Treatment of diseases caused by RNA viruses

Abstract

Caspase upregulation has been found in COVID-19 patients and also occurs in patients with diabetes, hypertension, metabolic syndrome, and other conditions. Such upregulation leads to the death and dysfunction of immune system cells, particularly certain types of lymphocytes, through a process called pyroptosis, and may contribute to poor clinical outcomes in patients with these diseases or disorders. Caspase inhibitors, such as caspase-1 inhibitors, or "pan-caspase" inhibitors (i.e., inhibitors of multiple caspase types, including caspase-1), can be used to treat COVID-19 patients or individuals at risk of infection by limiting immune system dysfunction. Caspase-1 or pan-caspase inhibitors are advantageously administered before or very early in infection with a positive-sense single-stranded RNA virus, such as SARS-CoV, MERS-CoV, or SARS-CoV-2. TIFF2023522339000012.tif82168

Description

[Technical Field]

[0001] Related Applications This application and the inventions described herein are related to U.S. Provisional Patent Applications Nos. 63 / 009,786, 63 / 014,109, 63 / 047,888, 63 / 055,310, 63 / 109,320, 63 / 109,867, and 63 / 161,905, filed April 14, 2020, April 22, 2020, July 2, 2020, July 22, 2020, November 3, 2020, November 4, 2020, and March 16, 2021, respectively, and the inventions described therein, each of which is incorporated by reference herein in its entirety for any and all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to the field of viral infections. In particular, the present invention relates to the prevention and treatment of viral infections, especially viral infections caused by positive-sense single-stranded RNA viruses, such as SARS-CoV-1, MERS, and SARS-CoV-2. [Background technology]

[0003] The novel coronavirus, SARS-CoV-2, was first identified in Wuhan, China, in December 2019. SARS-CoV-2 is contagious in humans and causes a respiratory illness called COVID-19 (coronavirus disease, 2019). The World Health Organization designated the Chinese SARS-CoV-2 outbreak a pandemic when it spread to many countries around the world, and COVID-19 now poses significant challenges to global public health, research, medicine, the economy, and society as a whole. Person-to-person spread is typically via respiratory droplets. This can cause severe upper and lower respiratory tract infections, potentially leading to death, especially in the elderly and those with underlying medical conditions. Symptoms are sometimes delayed or unnoticed, allowing for person-to-person spread.

[0004] Although the U.S. Food and Drug Administration has granted emergency use authorization for the drug candidate remdesivir (Gilead Sciences, Inc.), an antiviral compound that may be effective in shortening COVID-19 recovery time in hospitalized patients, no specific therapeutics have yet been approved by government regulatory agencies to prevent or treat SARS-CoV-2 infection. There remains a continuing need in the art to identify druggable targets that may reduce the severity of infection.

[0005] Currently, there are only a few potential therapies for COVID-19, including remdesivir and dexamethasone. The excessive inflammation and tissue damage associated with COVID-19 can lead to acute health problems (e.g., respiratory failure, sepsis, and ultimately death) or chronic health problems (e.g., fatigue, difficulty breathing, cough, joint pain, loss of smell), and the risk of these complications is higher among older populations, certain ethnic groups, and people with other comorbidities. Cellular caspases play a role in the pathophysiology of COVID-19.

[0006] Coronavirus disease 2019 (COVID-19) is the latest global health threat and, like the two preceding examples of coronavirus respiratory illnesses, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), poses significant challenges for the public health, research, and medical communities. Active research efforts are currently underway to develop a vaccine against SARS-CoV-2, the causative agent of COVID-19, while various investigational therapeutic approaches are also being investigated. Although the pathology of COVID-19 is now well described, the mechanisms underlying disease progression remain unclear. In clinical trials, dexamethasone reduced severe outcomes in critically ill patients, suggesting an inflammatory mechanism. However, although the use of specific anti-interleukin-6 antibody (anti-IL-6 Ab) treatment has been reported to attenuate the so-called "cytokine storm" associated with COVID-19, and eculizumab reduced inflammatory markers and C-reactive protein (CRP) in moderate to severe COVID-19, clinical benefit from such more targeted therapies has been minimal. These findings suggest a lack of understanding of the effector molecules involved in disease progression or a need for earlier intervention in the disease process.

[0007] Although the respiratory and gastrointestinal systems are the initial targets of SARS-CoV-2, the disease is clearly systemic in some individuals and appears to be driven by microemboli and inflammation, although the full impact of pathogenesis and clinical symptoms remains unclear. A marked impairment in type I interferon responses and rapid lymphopenia clearly play a role in disease severity. Better follow-up in natural progression studies will likely reveal additional postinfection sequelae. The severity of COVID-19 varies widely, from patients who exhibit no or only mild flu-like symptoms and rapidly recover, to those who experience persistent fever and persistent fatigue with postviral syndrome, to those with severe pulmonary complications resulting in hospitalization or the need for intubation and intensive care, and even death. This clearly highlights the need for novel therapeutics that take into account the mechanism(s) of infection, viral replication, and effector pathways that lead to COVID-19-related pathology.

[0008] Pyroptosis, also known as caspase-1-dependent cell death, is inflammatory in nature, triggered by various pathological stimuli (e.g., stroke, heart attack, cancer), and is important in controlling microbial infections. In stark contrast to the regulated death process of apoptosis, pyroptosis is characterized by rapid plasma membrane disruption and release of inflammation-induced intracellular contents.

[0009] COVID-19 primarily presents with respiratory symptoms but is in fact a systemic disease with a wide range of effects and post-infectious sequelae that are not yet fully understood. A range of symptoms is evident with COVID-19, with some people experiencing blood clots and neurological symptoms. A small number of children suffer from an autoimmune syndrome, suggesting that this is already a systemic disease for some people. The etiological implications and impact of clinical symptoms are not yet fully recognized. Some post-infectious sequelae will only be understood with good follow-up in natural progression studies. The range of severity of COVID-19 is very wide and in many ways unprecedented.

[0010] The role of caspases: apoptosis Inducible caspases are activated by the intrinsic and extrinsic apoptotic pathways, leading to the activation of other caspases, including executioner caspases, which carry out apoptosis by cleaving cellular components.

[0011] Apoptosis is a form of programmed cell death in which cells undergo morphological changes to minimize their impact on surrounding cells and avoid inducing an immune response. Cells shrink and condense, the cytoskeleton collapses, the nuclear membrane degrades, and genomic DNA is cleaved into fragments. As a result, cells form self-inclusion bodies called "blebs" to avoid the release of cellular components into the extracellular medium. Furthermore, the phospholipid content of the cell membrane changes, making dying cells more susceptible to phagocytic attack and removal.

[0012] Apoptotic caspases are subclassified as inducible caspases (caspases 2, 8, 9, and 10) and executioner caspases (caspases 3, 6, and 7). Once inducible caspases are activated, they initiate a chain reaction to activate several executioner caspases, which cleave over 600 cellular components to induce the morphological changes of apoptosis.

[0013] Examples of caspase cascades during apoptosis Intrinsic apoptosis pathway During cellular stress, mitochondrial cytochrome c is released into the cytosol. This molecule binds to an adaptor protein (APAF-1) that recruits inducible caspase-9 (via CARD-CARD interactions). This results in the formation of a caspase-activating multiprotein complex called the "apoptosome." Once activated, inducible caspases, such as caspase-9, cleave and activate other executioner caspases, resulting in the degradation of cellular components characteristic of apoptosis.

[0014] Extrinsic apoptosis pathwayThe caspase cascade can also be activated by extracellular ligands via cell surface death receptors. This occurs through the formation of a multiprotein death-inducing signaling complex (DISC), which recruits and activates procaspases. For example, Fas ligand binds to the FasR receptor on the extracellular surface of the receptor. This activates the death domain in the cytoplasmic tail of the receptor. The adaptor protein FADD recruits procaspase-8 via its DED domain (by death domain-death domain interaction). FasR, FADD, and procaspase-8 form the death-inducing signaling complex (DISC), where caspase-8 is activated. This can result in either downstream activation of the intrinsic pathway by inducing mitochondrial stress or direct activation of executioner caspases (caspase-3, caspase-6, and / or caspase-7) to degrade cellular components.

[0015] Pyroptosis Pyroptosis is a form of programmed cell death that inherently induces an immune response. It is morphologically distinct from other types of cell death, with cells swelling, bursting, and releasing proinflammatory intracellular contents. It occurs in response to a variety of stimuli, including microbial infections and heart attacks (myocardial infarction). In humans, caspase-1, caspase-4, and caspase-5 play key roles in inducing pyroptotic cell death, thereby limiting the lifespan and proliferation time of intracellular and extracellular pathogens.

[0016] Caspase-1-mediated pyroptosis Caspase-1 activation is mediated by a repertoire of proteins, allowing it to detect a variety of pathogenic ligands. Some mediators of caspase-1 activation are NOD-like leucine-rich repeats (NLRs), AIM2-like receptors (ALRs), pyrin, and IFI16.

[0017] These proteins enable caspase-1 activation by forming a multiprotein activation complex called the inflammasome. For example, the NOD-like leucine-rich repeat (NLRP3) senses potassium ion efflux from cells. This cellular ion imbalance leads to oligomerization of NLRP3 molecules to form a multiprotein complex called the NLRP3 inflammasome. Procaspase-1 is brought into close proximity with other procaspase molecules to dimerize and undergo autoproteolytic cleavage.

[0018] Some pathogenic signals that lead to caspase-1-mediated pyroptosis include: DNA in the host cytosol binding to AIM2-like receptors that induce pyroptosis; and the bacterial type III secretion system apparatus that binds to NOD-like leucine-rich repeat receptors called NAIPs (one in humans, four in mice).

[0019] Caspase-4 and caspase-5-mediated pyroptosis in humans Caspases 4 and 5 have the ability to induce direct pyroptosis when lipopolysaccharide (LPS) molecules (found in the cell walls of Gram-negative bacteria) are found in the cytoplasm of host cells. For example, caspase 4 acts as a receptor and is proteolytically activated without the need for the inflammasome complex or caspase-1 activation.

[0020] A key downstream substrate of caspases that initiate pyroptosis is gasdermin D (GSDMD).

[0021] Role in inflammation Inflammation is a defensive attempt by the organism to restore homeostasis after disruption from harmful stimuli such as tissue injury or bacterial infection. Caspase-1, caspase-4, caspase-5, and caspase-11 are considered "inflammatory caspases."

[0022] Caspase-1 is important in the activation of pro-inflammatory cytokines, including IL-1β and IL-18. These act as signals to immune cells, creating a favorable environment for their recruitment to the site of injury. Thus, caspase-1 plays a fundamental role in the innate immune system. This enzyme is involved in the processing and secretion of cytokines such as pro-IL-1β and pro-IL-18.

[0023] Caspases-4 and -5 in humans have a unique role as receptors that bind LPS, a molecule abundant in Gram-negative bacteria. This can result in the processing and secretion of IL-1β and IL-18 cytokines by activating caspase-1. The downstream effects are similar to those described above. It also leads to the secretion of another unprocessed proinflammatory cytokine, called pro-IL1α. There is also evidence for caspase-11, a proinflammatory caspase, that assists in cytokine secretion. It does this by inactivating a membrane channel that blocks IL-1β secretion.

[0024] Caspases can also induce inflammatory responses at the transcriptional level. There is evidence that they promote the transcription of nuclear factor-κB (NF-κB), a transcription factor that assists in the transcription of inflammatory cytokines such as IFN, TNF, IL-6, and IL-8. For example, caspase-1 activates caspase-7, which then cleaves poly(ADP) ribose and activates the transcription of NF-κB-regulated genes. Summary of the Invention

[0025] According to one aspect of the present invention, there is provided a method of prophylactically treating a human subject or patient to reduce morbidity and mortality associated with infection by a positive-sense single-stranded RNA virus (e.g., SARS-CoV-1, MERS, and SARS-CoV2 (which causes COVID-19)). Prophylactic or therapeutic treatment is achieved by administering an effective amount of a caspase inhibitor, advantageously an inhibitor of at least caspase 1, i.e., caspase 1 alone or an inhibitor of caspase 1 and one or more other caspases (e.g., caspase 3, 4, 5, 7, 8, 9, and 11), to a patient or subject known or suspected to be infected with or at risk of infection with a particular positive-sense single-stranded RNA virus. Advantageously, such treatment reduces pyroptosis of T cells in the patient.

[0026] According to another aspect of the present invention, there is provided a method of treating patients infected with positive-sense single-stranded RNA viruses, e.g., SARS-CoV-1, MERS, and SARS-CoV2, to reduce morbidity and mortality associated with the specific positive-sense single-stranded RNA virus. In certain preferred embodiments of this aspect, an effective amount of an inhibitor of caspase 1 (or of multiple caspases) is administered to a patient known or suspected to be infected with the specific positive-sense single-stranded RNA virus. Advantageously, such treatment reduces pyroptosis of T cells in the patient.

[0027] Another aspect of the present invention provides a method for predicting the severity of disease caused by positive-sense single-stranded RNA viruses, such as SARS-CoV-1, MERS, and SARS-CoV-2. In such a method, a blood sample from a patient known or suspected of being infected with a particular positive-sense single-stranded RNA virus is tested to determine the level of caspase activity, preferably caspase 1 activity, present in the sample. The resulting caspase activity level is compared to the caspase activity level in one or more control samples from healthy individuals. If the activity level of a particular caspase(s), e.g., caspase 1 and / or caspase 3, is statistically significantly higher than the caspase 1 and / or caspase 3 activity level in the one or more control samples, the patient is determined to be at high risk for experiencing a severe form of disease associated with the particular positive-sense single-stranded RNA virus (e.g., COVID-19 in the case of SARS-CoV-2).

[0028] According to yet another aspect of the present invention, a method for predicting the severity of disease caused by positive-sense single-stranded RNA viruses (e.g., SARS-CoV-1, MERS, and SARS-CoV-2) in multiple patients is provided. Multiple blood samples from multiple patients known or suspected to be infected with a particular positive-sense single-stranded RNA virus (e.g., SARS-CoV-2) are tested to determine the level of caspase 1 activity in each blood sample. The determined amount in each blood sample is compared to the level of caspase 1 activity in one or more control samples from healthy individuals. A patient is determined to be at high risk for experiencing a severe form of the disease if the level of caspase 1 activity is statistically significantly higher than the level of caspase 1 in one or more control samples. Conversely, a patient is determined to be at low risk for experiencing a severe form of the disease if the level of caspase 1 is not statistically significantly higher than the level of caspase 1 in one or more control samples. Higher-risk individuals can be treated with a caspase inhibitor.

[0029] These and other embodiments that will be apparent to those of skill in the art upon reading this specification provide the art with methods of treating those at risk of infection and those infected, particularly those at highest risk of experiencing significant morbidity and mortality. [The present invention 1001] 1. A method of treating a human subject to prevent or reduce T cell lymphopenia or pyroptosis of T cells, comprising the steps of: administering to the human subject an effective amount of a caspase inhibitor of at least caspase 1, wherein the human subject: (a) the subject is at risk of contracting a disease caused by a positive-sense single-stranded RNA virus and has one or more comorbidities that are associated with a negative outcome from the disease if the subject contracts the disease; or (b) infected with a positive-sense single-stranded RNA virus; said administering reducing T cell lymphopenia or T cell pyroptosis. [The present invention 1002] 1001. The method of claim 1001, wherein said subject has a condition selected from the group consisting of hypertension, psoriasis, heart disease, gout, arthritis, inflammatory bowel disease, gouty arthritis, type I and type II diabetes, vitiligo, autoimmune Addison's disease, cryopreservation syndrome, and metabolic syndrome. [The present invention 1003] 1001. The method of claim 1001, wherein said positive-sense single-stranded RNA virus is selected from the group consisting of SARS-CoV, MERS-CoV, and SARS-CoV-2. [The present invention 1004] The method of claim 1001, wherein the human subject is infected with SARS-CoV-2. [The present invention 1005] 1001. The method of claim 1001, wherein said human subject has a condition in which caspase-1 activity is elevated in the immune cells of said human subject. [The present invention 1006] 1001. The method of claim 1001, wherein said human subject has a condition in which expression of caspase 1 is elevated in the immune cells of said human subject. [The present invention 1007] 1001. The method of claim 1001, wherein said caspase inhibitor is selected from the group consisting of emricasan, CTS-2090, vernacasan (VX-765), pralnacasan (VX-740), and O-desethyl-vernacasan (VRT-043198). [The present invention 1008] 1001. The method of claim 10, wherein said inhibitor is a peptide. [The present invention 1009] 1001. The method of claim 1001, wherein said caspase inhibitor inhibits one or more of caspases 3, 4, 5, 7, 8, 9, and 11. [The present invention 1010] 1001. The method of claim 10, wherein said inhibitor is a pan-caspase inhibitor. [The present invention 1011] 1001. The method of claim 10, wherein said inhibitor is emricasan. [The present invention 1012] 1. A method of treating a human subject at risk for or infected with SARS-CoV-2 infection to reduce the risk of morbidity and mortality associated with SARS-CoV-2, comprising: administering to said human subject an effective amount of a caspase inhibitor of at least caspase 1. A method comprising: [The present invention 1013] 1013. The method of claim 1012, wherein said caspase inhibitor inhibits one or more of caspases 3, 4, 5, 7, 8, 9, and 11. [The present invention 1014] The method of claim 1012, wherein the human subject has a condition selected from the group consisting of hypertension, psoriasis, heart disease, gout, arthritis, inflammatory bowel disease, gouty arthritis, type I and type II diabetes, vitiligo, autoimmune Addison's disease, cryopreservation syndrome, and metabolic syndrome. [The present invention 1015] 10. The method of claim 12, wherein said subject has a condition in which caspase 1 activity is elevated in the subject's immune cells. [The present invention 1016] 10. The method of claim 12, wherein said subject has a condition in which expression of caspase 1 is elevated in the subject's immune cells. [The present invention 1017] 1012. The method of claim 1012, wherein said caspase inhibitor is selected from the group consisting of emricasan, CTS-2090, vernacasan (VX-765), pralnacasan (VX-740), and O-desethyl-vernacasan (VRT-043198). [The present invention 1018] 1013. The method of claim 1012, wherein said caspase inhibitor is a peptide. [The present invention 1019] 1012. The method of claim 1012, wherein said caspase inhibitor is a pan-caspase inhibitor. [The present invention 1020] 1012. The method of claim 1012, wherein said caspase inhibitor is emricasan. [The present invention 1021] 1. A method of treating a human subject infected with or suspected of being infected with SARS-Cov2, comprising the steps of: Administering an effective amount of a caspase inhibitor useful for treating COVID-19, optionally before the onset of COVID-19 symptoms or while the subject has mild or moderate severity of COVID-19. [The present invention 1022] 1021. The method of claim 1021, wherein said caspase inhibitor inhibits caspase-1 and caspase-3. [The present invention 1023] 1. A method of treating a human subject infected with SARS-Cov2, comprising the steps of: Administering effective amounts of a) a compound that inhibits caspases, (b) a compound that binds to the ACE-2 receptor, and (c) a compound that binds to the main protease of SARS-CoV-2, wherein said administering occurs before the onset of COVID-19 symptoms or while the patient has progressed beyond mild or moderate COVID-19 symptoms. [The present invention 1024] The method of any one of claims 1012, 1021, or 1023, further comprising administering a compound selected from the group consisting of dexamethasone, an antibody or antibody cocktail that specifically binds to SARS-CoV-2, an antibody against IL-6, and remdesivir. [The present invention 1025] 1. A test method for predicting the severity of SARS-Cov-2 disease, comprising: testing a blood sample from the subject to determine the amount of caspase 1 and comparing the amount of caspase 1 detected with the amount of caspase 1 in one or more control samples from healthy individuals; determining that the subject is at increased risk of experiencing a severe form of the disease if the amount of caspase 1 in the subject's blood sample is statistically significantly greater than the amount of caspase 1 detected in the one or more control samples; A method comprising: [The present invention 1026] 1. A test method for predicting SARS-Cov-2 disease severity in a plurality of human subjects, comprising: testing a plurality of blood samples from a plurality of subjects infected with SARS-Cov-2 to ascertain the amount of caspase 1 in each blood sample, and comparing the amount of caspase 1 from at least one subject blood sample to the amount of caspase 1 detected in one or more control samples from healthy individuals; determining that the subject has a high risk of experiencing a severe form of the disease if the amount of caspase 1 in the subject's sample is statistically significantly greater than the amount of caspase 1 detected in the one or more control samples, and determining that the subject has a low risk of experiencing a severe form of the disease if the amount of caspase 1 in the subject's sample is not statistically significantly greater than the amount of caspase 1 detected in the one or more control samples; A method comprising: [The present invention 1027] 1027. The method of claim 1025 or 1026, wherein said test uses an antibody test. [The present invention 1028] 1027. The method of claim 1025 or 1026, wherein said testing uses flow cytometry. [The present invention 1029] 1027. The method of any one of claims 1025 to 1026, wherein said assay uses one or more fluorescently labeled inhibitors of caspase-1. [The present invention 1030] 1027. The method of any one of claims 1025 to 1026, wherein said assay uses one or more fluorescently labeled substrates of caspase-1. [The present invention 1031] The method of any one of claims 1025 to 1026, further comprising treating a patient determined to be at high risk for severe disease with an inhibitor of caspase-1. [The present invention 1032] 1027. The method of claim 1025 or 1026, wherein the subject further has a condition selected from the group consisting of hypertension, psoriasis, heart disease, gout, arthritis, inflammatory bowel disease, gouty arthritis, type I and type II diabetes, vitiligo, autoimmune Addison's disease, cryopreservation syndrome, and metabolic syndrome. [The present invention 1033] 1031. The method of claim 1031, wherein said inhibitor is selected from the group consisting of emricasan, vernacasan (VX-765), pralnacasan (VX-740), and O-desethyl-vernacasan (VRT-043198). [The present invention 1034] 1032. The method of claim 1031, wherein said inhibitor is a peptide. [This invention 1035] 1032. The method of claim 1031, wherein said inhibitor is a pan-caspase inhibitor. [The present invention 1036] 1031. The method of claim 10, wherein said inhibitor is emricasan. [This invention 1037] 1. A method of treating a human subject having or at risk of having abnormal red blood cell aggregation or thrombosis, comprising the steps of: Administering an effective amount of a caspase-3 inhibitor to the human subject, wherein the human subject is (a) at risk of suffering from a disease caused by a positive-sense single-stranded RNA virus or (b) infected with a positive-sense single-stranded RNA virus, and wherein the administration prevents or reduces abnormal hemagglutination or thrombosis. [The present invention 1038] 1037. The method of claim 1037, wherein the positive-sense single-stranded RNA virus is a β-coronavirus. [This invention 1039] 1037. The method of claim 1037, wherein the positive-sense single-stranded RNA virus is selected from the group consisting of SARS-CoV, MERS-CoV, and SARS-CoV-2. [The present invention 1040] The method of claim 1037, wherein said human subject is asymptomatic with respect to the positive-sense single-stranded RNA virus. [This invention 1041] The method of claim 1037, wherein the human subject has a condition selected from the group consisting of hypertension, psoriasis, heart disease, gout, arthritis, inflammatory bowel disease, gouty arthritis, type I and type II diabetes, vitiligo, autoimmune Addison's disease, cryopreservation syndrome, and metabolic syndrome. [The present invention 1042] 1038. The method of claim 1037, wherein said subject has a condition in which caspase-3 activity is elevated in one or more cell types in said subject. [This invention 1043] 1038. The method of claim 1037, wherein said subject has a condition in which expression of caspase-3 is elevated in one or more cell types in said subject. [This invention 1044] 1037. The method of claim 1037, wherein said caspase inhibitor is a pan-caspase inhibitor. [This invention 1045] 1037. The method of claim 1037, wherein said caspase inhibitor is emricasan. [The present invention 1046] 1. A method of treating a human subject at risk for SARS-CoV-2 infection or a subject infected with SARS-CoV-2 to reduce the risk of morbidity and mortality associated with SARS-CoV-2, comprising: Administering an effective amount of a caspase-3 inhibitor to the human subject, wherein said administration reduces the risk of an adverse outcome associated with SARS-Cov-2. [This invention 1047] 1046. The method of claim 1046, wherein administering said effective amount of an inhibitor reduces pyroptosis of red blood cells. [This invention 1048] The method of claim 1046, wherein said subject has a condition selected from the group consisting of hypertension, psoriasis, heart disease, gout, arthritis, inflammatory bowel disease, gouty arthritis, type I and type II diabetes, vitiligo, autoimmune Addison's disease, cryopreservation syndrome, and metabolic syndrome. [This invention 1049] 1046. The method of claim 1046, wherein said caspase-3 inhibitor is a pan-caspase inhibitor. [The present invention 1050] 1046. The method of claim 1046, wherein said caspase-3 inhibitor is emricasan. [This invention 1051] 1. A method of treating a human subject having or at risk of developing late sequelae of Covid-19 infection, comprising: administering to said human subject an effective amount of a caspase inhibitor of at least caspase 1. A method comprising: [This invention 1052] 1051. The method of claim 1051, wherein said caspase inhibitor inhibits one or more of caspases 3, 4, 5, 7, 8, 9, and 11. [This invention 1053] 1051. The method of claim 1051, wherein said caspase inhibitor is emricasan. [Brief explanation of the drawings]

[0030] [Figure 1] Innate immune response. CD16+ monocytes show significant upregulation of CD11b, and pro-inflammatory CD14+CD16+ monocytes show upregulation of CD38. Approximately 15% of B cells show surface tetherin staining. [Figure 2] Humoral immune response. B cells show CD27+ memory and a marked loss of surface IgG and IgA. [Figure 3] T cell response. Despite elevated CD38 expression, most CD8 T cells were naive and showed slow and inefficient STAT-4 phosphorylation upon IL-12 stimulation. T cell CD38 upregulation may be an indicator of viral persistence, similar to the relationship between HIV persistence and T cell activation in peripheral blood. Despite relatively normal CD25+ T cell counts, T follicular helper cells were reduced and T-reg cells were completely absent. [Figure 4] Cytotoxic response. CD8 T cells show a decrease in the % of perforin-positive cells. No changes are observed in NK cell perforin / granzyme staining. Monocyte cytoplasmic IL6, MIP1α, β, and TNFα levels were generally normal compared to controls (see also Table 2). [Figure 5]Pyroptosis. Increased caspase-1 staining is observed in CD3+ T cells as well as CD45+CD3- cells. Caspase-3 staining was not different from controls (data not shown). [Figure 6A] Measurements of B cells and caspase-1 levels are shown. [Figure 6B] Measurements of B cells and caspase-1 levels are shown. [Figure 6C] Measurements of B cells and caspase-1 levels are shown. [Figure 6D] Measurements of B cells and caspase-1 levels are shown. [Figure 7] Percentage of CD20+CD317+ cells in healthy vs. Covid-19(+) samples. Elevated CD317 (tetherin) expression on B cells is indicative of an IFN-α response. [Figure 8] Caspase-1 expression as percent positive (%) and mean fluorescence intensity (MFI) in CD3 negative (B cells and NK cells), CD3+, and CD3+CD4+ cells. [Figure 9] Critically ill patients (in the intensive care unit (ICU)) have elevated expression of active caspase-1 in CD3-negative cells, CD3+ cells, and CD3+CD4+ cells, and this expression is lower than in less critically ill patients (non-ICU). [Figure 10] Caspase-1 activity in CD3+CD4+ T cells (see Example 4 below). [Figure 11] A, CD3+ T cell caspase-1 activity (see Example 5 below). B, CD4+ T cell caspase-1 activity (see Example 5 below). [Figure 12] "Red Ring" experimental results (see Example 6 below). [Figure 13] "Red Ring" experimental results (see Example 6 below). [Figure 14] "Red Ring" experimental results (see Example 6 below). [Figure 15] "Red Ring" experimental results (see Example 6 below). [Figure 16]"Red Ring" experimental results (see Example 6 below). [Figure 17] Caspase-1 activity in COVID-19(+) patient samples in response to emricasan (see Example 7 below). [Figure 18] Correlation of secreted human IL-18 (hIL-18) with CD3+ (A) and CD4+ (B) active caspase-1+ MFI in COVID-19 positive patient samples (see Example 8 below). [Figure 19] Panels AD: RNA-seq results of COVID-19 patients (see Example 9 below) for different caspases (Figures 19A-19C) and apoptosis-inducing (Figure 19D). [Figure 20] Panels A-G: Patient demographics (see Example 9 and Tables 1-3 below). Figures 20A-20D. Caspase 1 activity in different cell types from healthy subjects and non-ICU and ICU COVID-19 patients. Figures 20E-20G. Caspase 1 in CD4+ T cells correlated with other cell types. [Figure 21] Comparison of caspase-1 levels in adult and pediatric patients (see Example 9 below). [Figure 22] Panels A and B: Caspase 1 levels in nigericin-stimulated (FIG. 22B) and unstimulated (FIG. 22A) CD4 T cells (see Example 9 below). [Figure 23] Panels A and B: Caspase 1 levels in unstimulated (Figure 23A) CD4 T cells from healthy subjects and COVID-19 patients, and in nigericin-stimulated CD4 T cells from healthy subjects and COVID-19 patients (Figure 23B) in the presence of different caspase inhibitors (see Example 9 below). [Figure 24] Figure 24A: Absence and presence of a contaminating RBC layer in samples from healthy controls and COVID-19 patients (see Example 9 below). Figure 24B: Levels of caspase 3 in RBCs from healthy controls and COVID-19 patients (see Example 9 below). DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention The present invention relates to the prevention and treatment of viral infections, particularly viral infections caused by positive-sense single-stranded RNA viruses, such as SARS-CoV-1, MERS, and SARS-CoV-2.

[0032] The methods of the invention relate to treating infections caused by positive-sense single-stranded RNA viruses that infect humans, including any form or strain of SARS-CoV-2 that circulates among the human population, or that emerge as seasonal versions, mutated versions, versions resulting from genetic drift, etc. All such positive-sense single-stranded RNA viruses, and particularly SARS-CoV-2, are encompassed within the scope of the invention, so long as the virus upregulates caspase 1 activity and can then be treated according to the invention.

[0033] The methods of the present invention also relate to treating acute respiratory distress syndrome (ARDS) and hyperinflammatory disorders (e.g., secondary hemophagocytic syndrome, macrophage activation syndrome, macrophage activation-like syndrome of sepsis, and cytokine release syndrome), sometimes known as cytokine storm syndrome.

[0034] The proteolytic enzyme caspase-1 has been found to be overexpressed in lymphocytes of COVID-19 patients. Without being bound by any particular theory, this finding implicates pyroptosis among the possible mechanisms of T cell exhaustion and dysfunction of other lymphoid cell types in COVID-19 patients, as well as in other diseases caused by positive-sense single-stranded RNA viruses.

[0035] The present inventors have developed a method for treating patients before or during infection with a positive-sense single-stranded RNA virus, such as SARS-CoV-2. Specifically, caspase-1 has been identified as a druggable target for reducing morbidity and mortality associated with infection with such viruses. As described in Examples 1 and 2 below, CD45+ cells in immunocompromised SARS-CoV-2 victims were found to have elevated expression of caspase-1. The CD45 antigen (leukocyte common antigen) is expressed on nearly all hematopoietic cells except mature erythrocytes. Caspase-1 (formerly known as interleukin-1 converting enzyme (ICE)) proteolytically cleaves precursors of the inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18), as well as the pyroptosis-inducing factor gasdermin D, into active mature peptides.

[0036] The present invention relates to methods for treating human subjects or patients infected with positive-sense single-stranded RNA viruses, such as SARS-CoV-1 (which causes SARS), MERS-CoV (which causes MERS), and SARS-CoV2 (which causes COVID-19). Prophylactic or therapeutic treatment is achieved by administering an effective amount of at least one caspase inhibitor to a patient or subject known or suspected to be infected with, or at risk for, a particular positive-sense single-stranded RNA virus. As used herein, an "effective amount" is the amount required to reduce morbidity and mortality associated with infection. An "effective amount" of a caspase inhibitor is also an amount that reduces one or more biochemical activities of a particular caspase in an in vitro assay of caspase activity. An "effective amount" of a caspase inhibitor is also an amount that, when administered to a population of patients or subjects known or suspected to be infected with a positive-sense single-stranded RNA virus (e.g., SARS-CoV-1, MERS-CoV, or SARS-CoV2), causes a reduction in hospitalization and / or symptoms in such population compared to a similar population of patients or subjects not administered the inhibitor. Advantageously, such treatment reduces pyroptosis of T cells in the patient.

[0037] The data presented here link caspase-1 to COVID-19 with significant therapeutic implications. Rather than inhibiting the inflammatory response, preventing necrotic lymphocyte death, which stimulates inflammation, becomes more important. The later stages of COVID-19 are characterized by a viral sepsis-like clinical picture with a very high mortality rate. The methods of the present invention prevent progression to this end-stage disease by targeting the virus, pyroptosis, or both.

[0038] Indeed, the evidence for caspase involvement described herein sheds further light not only on the systemic nature of the disease, but also on the degree of chronicity observed in patients referred to as "long haulers."

[0039] Evidence is also accumulating for the role of both apoptotic and pyroptotic cell death in the disease progression seen in COVID-19. Pyroptosis results in the production of mature IL-18 and IL-1β as a result of cleavage of their precursors by inflammasome-activating caspase-1. Upon release from pyroptotic cells, IL-18 induces IFN-γ responses, while IL-1β induces neutrophil influx and activation, T and B cell activation, cytokine and antibody production, and promotes Th17 differentiation. High levels of IL-18, IL-1β, and other proinflammatory cytokines have been observed in the lungs and serum of COVID-19 patients.

[0040] The ultimate result of inflammasome activation is the release of IL-1β and IL-18 to enhance immunity against pathogens, but this process is expected to be driven not by immune cell pyroptosis but by dying infected cells, which send a danger signal to the immune system. Without wishing to be bound by any particular theory, T cell death, a hallmark feature of SARS-CoV-2, is thought to be a mechanism by which SARS-CoV-2 evades human immunity by promoting an uncontrolled inflammatory response through the release of cellular contents from both immune and non-immune cells and creating an adaptive immune defect. Furthermore, the upregulation of caspase-1, which is primarily observed in memory T helper cells, may explain why older people are more susceptible to more severe and complex disease processes compared to younger people.

[0041] Inflammasome activation is a normal part of the response to pathogens when building an immune response, but dysregulated processes occurring in the wrong cell types can have deleterious effects. Again, without wishing to be bound by any particular theory, T cell death, a hallmark of SARS-CoV-2, is thought to be a way for the virus to evade the human host by creating an adaptive immune deficiency while promoting an uncontrolled inflammatory response through the release of cellular contents that can function as danger signals. The end result is a self-damaging blockade of the immune system that further stimulates inflammation caused by viral infection, resulting in acute virus-induced immune deficiency (AVID). This relationship between caspase-1 and COVID-19 has significant therapeutic implications, as preventing pyroptotic lymphocyte death is a more desirable goal than inhibiting the inflammatory response. The failure of cytokine-targeted therapies may be due to the relative importance of adaptive immune dysfunction versus inflammatory responses in disease progression.

[0042] These findings helped elucidate some puzzling observations in COVID-19. For example, the fact that children generally perform better than adults can be explained by their much lower caspase-1 expression profile. Caspase-1 expression is targeted by memory T cells, which explains why older populations (with a higher proportion of memory T cells) are much more severely affected by SARS-CoV-2.

[0043] While the use of dexamethasone, remdesivir, and antibody cocktail therapy has shown early promise in at least some patient populations, none of them address the underlying disease mechanisms that lead to the acute complications or sequelae associated with COVID-19. There is accumulating evidence that an enhanced activation state of innate and adaptive immune cells is a risk factor for severe COVID-19, the course of which may be influenced by numerous comorbid conditions, as extensively detailed by the Centers for Disease Control.

[0044] While viral diseases typically affect lymphocyte function or life cycle, presenting either with lymphocytosis (e.g., CMV, influenza, chickenpox) or, more rarely, with lymphopenia (e.g., H5N1, H1N1, HIV), the finding of neutrophilia in the context of COVID-19 has been a common but intriguing finding. As described herein, the unique combination of inflammatory and apoptotic caspase upregulation, coupled with the timing of activation in the neutrophil response to SARS-CoV-2, may result in this unique laboratory finding in COVID-19.

[0045] Taken together, the findings described herein indicate that acute immune dysfunction induced by necrotic cell death of lymphocytes plays a key factor in COVID-19 progression. Without being bound by any particular theory, it is believed that the inflammatory response is secondary to the "danger signal" from necrotic cell death of immune system cells, resulting in more intense inflammation than that induced by dying tissue cells. The end result is self-damaging cell death and a subsequent cytokine storm, which further stimulates inflammation caused by viral infection, leading to AVID. The failure of cytokine-targeted therapies may be due to adaptive immune dysfunction having a greater impact than the inflammatory response in disease progression. This relationship between caspase-1 and COVID-19 has significant therapeutic implications, as preventing pyroptotic lymphocyte death rather than inhibiting the inflammatory response is a more desirable treatment option. An ideal approach would be to prevent progression to this end-stage disease through the use of therapeutic agents targeting the virus, pyroptosis, or both.

[0046] The caspase-mediated disease process in COVID-19 is not limited to caspase-1 and T cells, as we show that changes in RBCs (red blood cells, or erythrocytes) further involve the caspase-3 pathway.

[0047] Inflammatory microvascular thrombi are present in the lungs, kidneys, and heart and contain neutrophil extracellular traps associated with platelets and fibrin, along with altered RBC morphology. Therefore, RBCs are currently believed to be involved in the coagulation disorders observed in COVID-19 patients. While RBC layers contaminating PBMCs in COVID-19 blood samples are occasionally observed in other infections, such as influenza, COVID-19-associated upregulation of caspase-3 may result in RBC changes that mechanistically link RBCs to complications of this disease. In one embodiment of the present invention, a caspase inhibitor (e.g., one or more caspase inhibitors, such as an inhibitor of caspase 1 and / or 3) is used to treat patients who have or are at high risk for a thromboembolic event.

[0048] The findings described herein point to a treatment approach that uses caspase inhibition early in the course of viral infection to mitigate or prevent disease progression, particularly COVID-19 progression.

[0049] Although SARS-CoV-2 does not appear to infect immune system cells (with the possible exception of macrophages and / or dendritic cells), the consequences of T cell exhaustion in severe COVID-19 appear to be due to mechanisms similar to those seen in HI:caspase-1 activation. Furthermore, a better understanding of the impact of different coexisting conditions on baseline T cell caspase expression before exposure to SARS-CoV-2 may be important in the development of severe disease. Mounting evidence points to activated inflammasomes in a wide variety of disorders that overlap with high-risk conditions for severe COVID-19.

[0050] Therefore, strategies targeting the inflammasome / pyroptosis pathway, which is upstream of the production of effector cytokines, are effective approaches to reverse COVID-19-induced immune perturbations.

[0051] The present invention relates to a treatment approach that uses caspase inhibitors, advantageously early in the course of infection, to reduce or prevent disease progression in various viral diseases, particularly diseases caused by positive-strand RNA viruses such as beta coronaviruses, including SARS-CoV-2, SARS, and MERS.

[0052] RNA viruses Positive-sense single-stranded RNA viruses are those whose genomes are encoded by positive-sense single-stranded RNA molecules, meaning that the viral genome itself can function as messenger RNA (mRNA) that can be translated to produce encoded proteins in infected host cells. During replication (via a double-stranded RNA intermediate), such RNA molecules can serve as both mRNA and the genome of new virus particles. Positive-sense RNA viruses are responsible for many known viruses, including hepatitis C virus (HCV), West Nile virus (WNV), dengue virus, and various coronaviruses (e.g., SARS-CoV, MERS-CoV, and SARS-CoV-2), picornaviruses (e.g., poliovirus), retroviruses (e.g., HIV), and many human pathogens such as rhinoviruses (which cause the common cold) that can infect humans.

[0053] SARS-CoV-2 To date, SARS-CoV-2 is the seventh coronavirus known to infect humans, following 229E, NL63, OC43, HKU1, MERS-CoV (which caused the MERS (Middle East Respiratory Syndrome) epidemic in 2009), and the original SARS-CoV (which caused the initial SARS (Severe Acquired Respiratory Syndrome) epidemic in 2003). Like SARS-CoV and MERS-CoV, SARS-CoV-2 is a betacoronavirus.

[0054] Caspase inhibitors Several inhibitors of caspase-1 have been developed, including benlunasan (VX-765) and pralnasan (VX-740), as well as O-desethyl-benlunasan (VRT-043198). See U.S. Patent No. 7,807,659, the disclosure of which is expressly incorporated herein for purposes of describing caspase inhibitors. Additionally, peptide inhibitors have been developed for caspase-1. One such peptide inhibitor is Ac-YVAD-cmk (acetyl tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone). Another peptide inhibitor is carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethyl ketone (Z-VAD-FMK). Z-VAD-FMK is a cell-permeable pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases. These can be used at doses that effectively inhibit caspase-1. The caspase inhibitor may be specific for caspase-1, or may be more promiscuous and inhibit additional caspases; for example, the inhibitor may be a pan-caspase inhibitor (i.e., an inhibitor that inhibits the activity of one or more of two or more different types of caspases, e.g., an inhibitor that inhibits caspases 1 and 3). Unless otherwise specified, "caspase inhibitor" refers to any caspase inhibitor, including caspase inhibitors that inhibit a single caspase species, as well as caspase inhibitors that inhibit more than one caspase species, although the degree of inhibition of one species may differ from the degree of inhibition of one or more other caspase species. Yet another caspase-1 inhibitor is CTS-2090. Administration of these inhibitors may be oral, intravenous, intradermal, intratracheal, or other means compatible with proper processing and uptake of the drug or prodrug.

[0055] Most small-molecule caspase inhibitors are in early stages of development. NCX-1000, a small-molecule inhibitor that selectively inhibits caspases-3, -8, and -9 in the micromolar concentration range, was in Phase II clinical trials for the treatment of chronic liver disease. NCX-1000 is a steroid-based NO donor that covalently binds to thiol-containing moieties, including the catalytic cysteine ​​of caspases, presumably via S-nitrosylation, thereby causing enzyme inhibition. Results of a Phase II randomized, double-blind, dose-escalation study in patients with cirrhosis and portal hypertension revealed that NCX-1000 administration was safe. (MacKenzie SH1, Schipper JL, Clark AC. The potential for caspases in drug discovery. Curr Opin Drug Discov Devel. 2010 Sep;13(5):568-76.)

[0056] Emrikasan One pan-caspase inhibitor that can be used in the methods of the invention is emricasan (IDN-6556, PF-03491390; IUPAC name (3S)-3-{[(2S)-2-{[2-(2-tert-butylanilino)-2-oxoacetyl]amino}propanoyl]amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid). This is an irreversible, orally active pan-caspase inhibitor that is being investigated for the treatment of chronic HCV infection and liver transplant rejection. (MacKenzie SH1, Schipper JL, Clark AC. The potential for caspases in drug discovery. Curr Opin Drug Discov Devel. 2010 Sep;13(5):568-76.) See also U.S. Patent Nos. 6,197,750, 6,544,951, 7,053,056, 7,183,260, and 7,692,038, and published International Publication No. 2017117478(A1), each of which is expressly incorporated herein by reference for purposes of exemplifying caspase inhibitors. Emricasan can be administered, for example, in doses ranging from 5 mg to 100 mg, 10 mg to 75 mg, or 25 mg to 50 mg per day. The daily dose can be administered as a single dose or in divided doses throughout the day. Like other caspase inhibitors, emricasan can be administered prophylactically to prevent or reduce the risk of serious disease symptoms. Emricasan is particularly useful in high-risk groups, such as those over 60 years of age, immunocompromised individuals, and diabetics. Like other caspase inhibitors, emricasan can be administered at the onset of symptoms of COVID-19 (or seasonal forms of COVID-19) or later during the course of infection. Emricasan not only inhibits caspase-1 (and other caspases), but subsequent reports have shown that emricasan can also inhibit the activity of the SARS-CoV-2 main protease, as well as the binding activity of the ACE-2 receptor to SARS-CoV-2.Such multifactorial activity may prove useful in treating infections caused by positive-strand RNA viruses, such as SARS-CoV-2. It may be desirable to combine emricasan or other caspase inhibitors with other compounds that are inhibitors of ACE-2 receptor binding and / or essential viral-encoded protein activity (e.g., viral-encoded proteases, nucleic acid polymerases, etc.). Such combinations can form effective "cocktails" for treating diseases such as COVID-19, SARS, and MERS. Here, a "cocktail" refers to a composition or treatment containing multiple (i.e., two or more) chemically distinct molecules, e.g., two different drugs, mixed together in a single composition (or dosage form, e.g., a pill or capsule or lyophilized powder intended to be reconstituted prior to administration to a patient), or administered separately as different compositions but as part of the same treatment regimen. For cocktails containing different compositions, the different compositions can be administered simultaneously or sequentially. Similarly, the different compositions can be administered by different routes. Examples of drug "cocktails" are known in the art. For example, in the context of COVID-19, a known drug cocktail is the investigational "antibody cocktail" REGN-COV2 (Regeneron, Tarrytown, NY), which contains two different virus-neutralizing, non-competitive monoclonal antibody species (REGN10933 and REGN10987) specifically designed to block SARS-CoV-2 infectivity by binding to the receptor-binding domain of the spike protein of SARS-CoV-2. In the context of the present invention, a "drug cocktail" includes a caspase inhibitor (preferably a caspase 1 inhibitor) and at least one other drug (e.g., a protease inhibitor and / or a drug that inhibits ACE-2 receptor binding by SARS-CoV-2). As will be appreciated, in some contexts, a drug "cocktail" is also referred to as a combination therapy.

[0057] Formulation of pharmaceutical compositions The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more caspase inhibitors useful in the prevention, treatment, or amelioration of one or more conditions associated with or modulated by caspases, or one or more symptoms associated with or modulated by caspases, e.g., caspases as described elsewhere herein, and a pharmaceutically acceptable carrier. In advantageous embodiments, the caspase inhibitor is formulated for single administration. To formulate the composition, a weight fraction of the compound is dissolved, suspended, dispersed, or otherwise mixed in a selected vehicle at an effective concentration such that the condition being treated is alleviated or ameliorated. Pharmaceutical carriers or vehicles suitable for administering the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

[0058] Furthermore, the caspase inhibitor can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients. The caspase inhibitor is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without causing undesirable side effects to the treated patient. The therapeutically effective concentration can be empirically determined by testing the compound in in vitro and in vivo systems known in the art, and then extrapolated therefrom for dosages for humans.

[0059] The concentration of the caspase inhibitor in the pharmaceutical composition will depend on absorption, inactivation, and excretion rates of the compound, its physicochemical properties, administration schedule and dosage, as well as other factors known to those skilled in the art.

[0060] In some embodiments, the therapeutically effective amount or dose is from about 0.1 ng / ml to about 50 μg / ml to 100 μg / ml, from about 0.5 ng / ml to about 80 μg / ml, from about 1 ng / ml to about 60 μg / ml, from about 5 ng / ml to about 50 μg / ml, from about 5 ng / ml to about 40 μg / ml, from about 10 ng / ml to about 35 μg / ml, from about 10 ng / ml to about 25 μg / ml, from about 10 ng / ml to about 10 μg / ml, from about 25 ng / ml to about 10 μg / ml, or from about 50 The serum concentration of the caspase inhibitor should be about 1 ng / ml to about 10 μg / ml, about 50 ng / ml to about 5 μg / ml, about 100 ng / ml to about 5 μg / ml, about 200 ng / ml to about 5 μg / ml, about 250 ng / ml to about 5 μg / ml, about 500 ng / ml to about 5 μg / ml, about 1 μg / ml to about 50 μg / ml, about 0.1 ng / ml to about 5 ng / ml, about 1 ng / ml to about 10 ng / ml, or about 1 μg / ml to about 10 μg / ml. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.001 mg to about 2000 mg of the compound per kg of body weight per day, about 0.002 mg to about 1000 mg of the compound per kg of body weight per day, about 0.005 mg to about 500 mg of the compound per kg of body weight per day, about 0.005 mg to about 250 mg of the compound per kg of body weight per day, about 0.005 mg to about 200 mg of the compound per kg of body weight per day, about 0.005 mg to about 100 mg of the compound per kg of body weight per day, about 0.001 mg to about 0.005 mg of the compound per kg of body weight per day, about 0.01 mg to about 100 mg of the compound per kg of body weight per day, or about 0.02 mg of the compound per kg of body weight per day. The compound should be administered in a dose of about 100 mg to about 100 mg per kg of body weight per day, about 0.05 mg to about 100 mg per kg of body weight per day, about 0.1 mg to about 100 mg per kg of body weight per day, about 0.5 mg to about 100 mg per kg of body weight per day, about 0.75 mg to about 100 mg per kg of body weight per day, about 1 mg to about 100 mg per kg of body weight per day, about 1 mg to about 10 mg per kg of body weight per day, about 0.001 mg to about 5 mg per kg of body weight per day, about 200 mg to about 2000 mg per kg of body weight per day, or about 10 mg to about 100 mg per kg of body weight per day.Pharmaceutical unit dosage forms are prepared to provide from about 1 mg to about 1000 mg, from about 1 mg to about 800 mg, from about 5 mg to about 800 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 5 mg to about 100 mg, from about 10 mg to about 50 mg, from about 10 mg to about 100 mg, from about 25 mg to about 50 mg, and from about 10 mg to about 500 mg of the essential active ingredient or combination of essential ingredients per dosage unit form.

[0061] Pharmaceutical compositions containing caspase inhibitors can be administered at once or divided into several smaller doses administered at intervals. It is understood that the exact dose and duration of treatment are a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​may also vary depending on the severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0062] Compositions containing caspase inhibitors are intended to be administered by a suitable route, for example, orally, parenterally, rectally, topically, locally, by inhalation spray, nasally, buccally, vaginally, via an implanted reservoir, or via a nasogastric or orogastric tube. In some embodiments, administration is advantageously by the oral route. Capsules and tablets are preferred for oral administration.

[0063] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, and oil / water emulsions, containing an appropriate amount of the compound or its pharmaceutically acceptable derivatives. Pharmaceutically therapeutically active compounds and their derivatives are formulated and administered in unit dosage forms or multiple dosage forms. As used herein, unit dosage form refers to physically discrete units suitable for human and animal subjects and individually packaged as known in the art. Each unit dosage form contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, together with the required pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. A unit dosage form can be administered in fractions or multiples. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dosage forms. Examples of multiple dosage forms include vials and bottles of tablets or capsules that are not separated in packaging.

[0064] Active ingredient: 0.001% to 100% active ingredient, 0.002% to 100% active ingredient, 0.005% to 90% active ingredient, 0.01% to 100% active ingredient, 0.05% to 100% active ingredient, 0.05% to 90% active ingredient, 0.1% to 100% active ingredient, 0.1% to 1% active ingredient, 0.1% to 0.5% active ingredient, 0.1% to 100% active ingredient, 1% to 99% active ingredient, 1% to 98% active ingredient, 1% to 97% active ingredient, 1% to 100% active ingredient Dosage forms or compositions can be prepared that contain 5% to 96% active ingredient, 1% to 95% active ingredient, 5% to 95% active ingredient, 10% to 100% active ingredient, 10% to 95% active ingredient, 15% to 95% active ingredient, 20% to 95% active ingredient, 25% to 100% active ingredient, 50% to 100% active ingredient, 50% to 95% active ingredient, 60% to 95% active ingredient, or 75% to 100% active ingredient, with the remainder consisting of a non-toxic carrier. For oral administration, pharmaceutically acceptable non-toxic compositions can be formed by incorporating any commonly used excipient, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate, or saccharin sodium. Such compositions include, but are not limited to, solutions, suspensions, tablets, capsules, powders, and sustained-release formulations, such as implants and microencapsulated delivery systems, as well as biodegradable, biocompatible polymers (e.g., collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like). Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain 0.001%-100% active ingredient, and in one embodiment, 75-95% active ingredient.

[0065] Caspase inhibitors or compositions containing them can be prepared with carriers that protect the compounds against rapid elimination from the body, such as sustained-release formulations or coatings. Such compositions can also include other active compounds to achieve desired combinations of properties. The compounds provided herein, or pharmaceutically acceptable derivatives thereof as described herein, can also be advantageously administered, for therapeutic or prophylactic purposes, to a subject with one or more caspase-regulated conditions in combination with other pharmacological agents known in the general art to be beneficial in treating the same conditions. It should be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

[0066] Orally Administered Composition Oral pharmaceutical dosage forms are either solid, gel, or liquid.Solid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tablets, which may be enteric-coated, sugar-coated, or film-coated.Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

[0067] In certain embodiments, the formulation is a solid dosage form such as a capsule or tablet. The tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: diluents, disintegrants, lubricants, glidants, sweetening agents, and flavoring agents.

[0068] For oral administration, the caspase inhibitor can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. This composition can also be formulated in combination with an antacid or other such ingredient.

[0069] When the dosage unit is a capsule, in addition to the above-mentioned materials, it can contain a liquid carrier such as fatty oil.Furthermore, the dosage unit can contain various other substances that modify the physical form of the dosage unit (for example, sugar coating and other enteric agents).The compound can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum, etc.Syrup can contain, in addition to the active compound, sucrose as a sweetener and certain preservatives, dyes and coloring agents, and flavoring agents.

[0070] The active material can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is a compound described herein or a pharmaceutically acceptable derivative thereof. High concentrations of the active ingredient, up to about 98% by weight, can be included.

[0071] Pharmaceutically acceptable carriers contained in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents.

[0072] Enteric-coated tablets resist the action of stomach acid due to the enteric coating and dissolve or disintegrate in the neutral or alkaline intestines. Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied. Film-coated tablets are compressed tablets coated with polymers or other suitable coatings. Multiple-compressed tablets are compressed tablets made by two or more compression cycles using the aforementioned pharmaceutically acceptable substances. Coloring agents can also be used in the above dosage forms. Flavoring and sweetening agents are used in compressed tablets, sugar-coated tablets, multiple-compressed tablets, and chewable tablets.

[0073] Dosage and unit dosage form For human treatment, a physician will determine the appropriate dosage according to whether prophylactic or therapeutic treatment is required, as well as according to age, weight, stage of disease, and other factors specific to the subject being treated. Generally, dosages are about 1 to about 1000 mg / day, or about 5 to about 250 mg / day, or about 10 to 50 mg / day for adults. In specific embodiments, dosages are about 5 to about 400 mg / day or 25 to 200 mg / day for adults. Doses of about 50 to about 500 mg / day are also contemplated.

[0074] In certain embodiments, the amount of caspase inhibitor or composition that is therapeutically effective will vary depending on the nature and severity of the disease or condition and the route by which the active ingredient is administered. The frequency and dosage will also vary according to factors specific to each subject, depending on the particular treatment (e.g., therapeutic or prophylactic agent) being administered, the severity of the disorder, disease, or condition, the route of administration, and the age, weight, response, and past medical history of the subject. Effective amounts can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0075] Exemplary dosages of the compositions include milligram or microgram amounts of a caspase inhibitor per kilogram of subject or sample weight (e.g., about 0.001-1000 mg / kg, about 0.01-100 mg / kg, about 0.01-50 mg / kg, about 0.1-25 mg / kg, or about 0.1-10 mg / kg). In certain embodiments, the dosage administered to a subject is 0.20 mg / kg-2.00 mg / kg, or 0.30 mg / kg-1.50 mg / kg of the subject's body weight.

[0076] In certain embodiments, the recommended daily dose range for the caspase inhibitor is about 0.1 mg to about 1000 mg per day, given as a single, once-daily dose or in divided doses throughout the day. In some embodiments, the daily dose is administered twice daily in equally divided doses. Specifically, the daily dose range should be about 10 mg to about 200 mg per day, more specifically about 10 mg to about 150 mg per day, or even more specifically about 25 to about 100 mg per day. As will be apparent to those skilled in the art, in some cases it may be necessary to use dosages of the active ingredient outside the ranges disclosed herein. Furthermore, it should be noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate treatment in relation to the subject's response.

[0077] As can be easily understood by those skilled in the art, different therapeutically effective amounts may be applicable to different diseases and conditions.Similarly, the above-mentioned dosage and administration frequency schedule also encompasses an amount that is sufficient to prevent, manage, treat, or improve such disorders, but is insufficient to cause side effects associated with the compounds described herein, or is sufficient to reduce side effects.Furthermore, when administering multiple doses of the compounds described herein to a subject, not all doses need to be the same.For example, the dose administered to a subject can be increased to improve the preventive or therapeutic effect of the compound, or can be decreased to reduce one or more side effects experienced by a particular subject.

[0078] In some embodiments, the dose of caspase inhibitor administered to prevent, treat, manage, or ameliorate a disease, or one or more symptoms thereof, in a subject is 0.1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, or 15 mg / kg or more of the subject's body weight. In other embodiments, the dosage administered to prevent, treat, manage, or ameliorate a disease or disorder, or one or more symptoms thereof, in a subject is a unit dose of 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0079] In certain embodiments, treatment or prevention can be initiated with one or more loading doses of a caspase inhibitor, followed by one or more maintenance doses. In such embodiments, the loading dose can be, for example, about 60 to about 400 mg / day, or about 100 to about 200 mg / day, for one day to five weeks. The loading dose can be followed by one or more maintenance doses. Each maintenance dose can independently be about 10 mg to about 200 mg / day, more specifically, about 25 mg to about 150 mg / day, or even more specifically, about 25 mg to about 80 mg / day or about 25 mg to about 50 mg / day. The maintenance dose can be administered daily and can be administered as a single dose or in divided doses.

[0080] In certain embodiments, a dose of the caspase inhibitor can be administered to achieve a steady-state concentration of the active ingredient in the subject's blood or serum. The steady-state concentration can be determined by measurement according to techniques available to those skilled in the art or can be based on the subject's physical characteristics (e.g., height, weight, and age). In certain embodiments, a sufficient amount of a compound provided herein is administered to achieve a steady-state concentration in the subject's blood or serum of about 300 to about 4000 ng / mL, about 400 to about 1600 ng / mL, or about 600 to about 1200 ng / mL. A loading dose can be administered for 1 to 5 days to achieve a steady-state concentration in the blood or serum of about 1200 to about 8000 ng / mL or about 2000 to about 4000 ng / mL.

[0081] The maintenance dose can be administered to achieve a steady state concentration in the subject's blood or serum of about 300 to about 4000 ng / mL, about 400 to about 1600 ng / mL, or about 600 to about 1200 ng / mL.

[0082] In certain embodiments, administration of the same compound can be repeated, and administration can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, administration of the same prophylactic or therapeutic agent can be repeated, and administration can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

[0083] In certain preferred embodiments, a unit dose containing the compound or a pharmaceutically acceptable derivative thereof is provided in a form suitable for administration. Such forms are described in detail above. In certain embodiments, the unit dose contains 1-1000 mg, 5-250 mg, or 10-50 mg of the active ingredient. In certain embodiments, the unit dose contains about 1, 5, 10, 25, 50, 100, 125, 250, 500, or 1000 mg of the active ingredient. Such unit doses can be prepared according to techniques well known to those skilled in the art.

[0084] manufactured goods A caspase inhibitor or pharmaceutically acceptable composition useful in the practice of the present invention can be packaged as an article of manufacture comprising packaging material, a caspase inhibitor or a pharmaceutically acceptable composition comprising a caspase inhibitor for use in the treatment, prevention, or amelioration of a disease or condition modulated by a caspase or one or more symptoms associated with the disease or condition, and a label indicating that the caspase inhibitor or composition is used for the treatment, prevention, or amelioration of a particular disease or condition or one or more symptoms thereof. Examples of pharmaceutical packaging materials include blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material appropriate for the selected formulation and intended mode of administration and treatment.

[0085] kit Kits are provided for the use of one or more caspase inhibitors, alone or in combination with other therapeutic agents, to achieve a desired treatment. The kits can include a caspase inhibitor or a composition containing the caspase inhibitor(s), and instructions providing a healthcare provider with information regarding use to treat or prevent a disease or condition modulated by one or more caspases. The instructions can be provided in printed form, in the form of an electronic medium such as a CD or DVD, or in the form of a website address from which such instructions can be obtained. A unit dose can include a dose such that, when administered to a subject, a therapeutically or prophylactically effective plasma level of the compound or composition can be maintained in the subject for at least one day. Typically, the components of the kit are packaged separately. For example, a solid formulation and a diluent can be provided in separate containers or compartments. Similarly, different components of a cocktail can be provided separately for mixing immediately prior to administration. Alternatively, the drug and delivery device can be provided prefilled or packaged separately.

[0086] Combination therapy Combination therapy involving a caspase inhibitor can be a regimen in which two drugs are administered to the same patient in relation to one another. For example, the two drugs can be administered in a temporal relationship to one another (e.g., simultaneously or sequentially within a specific period of time). Alternatively, the two drugs can be administered in a biochemical or clinical relationship to one another. For example, one drug can be administered when the serum level of the other drug drops to a certain level. Alternatively, one drug can be administered when a specific clinical measurement, such as oxygen saturation, is achieved. When a common regimen is used, the two drugs can be administered by different routes.

[0087] Alternatively, combination therapy involving a caspase inhibitor can use a compound composition containing two different caspase inhibitors, which can be combined at the actual concentration for administration or in a concentrated form that can be diluted for use. The compound composition can be packaged in a single-dose form or can be packaged in multiple doses.

[0088] Like other caspase inhibitors, emricasan, whether administered orally, intravenously, or in other forms, can be administered alone or in combination treatment with one or more antiviral agents, such as remdesivir.

[0089] Like other caspase inhibitors, emricasan can also be administered in combination with convalescent plasma in the standard dosing regimens used for COVID-19.

[0090] Additionally, like other caspase inhibitors, emricasan can be administered in combination therapy with another biologic agent that targets the IL-6 inflammatory pathway or another inflammatory pathway.

[0091] Any inhibitor of the IL-6 inflammatory pathway can be used in combination with an inhibitor of caspase 1. IL-6 inflammatory pathway inhibitors that can be used include the IL-6 R neutralizing mAb tocilizumab (Actemra), anti-IL-6 antibodies, soluble forms of gp130 (sgp130) and sgp130Fc proteins.

[0092] Additionally, like other caspase-1 inhibitors, emricasan can be administered in combination with steroids such as dexamethasone and / or Covid-19 antibody cocktail therapy.

[0093] Timing of treatment Administering at least a caspase-1 (or pan-caspase) inhibitor early in the course of disease, particularly in those caused by betacoronaviruses (e.g., COVID-19, SARS, MERS), would be beneficial because the treatment functions by preventing acute immune dysfunction rather than inhibiting inflammatory responses and / or a "cytokine storm." Activation of caspase-1 in lymphocytes likely leads to lymphocyte death, which can be measured by a blood test called immunophenotyping. Lymphocyte death causes lymphopenia (lymphocyte counts below the normal range). Lymphopenia is a valid and reliable indicator of severity and hospitalization in COVID-19 patients. A lymphocyte count of less than 20% has been proposed as a cutoff point for severe disease. Patients with a lymphocyte count above 20% 10–12 days after symptom onset are classified as moderate disease and may recover quickly. Patients with a lymphocyte count below 20% are initially classified as severe disease. At the second time point, 17–19 days after onset, patients with lymphocyte percentages above 20% are recovering. Patients with lymphocyte percentages above 5% but below 20% remain at risk and require monitoring. Patients with lymphocyte percentages below 5% become critically ill with a high mortality rate and require intensive care. Ideally, patients should be initiated on caspase-1 inhibitors before lymphocyte levels reach below 20%. (See Tan L et al., “Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study,” Signal Transduction and Targeted Therapy (2020) 5:33.)

[0094] Treatment with caspase-1 inhibitors is advantageously initiated before CD4 T cells fall below 300 cells / mcl, CD8 cells below 100 cells / mcl, B cells below 20 cells / mcl, or NK cells below 15 cells / mcl, and before any functional deficiencies in these individual cells become apparent. As a wise measure, treatment with caspase-1 blockers (or pan-caspase inhibitors) can be initiated at the time of diagnosis or when a diagnosis of COVID-19 is suspected, such as upon exposure to an infected individual. In non-resistant populations, treatment can be initiated before a definitive diagnosis, especially if contact with an infected person is suspected. Because caspase-1 inhibitors do not act as antivirals, i.e., they do not reduce viral load, they should not affect viral testing performed within a few days of initiating treatment.

[0095] Once lymphopenia occurs, the body is unable to mount a protective immune response (either antibody- and / or cell-based) against the virus, leading to the patient's death. Therefore, caspase-1 inhibitors should be administered advantageously before the immune system becomes so compromised that it is unable to mount an immune response against the virus. For maximum benefit, caspase-1 inhibitors are administered prophylactically during a pandemic or epidemic, within hours of disease diagnosis, or before the onset of lymphopenia as measured by immunophenotyping. Given this mechanism, patient death from COVID-19 infection is secondary to immune deficiency caused by the virus, rather than due to the release of cytokines such as IL-1, IL-18, IL-6, and other cytokines and chemokines.

[0096] Those at higher risk of complications from the virus include the elderly, those with underlying cardiovascular or respiratory disease, diabetes, chronic kidney disease, or other comorbidities, and those with weakened immune systems (e.g., HIV-infected patients, transplant recipients, or patients undergoing cancer chemotherapy). Any person or group determined to be at high risk of severe disease can be advantageously treated prophylactically or before the onset of severe disease symptoms.

[0097] COVID-19 can range from mild to severe illness, with the latter including pneumonia, severe acute respiratory syndrome, multiple organ failure, and death. The incubation period for SARS-CoV-2 is thought to be as long as 14 days, with a median time from exposure to symptom onset of 4 to 5 days. Currently, no approved medications have been approved by the FDA to treat COVID-19. Clinical management includes symptomatic and supportive care, such as supplemental oxygen, mechanical ventilation, and extracorporeal membrane oxygenation (ECMO), as needed. The FDA has published a five-tiered classification system for COVID-19 severity (see, e.g., Developing Drugs and Biological Products for Treatment or Prevention Guidance for Industry, FDA, May 2020). When COVID-19 classifications are used herein, they are defined as provided in the table below.

[0098] (Table A) COVID-19 severity classification TIFF0007742357000001.tif170169

[0099] Treatment of COVID-19 according to the present invention involves administering a caspase inhibitor (an inhibitor of at least one or more of caspases 1, 3, 4, 5, 7, 8, 9, and 11) to a subject at any of the above stages / levels of COVID-19 severity.

[0100] Assay of caspase levels Patients at high risk for COVID-19 infection or severe COVID-19 can be tested for caspases, such as caspase-1 (see, e.g., Figures 6B-D). If elevated caspase-1 is detected, inhibitors of caspase-1 can be administered as either prophylaxis or treatment at the onset of febrile illness, particularly if COVID-19 is suspected. Measurement of other caspases can similarly indicate severe forms of COVID-19. High-risk groups are defined by the CDC as those with asthma and chronic lung disease, hypertension, obesity, diabetes, immunocompromised patients, adults over 65 years of age, elderly people in nursing homes and assisted living facilities, serious cardiac conditions, chronic kidney disease undergoing dialysis, and liver disease.

[0101] Late sequelae Certain COVID-19 patients develop sequelae and / or complications from COVID-19 that persist long after the acute phase of infection. As used herein, a "long-hauler" is a patient who experiences complications of the disease more than two weeks after the onset of symptoms. This is considered an immunological event because these long-hauler patients do not test positive for the virus during this chronic phase of the disease. It has been determined that caspase hyperactivity in blood cells (lymphocytes and erythrocytes) can be present several months after the end of infection, indicating that caspases are involved in the late sequelae of COVID-19. The etiology and pathophysiology of late sequelae are thought to reflect organ damage or inflammation from the acute infection phase, the development of a persistent hyperinflammatory state, ongoing viral activity associated with the host viral matrix, or an inadequate antibody or cellular immune response. In addition to the acute illness, factors that may further complicate the situation include physical ill-health at baseline or after a prolonged illness course, pre-COVID-19 comorbidities, and psychological sequelae after a prolonged or difficult illness course, as well as those related to lifestyle changes due to the pandemic.Likely, the persistent sequelae of COVID-19 represent multiple syndromes resulting from different pathophysiological processes along the disease spectrum.

[0102] Although information regarding the late sequelae of COVID-19 is limited, reports of persistent symptoms in individuals who have recovered from acute COVID-19 disease are emerging. The most commonly reported symptoms include fatigue, dyspnea, cough, joint pain, and chest pain. Other reported symptoms include cognitive impairment, depression, muscle pain, headache, fever, and palpitations. More severe complications, although less common, have also been reported. These complications include cardiovascular abnormalities such as myocardial inflammation and ventricular dysfunction, respiratory abnormalities such as pulmonary function abnormalities, renal abnormalities such as acute kidney injury, skin abnormalities such as rash and alopecia, neurological abnormalities such as olfactory and taste dysfunction, sleep dysregulation, cognitive changes, and memory impairment, and psychiatric abnormalities such as depression, anxiety, and mood changes.

[0103] Treatment or prevention of late sequelae (which occur in "long-haulers") can involve administration of caspase inhibitors at the onset of infection, during the acute phase of infection, or after the acute phase of infection. Caspase inhibitors (inhibitors of at least one of caspases 1, 3, 4, 5, 7, 8, 9, and 11) can be combined with steroids at a daily dose of 10-80 mg given either once daily or in two divided doses to prevent or treat long-term sequelae. For prevention of late sequelae, the drug can be administered for a short period (14 days) or a long period (1 month), depending on the severity of COVID-19 (e.g., mild (14 days), moderate (21 days), or severe (1 month)).

[0104] The above disclosure broadly describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0105] Example 1: Immunophenotyping This example presents the immune phenotype of an immunosuppressed patient who died rapidly from COVID-19, illustrating the uniqueness of the immunosuppressed population.

[0106] One patient with confirmed SARS-CoV-2 infection died from COVID-19.

[0107] Innate immune phenotyping of blood samples showed some evidence of antigen-presenting cell (APC) activation and the involvement of a type 1 interferon response, as indicated by upregulation of CD11b and CD38 on monocytes and Tetherin on B cells; however, further immunophenotyping demonstrated acute adaptive immune dysregulation (Figures 1–5). Notably, no memory B cells were found, and SARS-CoV-2-specific IgG and IgM were absent. The patient also exhibited severe T-cell lymphopenia, as well as the absence of T regulatory cells, low T follicular helper cells, overexpression of CD38 in CD8 T cells, and insufficient STAT-4 phosphorylation in CD4 T cells upon IL-12 stimulation. Finally, the overall increase in caspase-1 staining in lymphocytes suggested pyroptosis, which may explain the patient's inadequate immune response (6, 7). Taken together, this experience of immunosuppressed patients with COVID-19 can be characterized as a form of acute virus-induced immunodeficiency (AVID) (8, 9).

[0108] Example 2. Method SARS-CoV-2 Specimen Collection and Testing Nasopharyngeal clinical specimens for SARS-CoV-2 testing were obtained, handled, and processed via real-time polymerase chain reaction assay as previously reported ( 1 ).

[0109] Cell preparation and immunofluorescence staining Peripheral blood from venipuncture was drawn into EDTA- and heparin-coated vacutainer tubes (BD Bioscience) for immunophenotyping. Whole blood collected in EDTA tubes was immunostained according to a clinical standard immunophenotyping protocol (Amerimmune LLC, Fairfax, VA). Samples were stained with the antibody combinations shown in Table 1 for 30 minutes at 4°C. Red blood cells were lysed using BD FACS lysing solution (BD Bioscience, Jan Jose, CA) according to the manufacturer's instructions.

[0110] Peripheral blood mononuclear cells (PBMCs) were isolated from 2 mL of whole blood and diluted 1:1 with phosphate-buffered saline (PBS) pH 7.2 (Thermo Fisher Scientific, Carlsbad, CA) using Lymphoprep (Stem cell Technologies, Cambridge, MA) and Accuspin tubes (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. PBMCs were washed with PBS and resuspended in 0.4 mL of PBS. 100 μL of PBMCs were immunostained for 1 hour at 4°C with a mixture of antibodies listed in Table 1. Cells were washed and resuspended in PBS before acquisition.

[0111] Table 1: Immunological data of patients TIFF0007742357000002.tif204169 See Methods for a detailed description of immunological analysis. Items highlighted in red are outside the reference range.

[0112] Antibodies utilized from Thermo Fisher Scientific were: CD56 SB436, CD45 eF506, CD3 FITC, CD16 PE, CD8 PerCP-eF710, CD14 PE-CY7, CD4 APC, CD20 APC-eF780, Cd25 EF450, CD57 FITC, TCRγ-δPE, CD4 PerCP-eF710, CD3 PE-CY7, TCR αβAPC, HLA-DR AF700, CD8 APC-eF780, IgD SB436, IgA FITC, IgG PE, IgM PerCP-eF710, CD19 PE-CY7, CD27 APC, CD5 FITC, CD21 PE, CD27 PerCP-eF710, CD45RA FITC, CD45RO PerCP-eF710, CD294 APC, CD4 AF700, CD3 FITC, CD14 FITC, CD16 FITC, CD19 FITC, CD20 FITC, CD56 FITC, CD34, FITC, CD11c PE, HLA-DR PerCP-EF710, CD303a APC, CD4 SB600, CD45RA FITC, CD3 PE-CY7, CD8 AF700, CCR5 APC, CD25 APC, CD317 PE, IL-6 PE-CY7, and MIP1-β APC.

[0113] The antibodies used were HLA-DR BV480, CD38 PerCP-CY5.5, CD28 APC, CD45 APC H7, CD278 BV421, CXCR5 PerCP-CY5.5, CD127 BV480, CD45RO PerCP-CY5.5 CD20 APC-H7, CD11b BV421, CD16 FITC, MIP1-α PE, HLA-DR PerPC-Cy5.5, Perforin Alexa488, and Granzyme B PE from BD Bioscience. TNF-α BV421 was obtained from Biolegend (San Diego, CA).

[0114] Samples were processed according to Amerimmune's clinical safety SOPs. Appropriate PPE was used when processing samples.

[0115] Measurement of apoptosis and pyroptosis by flow cytometry Apoptosis and pyroptosis were measured by flow cytometry using the fluorescently labeled inhibitor of caspase probe assay, FLICA, according to the protocol (Immunochemistry Technologies, Minneapolis, MN). FAM-FLICA probes specific for caspase 1 and caspase 3 / 7 were added directly to 100 μl of PBMCs and incubated at 37°C for 1 hour. Cells were washed three times with wash buffer to remove unbound FLICA probes. Cells were stained with CD45 PE-CY7, CD3 AF700, CD4 PE, and CD45RO PerCP-EF710.

[0116] Measurement and flow cytometry acquisition Samples were acquired on a three-laser BD FACS Canto 10. CS&T beads (BD Bioscience, San Jose, CA) were acquired daily to ensure consistent performance of the Canto 10. After sample acquisition, the BD FACS Canto 10 was washed with 10% bleach and water for 10 minutes. The CANTO 10 utilized for this study has been validated for clinical diagnostic testing of T, B, NK, and dendritic cell immunophenotyping.

[0117] Example 3 Caspase-1 in non-immunosuppressed COVID-19 patients is higher in CD3-, CD3+, and CD3+CD4+ cells than in controls Although an exaggerated immune response and a "cytokine storm" have been hypothesized as possible reasons for the complications seen from COVID-19, some perplexing findings, such as low levels of IFN-α production and effector responses (see Figure 7 below), severely ill patients with higher antibody production, the failure of clinical trials investigating attempts to block effector cytokine and chemokine molecules, and the characteristic finding of T-cell lymphopenia, all point to alternative mechanisms involving immune cell death and subsequent associated complications.

[0118] Recent studies in HIV demonstrated upregulation of caspase-1 as a mechanism of T helper cell death (3). Following this, caspase-1 levels were measured in hospitalized patients with COVID-19. These patients were found to have significantly higher baseline and nigericin-induced caspase-1 expression compared to healthy controls. See Figure 8, which shows caspase-1 expression in CD3 (B cells and NK cells), CD3, and CD3CD4 cells as percent positive (%) and mean fluorescence intensity (MFI).

[0119] Figure 8 clearly shows that COVID-19 patients have elevated caspase-1 expression both at baseline and after nigericin stimulation.

[0120] Although there were no differences in serial samples from individual patients, patients with severe disease had lower baseline caspase-1 levels compared with non-severe disease patients. Nigericin-induced caspase-1 expression was not statistically different between the two groups (severe and non-severe disease), indicating functional deterioration of the cells. These caspase-1 findings were also observed in non-T cells (B cells and NK cells).

[0121] When patients were stratified by disease severity (ICU vs. non-ICU), clear statistical differences were observed between ICU and non-ICU patients, with elevated caspase-1 expression being consistently lower in severely ill ICU patients (Figure 9).

[0122] In all cases, the statistical differences between samples from healthy control patients and samples from both ICU and non-ICU patients remain.

[0123] Example 4 Older patients have higher elevated caspase-1 expression in CD3+CD4+ cells Age is a significant risk factor for COVID-19 severity and complications. As shown in Figure 10, a correlation was found between age and caspase-1 activity in CD3+CD4+ T cells.

[0124] Example 5 CD4+ memory T cells correlate with elevated caspase-1 activity (B). Age is a significant risk factor for COVID-19 severity and complications. As shown in Figures 11A and 11B, a correlation was found between age and caspase-1 activity in CD3+ and CD4+ T cells.

[0125] Elevated CD4+ T cell caspase-1 activity correlated with memory CD4+ T cell subpopulations (see Figure 11B), which also explains the age-related increase in caspase-1 activity in elderly patients, who tend to have higher memory CD4+ T cell populations.

[0126] Example 6 Red Ring One observation made during the preparation of peripheral blood mononuclear cells (PBMCs) for some of the experiments described herein was the presence of a contaminating red blood cell (RBC) layer within the PBMC layer, which we termed the "red ring." This "red ring" was present in all COVID-19 patient samples, with no difference between critically ill patients and those receiving only supportive care outside of an ICU setting (see Figure 12A).

[0127] When examined under a microscope, the RBCs in the "red ring" showed clumping and aggregation, despite the blood being collected in heparinized or EDTA-treated tubes, and all patients were treated with heparin (see Figure 13).

[0128] The "red ring" observed in COVID-19 patient samples was absent in non-COVID-19 patient samples. See Figures 14 and 15.

[0129] When PBMCs isolated from COVID-19 patient samples were incubated with emricasan (1 micromolar) for 17 hours at room temperature, a reduction in the "red ring" was observed. See Figure 16.

[0130] Because emricasan reduces and possibly prevents RBC clumping, the results support the use of emricasan to treat COVID-19.

[0131] Example 7 Emricasan-mediated inhibition of caspase activity Whole blood from COVID-19(+) patients was incubated for 3.5 hours at room temperature with (A) no emricasan (untreated) or (B) emricasan (Sigma Aldrich, MO, SML2227-5MG) at a final concentration of 1 μM. After incubation with or without emricasan, PBMCs were purified using Accuspin System-Histopaque 1077 (Sigma Aldrich, MO, A6929) according to the manufacturer's instructions. Purified treated and untreated PBMCs were incubated with nigericin (Immunochemistry Technologies, MN) for 2 hours according to the manufacturer's instructions. A Fam-FLICA probe specific for caspase 1 was added directly to 50 μl of PBMCs and then incubated at 37°C for 1 hour. PBMCs were washed with cell wash buffer (Immunochemistry Technologies, MN) to remove unbound FLICA probe. Washed PBMCs were FACS-sorted using CD45 PE-CY7[HI30], CD3 AF700[UCHT1], CD4 PE[RPA-T4], CD45RO PerCP-EF710[UCHL1], and Viability Dye 780 (Thermo Fisher Scientific, Carlsbad, CA) to identify viable CD3- cells, as well as CD3+ and CD3+CD4+ T cells. Lymphocytes were identified using a standard gating scheme incorporating gating on lymphocytes on an FSC / SSC plot and singlets on an FSC-A / FSC-H plot. Lymphocytes were further identified as CD45+ on a CD45 / SSC plot, and CD3-, CD3+, and CD3+CD4+ cells were then identified on a CD45+CD3 / CD4 plot. Figure 17 shows active caspase-1 MFI and % positive data for the three cell populations indicated. Emricasan-treated COVID-19-positive (COVID-19(+)) whole blood showed a significant decrease in both active caspase-1 MFI and active caspase-1 % positive cells compared to untreated COVID-19-positive whole blood (p≦0.005) (Prism, paired t-test).

[0132] In a separate experiment, whole blood was incubated with 1 μM emricasan on a rocker at room temperature for 17 hours (overnight, from 5 PM to 10 AM). The same experiment as above was performed to examine active caspase-1 in PBMCs. In three samples from COVID-19 patients, nigericin-induced inhibition of caspase-1 activity was observed in only one-third of the samples.

[0133] Example 8 Correlation between IL-18 and caspase-1 activity Whole blood was centrifuged at 960 rcf to separate plasma from RBCs and WBCs. Plasma was removed and frozen in vapor-phase liquid nitrogen until testing. The remaining sample was processed for PBMCs using Accuspin System-Histopaque 1077 (Sigma Aldrich, MO, A6929) according to the manufacturer's instructions. A Fam-FLICA probe specific for active caspase-1 (see Example 7 above) was added directly to 50 μl of PBMCs, followed by incubation at 37°C for 1 hour. PBMCs were washed with cell wash buffer (Immunochemistry Technologies, MN) to remove unbound FLICA probe. Washed PBMCs were FACS-sorted using CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4], CD45RO PerCP-EF710 [UCHL1], and Viability Dye 780 (Thermo Fisher Scientific, Carlsbad, CA) to identify viable CD3- cells, as well as CD3+ and CD3+CD4+ T cells. Lymphocytes were identified using a standard gating scheme incorporating gating on lymphocytes on an FSC / SSC plot and singlets on an FSC-A / FSC-H plot. Lymphocytes were further identified as CD45+ on a CD45 / SSC plot, and CD3-, CD3+, and CD3+CD4+ cells were then identified on a CD45+CD3 / CD4 plot.

[0134] Secreted IL-18 was assayed using a human IL-18 ELISA kit (Abcam, ab215539) according to the manufacturer's instructions. Samples were tested in duplicate and triplicate when sufficient sample was available. Results were analyzed using GraphPad Prism v8. A four-parameter curve fit was performed and IL-18 concentrations were interpolated using the Prism interpolation function. See Figures 18A and 18B.

[0135] Figures 18A and 18B show active caspase-1 MFI for both CD3+ and CD4+ cell populations correlated with secreted hIL-18. Analysis was performed in GraphPad Prism v8 using the Spearman correlation algorithm. P values ​​are indicated on the graphs.

[0136] Although COVID-19 patients exhibit acute immune hyperactivation, which has prompted the use of targeted and non-targeted immunosuppressants, there is evidence of profound adaptive immune dysfunction, accompanied by loss of T cells and pyroptosis of other lymphoid cell compartments. As the data presented herein demonstrate, pyroptosis of lymphocytes and RBCs leads to immune dysfunction and coagulopathy, respectively. Other cell types (e.g., endothelial cells) in patients infected with SARS-CoV-2 may also undergo pyroptosis, which may further contribute to the "cytokine storm" described in the literature.

[0137] Overall, these findings draw attention to SARS-CoV-2-induced acute immune deficiency resulting from necrotic cell death of lymphocytes. Without being bound by any particular theory, the inflammatory response observed in COVID-19 patients is secondary to danger signals from necrotic cell death of immune system cells, resulting in more intense inflammation than processes induced by tissue cell death. The end result is a self-damaging blockade of the immune system that further stimulates inflammation caused by viral infection, resulting in acute virus-induced immune deficiency.

[0138] References for Examples 1-8 The disclosure of each cited reference is expressly incorporated herein. TIFF0007742357000003.tif198148

[0139] Example 9 Caspases and the therapeutic potential of caspase inhibitors in COVID-19 disease and sequelae In this study, we evaluated the transcriptional status of caspases in immune cells from COVID-19 patients and used flow cytometric profiling of cellular caspases to examine immune cells and erythrocytes from a series of COVID-19 patients hospitalized with acute illness or in the convalescent stage. Gene expression levels of several caspases were elevated in an in vitro SARS-CoV-2 infection model, and single-cell RNA-Seq data from peripheral blood samples from COVID-19 patients showed distinct patterns of caspase expression in T cells, neutrophils, and dendritic cells. Flow cytometric assessment of CD4 T cells demonstrated upregulation of caspase-1 in hospitalized COVID-19 patients compared with unexposed controls, except in a subset of patients with asthma and chronic rhinosinusitis (CRS). Convalescent COVID-19 patients with prolonged symptoms ("long-haulers") showed persistent upregulation of caspase-1 in CD4 T cells that was attenuated ex vivo after coculture with selected pan-caspase inhibitors. Furthermore, elevated caspase 3 levels were observed in erythrocytes from COVID-19 patients compared with controls in response to caspase inhibition. Taken together, these results reveal a significant caspase response in COVID-19 that promotes immune-related pathological processes leading to severe outcomes. Pan-caspase inhibition is a treatment strategy to ameliorate, reduce, or prevent severe COVID-19 outcomes.

[0140] The research described herein involves analyses to investigate the expression of inflammatory caspases, as well as inducible and executioner caspases, across the spectrum of COVID-19 disease in multiple immune cell types. The discovery of increased caspase expression in red blood cells (RBCs), such as caspase-3, beyond caspase-1, has led to further definition of the complete caspase expression profile of immune system cells. The influence of unique caspase expression profiles in a given cancer cell population may influence specific outcomes, such as RBC involvement in coagulopathy in COVID-19 infection, and determine relationships with parameters of disease progression.

[0141] Materials and Methods Patient population Patient samples for immunophenotyping were obtained during patient visits or hospitalizations. Patients were defined as 1) not hospitalized and without COVID-19 symptoms, or 2) hospitalized with COVID-19 symptoms. Peripheral blood via venipuncture was collected for immunophenotyping into EDTA- and heparin-coated vacutainer tubes and processed within 48 hours of collection.

[0142] Flow cytometry Whole blood was stained according to a clinical standard immunophenotyping protocol (Amerimmune LLC, Fairfax, VA). Samples were stained with a combination of antibodies for 30 minutes at 4°C. RBCs were lysed using BD FACS lysing solution (BD Bioscience, San Jose, CA) according to the manufacturer's instructions. Briefly, freshly obtained peripheral blood mononuclear cells (PBMCs) were isolated from 2 mL of whole blood within 24 hours of collection and diluted 1:1 with phosphate-buffered saline (PBS) pH 7.2 (Thermo Fisher Scientific, Carlsbad, CA) using Lymphoprep (Stem cell Technologies, Cambridge, MA) and Accuspin tubes (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. PBMCs were washed with PBS and resuspended in 0.5 mL of PBS. 100 μL of PBMCs were immunostained with the antibody mixture for 1 hour at 4°C. Cells were washed and resuspended in PBS before acquisition.

[0143] Apoptosis and pyroptosis were measured by flow cytometry using fluorescently labeled inhibitor of caspase probe assay (FLICA; Immunochemistry Technologies, Minneapolis, MN). As a control, PBMCs were stimulated with nigericin for 2 hours. A FAM-FLICA probe specific for caspase-1 was added to 50 μl of PBMCs and incubated at 37°C for 1 hour. Cells were then washed and stained with CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4], CD45RO PerCP-EF710 [UCHL1], and Viability Dye 780 (Thermo Fisher Scientific, Carlsbad, CA). Samples were acquired on a three-laser BD FACS Canto 10. CS&T beads (BD Bioscience, San Jose, CA) were acquired daily to ensure consistent performance of the Canto 10. The CANTO 10 utilized for this study has been validated for clinical diagnostic testing of T, B, NK, and dendritic cell immunophenotyping. Denovo FCS Express v6 Clinical Edition (De Novo Software, Pasadena, CA) was used for flow cytometry analysis.

[0144] Gating strategies for T cells, B cells, and NK cells Monocytes were identified using a standard gating strategy utilizing CD14, CD16, and HLA-DR to identify classical, intermediate, and non-classical monocytes. CD38 and CD11b MFI and percent positivity were determined from CD14+CD16+ intermediate monocytes. Lymphocytes were identified using the following gating schemes: singlets (FSC-A / FSC-H); CD45+ (CD45 / SSC plot); CD45+CD3+ (CD3 / SSC plot); and CD4+ and CD8+ T cells (CD4 / CD8 CD3+ gating). Subsequent T cell subpopulations, as indicated, were identified from CD45+CD3+CD4+ or CD8+ cells. From the lymphocyte gating plot, B cells were identified as CD45+; CD20+ (CD20 / SSC plot). All B cell subpopulations were identified from the CD45+CD20+ population.

[0145] IL-18 ELISA Plasma IL-18 was assayed using a human IL-18 ELISA kit (Abcam, ab215539) according to the manufacturer's instructions. Samples were tested in duplicate and triplicate, if sufficient sample was available. A four-parameter curve fit was performed, and IL-18 concentrations were interpolated using the GraphPad Prism version 8.0 (Graphpad Software Inc., CA, USA) interpolation function.

[0146] Plasma experiments Plasma was separated from whole blood after centrifugation at 960 RCF. Cells (RBCs and WBCs) were incubated at 37°C alone or in the presence of trypsin for 1 hour and then washed with 10 packed cell volumes of RPMI 1640 incomplete medium. Plasma was either kept at room temperature (18-25°C) or heat-inactivated at 56°C for 1 hour. Plasma was added back to the RBCs / WBCs in a 1:1 ratio and incubated overnight at room temperature with shaking.

[0147] Official SARS-CoV-2 and COVID-19 Transcriptome Analysis: Single-cell RNA-seq data from three ventilated COVID-19 participants diagnosed with acute respiratory distress syndrome 2–16 days after symptom onset and six healthy controls were accessed from GEO (27). RNA-seq data from cell lines infected with SARS-CoV-2 in vitro were obtained from GEO:GSE147507. Caspase gene expression values ​​were normalized using DESeq2.

[0148] Ex vivo stimulation test Active caspase-1 in COVID-19+ patient samples Whole blood from COVID-19-positive patients was either (A) untreated or (B) treated with emricasan (1 μM, Sigma-Aldrich, MO, SML2227-5MG) or the selective caspase-1 inhibitor VX765 overnight at 37°C in a water bath. PBMCs were then purified (Accuspin System-Histopaque 1077; Sigma-Aldrich, MO, A6929) and incubated with nigericin (Immunochemistry Technologies, MN) for 2 hours. A Fam-FLICA probe specific for active caspase-1 was added to 50 μl of PBMCs and then incubated at 37°C for 1 hour. PMBCs were washed with cell wash buffer (Immunochemistry Technologies, MN) and stained with CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4], CD45RO PerCP-EF710 [UCHL1], and Viability Dye 780 (Thermo Fisher Scientific, Carlsbad, CA). Lymphocytes were identified using a standard gating scheme incorporating gating on lymphocytes on an FSC / SSC plot and singlets on an FSC-A / FSC-H plot. Lymphocytes were further identified as CD45+ on a CD45 / SSC plot, and CD3-, CD3+, and CD3+CD4+ cells were then identified on a CD45+CD3 / CD4 plot. Active caspase-1 MFI and % positivity for the three cell populations indicated are shown.

[0149] statistical analysis Demographic and COVID-related characteristics were described using median, first quartile (Q1), and third quartile (Q3) for continuous variables and frequencies for categorical variables. Differences between continuous variables were assessed by either the Mann-Whitney test or the Cluster-Wallis test with Dunn's multiple comparisons. Relationships between parameters were examined by Pearson correlation for continuous variables. All statistical tests were performed using GraphPad Prism version 8.0 (Graphpad Software Inc., CA, USA). Statistical significance was determined by the mean of the mean. * p<0.05, ** p<0.01, *** p<0.001, **** P values ​​< 0.100 are not significant but are recorded as a statistical trend.

[0150] result 1. Transcriptional profiles of multiple caspases in immune cells during COVID-19 infection To follow up on the findings of elevated caspase-1 expression in T cells from patients with COVID-19, we assayed multiple caspases in different immune cell types from blood samples of patients with moderate to severe COVID-19. Caspase gene expression levels were examined in public transcriptome profiling datasets of in vitro SARS-CoV-2 infection and single-cell RNA sequencing of immune cells from individuals with COVID-19 (Figure 1). As expected, caspase-1 was found to be upregulated in CD4 T helper cells. We also found evidence of altered transcriptome levels of caspase genes in natural killer cells and a highly dramatic increase in neutrophils. Interestingly, plasmacytoid DCs were the only immune cell type to show upregulation of caspase-9. Neutrophils displayed a unique profile with upregulated caspase-5, a pro-inflammatory caspase, but also a similar increase in caspase-7, a pro-apoptotic molecule. IFN-stimulated CD4 T helper cells showed significant upregulation of caspases-7 and -9, indicating that multiple cell death mechanisms play a role in addition to the caspase-1 pathway.

[0151] For comparison, we analyzed intracellular levels of active caspase-1 in T cells from COVID-19 participants (non-ICU and ICU) and healthy controls (Figure 2A; participant demographics are detailed in Supplementary Table 1). The frequency of intracellular caspase-1-positive CD4 T cells was significantly elevated at baseline in non-ICU and ICU COVID-19 patients compared to baseline and nigericin-stimulated healthy participants (all p-values ​​< 0.0001).

[0152] Although RNA analysis did not reveal increases in IL-18 and IL-1β, serum levels of IL-18 were elevated in individuals with moderate to severe COVID-19 and showed a positive correlation with the expression of helper T cell caspase-1 (Figure 2B). Caspase-1 expression was primarily present in the memory CD45RO population and showed a weak correlation with older age, a finding that may potentially explain older age as one of the greatest risk factors for poor outcomes in COVID-19 (Figure 2C and D). Furthermore, helper T cell caspase-1 levels in COVID-19 patients correlated with CRTH2+ T cells, γ / δ T cells, and plasmacytoid dendritic cells (Supplementary Table 2). Such correlations point to complex cellular interactions involved in COVID-19. It is also important to note that there was no statistical difference in the expression of the Fas receptor on T helper cells (Figure 2F), suggesting that Fas-dependent cell death is not a contributing factor to T cell exhaustion in COVID-19.

[0153] 2. Increased caspase-1 in T helper cells is seen in a subset of patients undergoing clinical allergy and immunology care To assay caspase-1 levels in patients presenting to an allergy / immunology clinic, a laboratory-developed test (LDT) was designed to stain intracellular active caspase-1 in T helper cells and analytically validated in a CLIA-certified and CAP-accredited flow cytometry laboratory. The normal range of the assay is indicated by the gray shaded area (Figure 3). Data from 102 adult and pediatric subjects are shown for patients with chronic sinusitis, moderate to severe asthma, chronic idiopathic urticaria, and immunodeficiency. Because the study is CLIA / CAP-approved, the assay was performed as part of patient care in immunology settings. Participant demographics are shown in Supplementary Table 3. There was no significant correlation between comorbid conditions defined by the Centers for Disease Control and Prevention and high caspase-1 expression. However, given their low frequency in this patient cohort, it is difficult to draw definitive conclusions. However, asthma patients showed baseline elevations of caspase-1.

[0154] 3. Caspase-1 upregulation is not limited to the acute phase of COVID-19 Up to 87% of hospitalized patients and 35% of outpatients who recovered from COVID-19 reported at least one persistent symptom, particularly fatigue and dyspnea [26, 27]. Preliminary reports have described this new feature as the "post-COVID syndrome," but its mechanism and natural history remain unknown. Caspase-1 expression was analyzed in T helper cells of healthcare workers (HCWs) with persistent symptoms for at least 90 days after SARS-CoV-2 infection (Supplementary Table 4). Only in symptomatic "post-COVID-19" HCWs, also known as long-haulers, was there a significant upregulation of baseline and nigericin-stimulated T helper cell caspase-1 levels (Figure 4). Interestingly, PCR-negative HCWs with a history of influenza-like illness in early 2020 and HCWs with positive IgG for SARS-CoV-2 also exhibited elevated caspase-1 expression. Nigericin-stimulated caspase-1 expression levels were comparable to those in patients with active infection, but baseline caspase-1 levels were lower in these long-haulers (Figure 2). Unexposed control subjects did not demonstrate overexpression of T cell caspase-1.

[0155] 4. Pan-caspase inhibitors suppress elevated caspase-1 activity in CD4 T cells from patients with moderate to severe COVID-19. To assess whether T cell caspase-1 activity could be suppressed by caspase inhibitors, whole blood samples were incubated with either the pan-caspase inhibitor emricasan (EMR) or the selective caspase-1 inhibitor VX765, followed 24 hours later by incubation with or without nigericin stimulation. While EMR suppressed T helper cell caspase-1 expression in COVID-19 samples or prevented its upregulation in healthy subjects (Figure 5), VX765 did not exhibit a similarly robust inhibitory effect.

[0156] 5. Erythrocytes show increased caspase-3 in COVID-19 infection and are inhibited by pan-caspase inhibitors. Cellular caspases are not limited to immune cells. Recent reports have suggested abnormalities in RBCs in COVID-19 patients [28-30]. During Ficoll separation, a layer of RBCs contaminating the PBMC layer was observed, and this layer was universally present in all samples from COVID-19 individuals (Figure 6A). This finding was also present in up to 80% of COVID-19 convalescent subjects. Plasma from acutely infected subjects induced similar findings when incubated overnight with plasma-depleted whole blood from healthy patients. Treatment of plasma samples with trypsin, DNAse, or heat inactivation did not abolish this effect. RBCs do not express caspase-1 but have detectable caspase-3, which is increased in various disorders. RBCs from acute COVID-19 subjects were found to have significantly upregulated caspase-3 compared to healthy controls (Figure 6B). Plasma from these patients also upregulated caspase-3 in RBCs from healthy subjects. This effect was not observed when RBCs from healthy subjects were incubated with plasma from influenza-infected patients, although similar RBC contamination was observed in these samples after Ficoll separation. Furthermore, EMR suppressed the upregulation of caspase-3 in samples incubated in COVID-19 plasma but did not alter baseline expression levels in samples incubated in influenza plasma.

[0157] References for Example 9 The disclosure of each cited reference is expressly incorporated herein. TIFF0007742357000004.tif182160TIFF0007742357000005.tif217161TIFF0007742357000006.tif209158TIFF0007742357000007.tif203161TIFF0007742357000008.tif203160TIFF0007742357000009.tif210160TIFF0007742357000010.tif188161

Claims

1. 1. A pharmaceutical composition for treating a human subject to reduce T-cell lymphopenia or T-cell pyroptosis, comprising an effective amount of a caspase inhibitor of at least caspase 1, wherein the caspase inhibitor is selected from the group consisting of emricasan, CTS-2090, vernacasan (VX-765), pralnacasan (VX-740), and O-desethyl-vernacasan (VRT-043198), and wherein the human subject is infected with the positive-sense single-stranded RNA virus SARS-CoV-2.

2. 10. The pharmaceutical composition of claim 1, wherein the subject has a condition selected from the group consisting of high blood pressure, psoriasis, heart disease, gout, arthritis, inflammatory bowel disease, gouty arthritis, type I and type II diabetes, vitiligo, autoimmune Addison's disease, cryopreservation syndrome, and metabolic syndrome.

3. The pharmaceutical composition of claim 1, wherein the activity of caspase 1 is elevated in immune cells of the human subject.

4. The pharmaceutical composition of claim 1, wherein the expression of caspase 1 is elevated in immune cells of the human subject.

5. 2. The pharmaceutical composition of claim 1, wherein the caspase inhibitor inhibits one or more of caspases 3, 4, 5, 7, 8, 9, and 11.

6. The pharmaceutical composition of claim 1 , wherein the inhibitor is a pan-caspase inhibitor.

7. 2. The pharmaceutical composition of claim 1, wherein the inhibitor is emricasan.

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