly vortexed and plated on Vero 76 cells for VYR titration. Duplicate wells were used for virus control and cell controls.

Determination of virus titers from each treated cell culture: At day 3 and day 5, virus released into the apical compartment was harvested by the addition of 0.4 ml culture media. The virus titer was then quantified by infecting Vero76 cells in a standard endpoint dilution assay and virus dose that was able to infect 50% of the cell cultures (CCID50 per ml) was calculated (Reed and Muench, 1938, Am J Hygiene 27: 493-497, doi: 10.1093/oxfordjournals.aje.a1 18408). To determine the EC50 and EC₉₀ values, the CCID₅o/ml values were normalized to that of no drug control as a percentage of inhibition and plotted against compound concentration in GraphPad Prism software by using four-parameter logistic regression. Untreated, uninfected cells were used as the cell controls.

Results: Dose-dependent viral inhibition was demonstrated for both binimetinib (ARRY- 162) and remdesivir at day 3 and day 5 as shown in Figure 1 . Binimetinib EC50 at day 3 was 258.4 nm. EC50 at day 5 was not calculated but was estimated at around 10 nM. In comparison, remdesivir EC50s at day 3 and day 5 were 5.3 nM and 6.9 nM, respectively.

Example 2 - In vivo assay to determine the effect of binimetinib on SARS-CoV-2 in MA-SARS- CoV-2 mouse infection model

To determine the effect of ARRY- 162 (binimetinib) as an inhibitor of SARS-CoV-2 infection in the MA-SARS-CoV-2 mouse infection model, a typical study will include the following groups for evaluation: 1) vehicle alone, 2) binimetinib at dose 3 mg/kg BID, 3) binimetinib at dose 10 mg/kg BID, 3) binimetinib at dose 30 mg/kg BID and 4) optionally a positive control group, dosed with a treatment such as a 3CL protease inhibitor Six animals per group will receive either treatment dose or positive control dosed orally or vehicle only daily during the duration of the study. Six animals in vehicle control group will receive vehicle only.

For virus challenge, mice will be anesthetized by intraperitoneal injection of ketamine/xylazine (50 mg/kg/5 mg/kg) prior to challenge by dosing intranasally with 1 x 10⁵ CCIDso of SARS-CoV-2-MA-10 (mouse adapted MA 10 virus), in a 90 pL inoculum volume. Animals will then be treated with the treatment dose or vehicle daily beginning four hours post infection by per oral administration of a 0.1 mL volume of drug, or vehicle, or optionally with a positive control dosed as needed. For oral administration drug will be solubilized in an appropriate vehicle such as 0.5% methylcellulose in water, containing 2% Tween80. Six animals per group will be euthanized on study day 4 by isoflurane inhalation and the right lung lobes will be fixed in 4% paraformaldehyde at 4°C for histopathology and the left lung lobes placed in 1 mL phosphate buffered saline (PBS) with sterile glass beads at -80°C to evaluate lung virus titers.

Virus lung titers will be determined using a standard CCID50 assay in Vero 76 cells. MA- SARS-CoV-2 lung titers will be quantified by homogenizing mouse lungs in 1 mL PBS using 1.0 mm glass beads and a Beadruptor. Endpoint dilution method will be used for virus titration as follows: Serial log™ dilutions of lung tissue homogenate will be plated in quadruplicate wells of 96-well microplates containing confluent monolayers of Vero 76 cells. The plates will be incubated at 37°C and 5% CO₂ for 6 days. The plates will then be scored by visual observation under a light microscope for the presence of cytopathic effect using a light microscope. Virus titer for each sample will be calculated by linear regression using the Reed-Muench method (Reed & Muench, 1938, Epidemiol'. 27, 493-497).

The fixed lung lobes will be shipped to an external histology laboratory for processing as follows: Group samples (n = 6) will be processed as one H&E-stained slide from each lung specimen. Blinded evaluation of each lung sample will be conducted by an experienced veterinary pathologist using a semi-quantitative analysis using four parameters: perivascular inflammation, bronchial or bronchiolar epithelial degeneration or necrosis, bronchial or bronchiolar inflammation, and alveolar inflammation. A 5-point scoring system for assessment of epithelial degeneration/necrosis and inflammation will be utilized (0-with normal limits; 1-mild, scattered cell necrosis/vacuolation, few/scattered inflammatory cells; 2-moderate, multifocal vacuolation or sloughed/necrotic cells, thin layer of inflammatory cells; 3-marked, multifocal/segmental necrosis, epithelial loss/effacement, thick layer of inflammatory cells; 4- severe, coalescing areas of necrosis, parenchymal effacement, confluent areas of inflammation. A total pathology score will be calculated for each mouse by adding the individual histopathological scores.

Example 3 - Protocol

Background

Multifocal interstitial pneumonia represents the most common cause of admission in intensive care units and death in SARS-CoV-2 infections. In our hospital up to 25% of admitted patients with pneumonitis require mechanical ventilation or oro-tracheal intubation within 5-10 days.

Although information about pathological and pathophysiological features of alveolar- interstitial damage is very limited, available data, mostly collected on other coronavirus infections with similar clinical presentation (SARS1 , MERS), seem to indicate as the primary pathogenic mechanism an intense cytokine storm with a consequent inflammatory infiltrate of the pulmonary interstitium, macrophage activation, giant cells formation and subsequent extended alveolar damage.

Preliminary evidence is accumulating about the efficacy of an aggressive treatment of coronavirus-induced inflammation, and case series have shown the effectiveness of anti-IL6 strategies in reducing the severity of multifocal interstitial pneumonia in patients affected by SARS-CoV-2, implying a major role of IL-6 in the pathogenesis of lung damage in these patients.

Based on the above rationale, we believe that inhibition of MEK will be clinically beneficial in down-regulating cytokine-driven, in particular, IL-6, TNF and IL- 1 , inflammation in patients with SARS-CoV-2 infection. The nonclinical data in models of viral disease may also to translate to beneficial anti-viral activity of MEK inhibition.

Aims

1) To decrease the virus-induced cytokine storm and prevent the developing of severe pulmonary function deterioration and multiple organ dysfunction, as monitored by the levels of key inflammatory cytokines (e.g., IL-6, IL-1 , TNF). 2) To reduce the rate of patients who need mechanical ventilation and/or oro-tracheal intubation (primary outcome). 3) To reduce infection- related mortality. 4) To obtain a preliminary assessment of anti-viral activity. Study Design: Prospective, single cohort, open-label pilot study

It is expected that 25% of patients will reguire mechanical ventilation within 7-10 days after hospital admission for SARS-CoV-2 related pneumonitis. To test the hypothesis that early administration of binimetinib will reduce this rate to <5%, 50 patients will be enrolled in the study (power 80%, alpha 0.05, excluded from analysis 5%). The planned study duration is 10 weeks.

Inclusion Criteria

Able to provide written informed consent; patients under guardianship may participate with the consent of their legally authorized guardian if permitted by local regulations. Age 18-80 years. SARS-CoV-2 infection diagnosed by rt-PCR or suspected SARS-CoV-2 infection with results of SARS-CoV-2 assay not available may be enrolled with prior Sponsor approval. Radiographic scan-confirmed interstitial pneumonia. Hospital admission within the previous 24 hours.

Adeguate bone marrow, organ function and laboratory parameters: Aspartate transaminase (AST) and alanine transaminase (ALT) < 2.5 x upper limit of normal (ULN); total bilirubin < 1.5 x ULN; serum creatinine < 1.5 x ULN or calculated creatinine clearance > 50 mL/min by Cockroft-Gault formula or estimated glomerular filtration rate > 50 mL/min/1 .73 m² using the Modification of Diet in Renal Disease Study (MDRD) Eguation; female patients must have negative serum or urine pregnancy test priorto enrollment; agreement to use effective contraception for 30 days for males and females (of childbearing potential) after last dose of study treatment.

Exclusion Criteria

Reguirement for mechanical ventilation at time of admission; history of thromboembolic or cerebrovascular events < 12 weeks prior to the first dose of study treatment (e.g., transient ischemic attacks, cerebrovascular accidents, hemodynamically significant deep vein thrombosis or pulmonary emboli); pregnancy or breastfeeding; known history of retinal degenerative disease, retinal vein occlusion or uncontrolled glaucoma reguire careful consideration of the risk:benefit of binimetinib treatment and prior investigator/sponsor approval.

Impaired cardiovascular function or clinically significant cardiovascular disease including, but not limited to, any of the following: history of acute coronary syndrome (ACS) within the last 6 months or active congestive heart failure (CHF) (i.e. New York Heart Association (NYHA) 3 or greater) or active uncontrolled hypertension (150/100 or greater) reguire a careful consideratiom of the risk: benefit and prior investigator/sponsor approval; LVEF < 50% as determined by multigated acguisition scan (MUGA) or extracorporeal membrane oxygenation (ECHO); uncontrolled hypertension defined as persistent systolic blood pressure > 150 mmHg or diastolic blood pressure > 100 mmHg despite optimal therapy; history of or current serious arrhythmia (atrial fibrillation (AF) and paroxysmal supraventricular tachycardia (PSVT) are allowed if controlled); baseline QTc interval > 480 msec or a history of prolonged QT syndrome.

Outcome

Primary Outcomes: Rate of patients needing mechanical ventilation to maintain SO2 >92%; rate of patients needing admission to the intensive care unit for oro-tracheal intubation and/or evidence of multiple organ dysfunction.

Secondary Outcomes: Rate of patients with evidence of pulmonary function deterioration, defined as worsening of SO₂ > 3 percentage points (with stable FiO₂) or decrease of PaO₂ >10% or decrease of PaO₂/FiO₂ ratio >50%; duration of hospitalization, measured in days; duration and incidence of new non-invasive ventilation or high flow oxygen use; duration and incidence of new oxygen use; duration and incidence of new ventilator or ECMO use; number of non-invasive ventilation/high flow oxygen free days; number of oxygenation free days; subject 14-day mortality; date and cause of death (if applicable); subject 28-day mortality; date and cause of death (if applicable); ventilator/ECMO free days.

Outcome Assessments

Patients will be evaluated at baseline (time 0) and followed for 14 days or until discharge. At baseline and every 24 hours (unless otherwise indicated), the following will be assessed: hemodynamic and respiratory parameters; changes in hematology, chemistry or coagulation parameters (every other day); arterial blood gases; physical exam (including mental status); viral load.

At baseline and at day +7 and +14, 7 ml of serum will be stored to evaluate the serum levels of cytokines and other exploratory analyses. Patients who require mechanical ventilation or ICU transfer within 24 hours from hospital admission will be excluded from analysis.

Experimental Intervention

Patients will receive binimetinib 45 mg orally BID. For patients with moderate or severe hepatic impairment the recommended dose is 30 mg orally BID. Treatment will be started within 12 hours from admission and maintained for 14 days. Dose modifications for toxicities associated with binimetinib should be made in accordance with the local prescribing information.

Concomitant Treatments

All patients should be treated with hydroxychloroquine (400-600 mg/day) and low molecular weight heparin subcutaneously as per local guidance. Other treatments such as antivirals, antibiotics, or other supportive therapies are permitted and may be administered as per local guidance.

Rescue Therapy

In patients who require mechanical ventilation, binimetinib treatment can be stopped and rescue therapy started according to institutional standards. Safetv Evaluations

Patients will be evaluated for adverse events every day by clinical examination. Blood examinations will be performed every alternate day.

Important potential adverse effects associated with the administration of binimetinib in combination with encorafenib identified primarily from safety data from the COLUMBUS study (BRAF mutation-positive melanoma) and, where indicated, from other studies of the combination, include:

1 . Left ventricular dysfunction: Symptomatic or asymptomatic decreases in ejection fraction occurred in 7% of patients, with Grade 3 left ventricular dysfunction occurring in 1 .6% of patients.

2. Hemorrhage: Hemorrhage occurred in 19% of patients, with events > Grade 3 occurring in 3.2% of patients. Fatal intracranial hemorrhage in the setting of new or progressive brain metastases occurred in 1 .6% of patients. The most frequent hemorrhagic events were gastrointestinal, including rectal hemorrhage (4.2%), hematochezia (3.1 %), and hemorrhoidal hemorrhage (1 %).

3. Venous thromboembolism: Occurred in 6% of patients, including 3.1 % of patients who developed pulmonary embolism.

4. Ocular toxicities: Serous retinopathy is a class effect of MEK inhibitors. It is generally asymptomatic or mildly symptomatic and reversible (Urner-Bloch et al. 2016, Eur J Cancer 65: 130-138). Serous retinopathy occurred in 20% of patients. Symptomatic serous retinopathy occurred in 8% of patients with no cases of blindness. The median time to onset of the first event of serous retinopathy (all grades) was 1 .2 months. RVO is a known class-related adverse reaction of MEK inhibitors and may occur in patients treated with binimetinib in combination with encorafenib. In patients with BRAF mutation-positive melanoma across multiple clinical trials, 0.1 % of patients experienced RVO.

5. Pneumonitis/lnterstitial Lung Disease: Pneumonitis occurred in 0.3% of patients with BRAF mutation-positive melanoma across multiple clinical trials.

6. Hepatotoxicity: The incidence of Grade 3 or 4 increases in liver function laboratory tests was 6% for alanine aminotransferase (ALT), 2.6% for aspartate aminotransferase (AST), and 0.5% for alkaline phosphatase. No patient experienced Grade 3 or 4 serum bilirubin elevation.

7. CK Elevation/Rhabdomyolysis: Asymptomatic elevations of laboratory values of serum

CK occurred in 58% of patients. Rhabdomyolysis was reported in 0.1% of patients with BRAF mutation-positive melanoma across multiple clinical trials.

8. Embryo-Fetal Toxicity: Binimetinib can cause fetal harm when administered to pregnant women. Any event will be recorded on patient’s documentation and case report form (CRF). All adverse events (AEs), serious and nonserious (including the exacerbation of a pre-existing condition) and regardless of causality to study drug, will be fully recorded on the appropriate eCRF. For each AE, the Investigator must provide its duration (start and end dates or ongoing), severity (intensity), assessment of causality and whether specific action or therapy was required and whether action was taken with regard to study drug treatment.

Stopping Rule Enrollment will be suspended for detailed case review if more than two serious treatment-related adverse events are reported.

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention.

Claims

- 27 - We Claim:

1 . A method of treating a patient infected with SARS-CoV-2 comprising administering to the patient in need of such treatment a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 comprising administering 45 mg of binimetinib orally, twice daily, or an equivalent amount of binimetinib in the form of a pharmaceutically acceptable salt thereof.

3. The method of claim 1 comprising administering 30 mg of binimetinib orally, twice daily, or an equivalent amount of binimetinib in the form of a pharmaceutically acceptable salt thereof.

4. The method of claim 1 comprising administering 60 mg of binimetinib orally, twice daily, or an equivalent amount of binimetinib in the form of a pharmaceutically acceptable salt thereof.

5. The method of any of claims 1-4, comprising administering binimetinib in combination with a protease inhibitor.

6. The method of any of claims 1-5, wherein one or more cytokine selected from the list of consisting of tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin- 8 (IL-8) levels, is reduced for at least 4 hours after administering binimetinib.

7. The method of claim 6, wherein the cytokine is reduced by at least 40%.

8. The method of claim 6, wherein the cytokine is reduced by at least 80%.

9. A method of treating a patient infected with SARS-CoV-2 comprising administering to the patient in need of such treatment a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a 3CL protease inhibitor.

10. The method of claim 9, wherein the 3CL protease inhibitor is PF-07321332 or a pharmaceutically acceptable salt thereof.

11 . The method of claim 9, wherein the 3CL protease inhibitor is PF-07304814 or PF- 00835231 or a pharmaceutically acceptable salt thereof.

12. The method of claim 10, comprising administering a dose of 300 mg of PF-07321332 twice per day, or an equivalent amount of PF-07321332 in the form of a pharmaceutically acceptable salt thereof.

13. The method of claim 10, further comprising administering to the patient a therapeutically effective dose of ritonavir, or a pharmaceutically acceptable salt thereof.

14. The method of any of claims 12 - 13, further administering a dose of 150 mg of ritonavir twice per day, or an equivalent amount of ritonavir in the form of a pharmaceutically acceptable salt thereof.

15. The method of any of claims 12 -14, wherein ritonavir, or a pharmaceutically acceptable salt thereof, is co-administered with PF-07321332, or a pharmaceutically acceptable salt thereof.

16. The method of any one of claims 1-1 1 , wherein the patient is symptomatic or asymptomatic from the coronavirus infection.

17. The method of claim 12, wherein the patient is symptomatic from the coronavirus infection with one or more symptom selected from the group consisting of fever, dry cough, diarrhea, headache, lymphopenia, hypoxia, shortness of breath, transaminitis, pulmonary edema, acute hypoxemic respiratory failure, kidney failure, respiratory failure, pneumonia, macrophage activation syndrome (MAS), cytokine storm, severe acute respiratory syndrome (SARS), acute respiratory distress syndrome (ARDS), and cardiac failure.

18. A method for treating severe acute respiratory syndrome (SARS) or acute respiratory distress syndrome (ARDS) comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

19. A method for reducing a risk that the patient will develop severe acute respiratory syndrome (SARS) or acute respiratory distress syndrome (ARDS) comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

20. The method of claim 15, wherein the patient has tested positive for infection by SARS- Cov-2.

21. The method of any of claims 14-16, wherein the patient has one or more symptoms selected from the group consisting of fever, dry cough, diarrhea, headache, lymphopenia, hypoxia, shortness of breath, transaminitis, pulmonary edema, acute hypoxemic respiratory failure, kidney failure, respiratory failure, pneumonia, MAS or cytokine storm.

22. The method of any of claims 14-17, wherein the method further comprises administering a therapeutically effective amount of additional therapeutic agent that is an anti-viral agent, an anti-cytokine agent, a steroid, or an anti-inflammatory agent.

23. The method of claim 18, wherein the additional therapeutic agent is a 3CL protease inhibitor.

24. The method of claim 19, wherein the 3CL protease inhibitor is PF-07321332 or a pharmaceutically acceptable salt thereof.

25. The method of claim 19, wherein the 3CL protease inhibitor is PF-07304814 or PF- 00835231 or a pharmaceutically acceptable salt thereof.

26. The method of claim 24, comprising administering a dose of 300 mg of PF-07321332 twice per day, or an equivalent amount of PF-07321332 in the form of a pharmaceutically acceptable salt thereof.

27. The method of claim 23, further comprising administering to the patient a therapeutically effective dose of ritonavir, or a pharmaceutically acceptable salt thereof.

28. The method of any of claims 24 - 27, further administering a dose of 150 mg of ritonavir twice per day, or an equivalent amount of ritonavir in the form of a pharmaceutically acceptable salt thereof.

29. The method of any of claims 25 - 27, wherein ritonavir, or a pharmaceutically acceptable salt thereof, is co-administered with PF-07321332, or a pharmaceutically acceptable salt thereof.

30. The method of claim 18, wherein the anti-cytokine agent is selected from an anti-IL-6 agent, anti-IL-1 agent, and an anti-TNF agent.

31. A method of reducing viral load in a patient infected with SARS-CoV-2 comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

32. A method of reducing morbidity and mortality in a patient infected with SARS-CoV-2, wherein the patient has active pneumonia, comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.

33. A method of mitigating lung injury leading to ARDS in a patient infected with SARS-CoV- 2, comprising administering to a patient in need thereof a therapeutically effective amount of binimetinib or a pharmaceutically acceptable salt thereof.