Compositions and methods for treating coronavirus-induced acute respiratory distress syndrome (ARDS) and inflammatory disorders

Abstract

Methods and compositions containing phorbol esters or derivatives of phorbol esters are provided for preventing or treating sudden acute respiratory syndrome (SARS) coronavirus infections, including SARS-CoV-2 infections and the associated COVID-19 disease. Methods and compositions are also provided for preventing and treating acute inflammatory conditions and associated pathogenic insults, including acute respiratory distress syndrome (ARDS) and cytokine storm syndrome (CSS), seen in severe SARS-CoV-2 / COVID-19 cases.

Description

[Technical Field]

[0001] The present invention relates to therapeutic and prophylactic compositions and methods for treating pulmonary viral infections and associated inflammatory and immunological disease states of the lungs and other organs mediated by viral infections and other agents. In certain embodiments, the present invention relates to methods and compositions for treating Acute Respiratory Distress Syndrome (ARDS) or Severe Acute Respiratory Syndrome (SARS), caused by viral pathogens, bacterial pathogens, caustics, trauma, burns, or other sources of lung or immune system injury, or other sources of inflammatory injury or stimuli. In more specific embodiments, the present invention relates to compositions and methods for treating and preventing pulmonary viral infections and the associated pulmonary and inflammatory disease symptoms, ARDS / SARS, caused by SARS coronavirus or other respiratory viruses. [Background technology]

[0002] Background of the Invention Acute respiratory distress syndrome (ARDS), also known as "adult respiratory distress syndrome," is a severe condition that often results in lung failure, hospitalization, and critical care. ARDS can be caused by a variety of pulmonary and extrapulmonary causes, including viral or bacterial pneumonia, aspiration of gastric contents, inhalation of hot gases or caustic agents, pulmonary contusion or other pulmonary trauma, sepsis, acute pancreatitis, and other causes. The cause of ARDS currently receiving the most attention is emerging from the ongoing COVID-19 disease pandemic caused by the SARS coronavirus SARS-CoV-2.

[0003] Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses. They have the largest genomes (26–32 kb) of any known RNA virus. Coronaviruses infect a wide range of avian and mammalian species, including humans. Of the six known human coronaviruses, four (HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63) circulate annually within human populations worldwide and generally cause mild respiratory illness, although the disease may be more severe in infants, the elderly, and immunocompromised individuals.

[0004] In contrast, several recent coronavirus outbreaks have had devastating health and economic consequences for people. These “novel” coronaviruses have all induced ARDS in severe cases (specifically, in the context of coronavirus disease, the disease is called “severe acute respiratory syndrome” (SARS)). Coronaviruses capable of causing ARDS / SARS include the original “SARS” virus (SARS-CoV), which emerged in Guangdong Province, China in 2002, the COVID-19 (SARS-CoV-2) coronavirus (also known as the “COVID-19 virus”), which first emerged and was identified in Wuhan, and the Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV). All SARS / MERS coronaviruses induce ARDS as a proximate cause of the most severe cases, which are associated with debilitating respiratory tract infections.

[0005] Within the past two decades, each of the SARS coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) has been transmitted zoonotically from other mammalian species to humans, causing deadly outbreaks. While horseshoe bats are believed to be the primary reservoir for these novel coronaviruses, intermediate hosts for transmission of the viruses to humans have been identified: palm civets in the case of SARS-CoV and dromedary camels in the case of MERS-CoV. In the case of SARS-CoV-2, recent metagenomic studies strongly suggest that this most recent SARS coronavirus was transmitted to the human population from Malayan pangolins (Manis javanica), which were smuggled / traded in South China wet markets (Lam et al., 2020). Given the high pathogenicity and airborne transmissibility of SARS-CoV and MERS-CoV, concerns about the potential for a new coronavirus pandemic have been raised for years, culminating in the current COVID-19 pandemic.

[0006] Since December 2019, the COVID-19 pandemic has been rapidly spreading through populations worldwide. The COVID-19 virus is now known to be highly contagious via airborne transmission (exhaled droplets) and contact, with transmission by pre-symptomatic and asymptomatic carriers also possible. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. As of May 17, 2020, over 4.75 million cases of COVID-19 had been identified, causing 314,000 deaths in approximately 213 countries.

[0007] The clinical course of COVID-19 pneumonia exhibits a wide spectrum of severity and progression patterns. Some patients develop dyspnea (shortness of breath) within a median of 8 days (range 5–13 days) after illness onset, whereas others may not experience respiratory distress. Approximately 3–30% of patients require hospitalization for intensive care. Severely ill patients may experience rapid progression to multiple organ dysfunction and even death. Patients presenting with shortness of breath and hypoxemia may rapidly progress to acute respiratory distress syndrome (ARDS), severe sepsis with shock, and multiple organ dysfunction within just a few days, usually within about 1 week. ARDS has been described in approximately 15–30% of hospitalized COVID-19 patients, appearing on average 8 days after symptom onset (Tu et al., 2020).

[0008] While COVID-19 disease may be moderate or even asymptomatic in most people, approximately 20% of infected subjects develop severe symptoms. Morbidity and mortality from COVID-19 are particularly high among the elderly and subjects with underlying lung disease, heart disease, diabetes, cancer, or other serious health conditions. A small number of patients who present with ARDS / SARS become fatal, resulting in respiratory failure and multiple organ failure, often accompanied by septic shock.

[0009] The pathogenesis of ARDS / SARS involves inflammatory, immunological, and tissue / organ injury, including lung parenchymal inflammation, neutrophil infiltration into alveolar air spaces, oxidative stress, disruption of the endothelial and epithelial barriers, and damage to the epithelial lining with subsequent pulmonary fibrosis.

[0010] Despite our considerable knowledge of the mechanisms and pathogenesis of ARDS, over 20 years of extensive clinical research have failed to yield effective treatments to prevent ARDS or substantially reduce its mortality (Fanelli et al., 2013). First-line ARDS treatment remains primarily supportive, consisting of administering oxygen or mechanical ventilation to the patient and closely managing the patient's fluid balance.

[0011] In the case of COVID-19 disease, our research and that of others have pointed to a critical inflammatory mechanism that causes ARDS in severe COVID-19 patients. Common symptoms of severe COVID-19 disease include (1) rapid worsening of the disease after 1–2 weeks, (2) a significant decrease in circulating lymphocytes, particularly natural killer (NK) cells, (3) elevated levels of proinflammatory cytokines (IL-6, TNFα, IL-8, etc.) and other inflammatory factors, such as C-reactive protein (CRP), and (4) severe immune dysfunction characterized by atrophy of the spleen and lymph nodes and a decrease in lymphocytes in lymphoid organs. Other sequelae commonly observed in severe COVID-19 patients include infiltration of monocytes and macrophages with minimal lymphocytic infiltration into lung lesions, as well as a form of vasculitis accompanied by hypercoagulability and multiple organ damage.

[0012] The emergence of a hyperinflammatory state, now termed Pediatric Inflammatory Multisystem Syndrome (PIMS), has now been identified in a small subset of children infected with COVID-19. This Kawasaki-like illness has emerged in the midst of the global COVID-19 pandemic, as evidenced by initial reports in Italy and parallel reports of similar cases in New York, New Jersey, Massachusetts, and the United Kingdom. Although rare, the effects of PIMS can be severe and potentially life-threatening.

[0013] Emerging data reveal that PIMS is an abnormal immune response to SARS-CoV-2 infection, causing a Kawasaki disease-like illness in genetically predisposed pediatric patients. Common symptoms of COVID-19-associated PIMS include fever, rash, bloodshot eyes, dry or cracked mouth, redness in the palms and soles of the feet, and enlarged glands. Approximately one-quarter of children have cardiac complications. The abnormal immune response in COVID-19-associated PIMS leads to inflammation and swelling of blood vessels, sometimes accompanied by coronary artery aneurysms.

[0014] The first observational cohort study in Italy (published May 13, 2020, in The Lancet by Verdoni et al.) found that 8 of 10 children diagnosed with PIMS between March 17 and April 14, 2020, tested positive for COVID-19 (it is highly likely that the remaining 2 subjects had false-negative SARS-CoV-2 tests). The Italian study compared data on this COVID-19 cohort with data on 19 children diagnosed with Kawasaki disease over the previous 5 years before the pandemic. On average, before the pandemic, one Kawasaki disease case was identified every 3 months in the same jurisdiction, representing a 30-fold increase in PIMS over the study period during the COVID-19 pandemic.

[0015] Children with PIMS in an Italian study diagnosed during the COVID-19 pandemic experienced more severe symptoms than children treated for classic Kawasaki disease within the past five years. Six of 10 new patients (60%) developed cardiac complications, compared with only 2 of 19 previously treated Kawasaki disease subjects (11%). Five of 10 children (50%) in the Italian study diagnosed during the COVID-19 pandemic developed symptoms of toxic shock syndrome (TSS) and required fluid resuscitation to correct hypotension, whereas none of the 19 Kawasaki disease subjects diagnosed before the COVID-19 outbreak developed this complication. TSS is a rare, life-threatening illness characterized by high fever, low blood pressure, and a rash, usually caused by a bacterial infection.

[0016] Treatment of PIMS patients in the Italian study included immunoglobulin therapy, and 8 of 10 patients also required steroid treatment (compared to only 3 of 19 in the pre-COVID-19 Kawasaki disease study group). Children hospitalized during the pandemic in Italy were also older than those diagnosed earlier (mean age 7.5 years vs. 3 years). Seven of 10 children were boys.

[0017] The U.S. Centers for Disease Control and Prevention (CDC) now recognizes a link between PIMS and COVID-19, and the New York City Department of Health has identified 145 cases of PIMS (also known as multi-system inflammatory syndrome in children, or MISC) in children to date.

[0018] Understanding the mechanisms involved in PIMS will help elucidate the human SARS-CoV-2 immune response more broadly, in both adults and children, and will help guide research and development of anti-inflammatory treatment tools and methods that may help alleviate the diverse COVID-19 disease symptoms in both populations.

[0019] The excessive activity of proinflammatory cytokines induced in severe cases of COVID-19 disease is commonly referred to as a "cytokine storm." "Cytokine storm syndrome" (CSS) (also known as cytokine release syndrome, or CRS) generally refers to the unrestrained and excessive release of proinflammatory cytokines and other inflammatory factors, leading to potentially life-threatening excessive inflammation. CSS has been shown to correlate with various infectious, rheumatic, and autoimmune disorders, as well as in some subjects receiving immuno-oncology therapy.

[0020] In infectious diseases, CSS usually arises from a localized area of ​​infection but can rapidly spread throughout the body. COVID-19 and other SARS coronaviruses (SARS-CoV and MERS) manifest in severe cases with rapid viral replication, widespread inflammatory cell infiltration, and CSS, leading to ARDS / SARS (acute lung inflammation and injury, resulting in pulmonary failure and death in the most severe cases). CSS associated with COVID-19 disease can also result in systemic inflammation, vasculitis, multiple organ failure, and hypercoagulation, potentially leading to stroke in severe cases.

[0021] Severe COVID-19 patients exhibit an elevated proinflammatory cytokine profile similar to the CSS symptoms previously observed in SARS-CoV and MERS subjects. Huang et al. (2020) reported that a diverse array of proinflammatory cytokines and other inflammatory factors are elevated in patients with severe COVID-19 disease. Huang and colleagues reported increased levels of interleukin (IL)-1B, IL-1RA, IL-7, IL-8, IL-9, IL-10, fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFNγ, granulocyte-colony-stimulating factor (G-CSF), interferon-γ-inducible protein (IP10), monocyte chemoattractant protein (MCP1), macrophage inflammatory protein 1 alpha (MIP1A), platelet-derived growth factor (PDGF), tumor necrosis factor (TNFα), and vascular endothelial growth factor (VEGF) in 41 hospitalized COVID-19 patients (13 ICU and 28 non-ICU). Huang and colleagues reported that IL-2, IL-7, IL-10, G-CSF, IP10, MCP1, MIP1A, and TNFα were higher in more severely ill ICU patients than in non-ICU patients (Huang et al., 2020; Conti et al., 2020). Of note, Huang et al.'s study reported no difference in serum IL-6 levels between ICU and non-ICU patients.

[0022] Proinflammatory cytokines, such as interleukin-1 (IL-1), are important mediators of localized and systemic inflammation. When stimulated by viral infection, IL-1 plays a fundamental role in tissue inflammation, fever, and fibrosis. IL-1 activates macrophages, which phagocytose infected and dead cells and release other inflammatory factors. Therefore, cytokine-induced macrophages play a central role in the excessive inflammation and associated lung pathogenesis of COVID-19.

[0023] Other proinflammatory cytokines have been implicated in the clinical progression of CSS-mediated SARS-CoV and COVID-19 disease. Among these cytokines, interferon-alpha (IFNα), tumor necrosis factor (TNF), and IL-1 have long been considered to play important roles (Ho et al., 2003; Auyeung et al., 2005; Chousterman et al., 2017). 2,18-19 Other proinflammatory cytokines suggested to potentially mediate SARS-associated CSS include IL-8, IL-6, and others.

[0024] IL-1 is the most thoroughly studied cytokine, and its properties are associated with inflammatory diseases, such as those caused by viral infections. Following binding of COVID-19 to Toll-like receptors (TLRs), regulated synthesis and release of IL-1 occur. Activation of this receptor triggers a proinflammatory "cascade." This cascade begins with the synthesis of pro-IL-1, which is then cleaved by caspase-1, followed by inflammasome activation (Chen et al., 2006). High levels of adenosine triphosphate (ATP) correlate with activation of the P2X7 receptor (purinogenic 2 receptor), which mediates inflammasome activation. Pro-caspase-1 and other factors drive the synthesis of IL-1b in lysosomes, which is then secreted out of macrophages and mediates lung inflammation, fever, and fibrosis associated with severe respiratory distress in susceptible COVID-19 patients. Immune cells are attracted to the site of infection by the chemokine IL-8, which is produced at the site of inflammation, further exacerbating the cytokine "storm" in severe cases.

[0025] Inflammation is an adaptive response that normally develops to combat injury and defend against foreign substances and pathogens introduced into the body. In contrast, excessive inflammation in cases of CSS associated with COVID-19 disease and other inflammatory conditions can be extremely harmful if not managed. The described association between proinflammatory cytokine levels and viral replication and disease severity in COVID-19 and SARS-CoV has led many researchers to theorize that anti-inflammatory cytokines may be useful therapeutic agents to combat these diseases and conditions. The primary "anti-inflammatory cytokines" envisioned in this context include interleukin (IL)-1 receptor antagonist (IL-1Ra), IL-4, IL-10, IL-11, and IL-13. Specific cytokine receptors for IL-1, tumor necrosis factor-alpha, and IL-18 are also characterized as "anti-inflammatory" factors that function as inhibitors of proinflammatory cytokines. Therefore, IL-1Ra, IL-37 or IL-13 have been proposed as candidates to alleviate both pulmonary and systemic inflammation and fever in severe COVID-19 disease.

[0026] The high mortality rate, complex pathogenesis and epidemiology, and lack of vaccines or therapeutic tools against coronaviruses have created an urgent need for effective prophylactic and therapeutic tools, vaccines, and related therapeutic agents to prevent and treat infections and diseases mediated by these pathogens.

[0027] Efforts to develop effective drugs and methods for preventing or treating COVID-19 disease have been hampered, in part, by the complexity of the SARS virus genome and its complex disease pathogenesis. SARS-CoV, MERS-CoV, and SARS-CoV-2 each have a large, single-stranded, positive-sense RNA genome (27–32 kb) encoding six to ten genes. The gene order is generally highly conserved, with replication and transcription-related genes first, followed by structural genes. The replication and transcription-related genes are translated into two large nonstructural polyproteins by overlapping open reading frames (ORFs) translated by ribosomal frameshifting. The structural genes, including the spike (S), envelope (E), and membrane (M) proteins that make up the viral coat, and the nucleocapsid (N) protein that packages the viral genome, are all translated from subgenomic RNA. Some of these proteins undergo glycosylation in the host Golgi apparatus to form glycoproteins.

[0028] The SARS coronavirus spike (S) glycoprotein mediates viral binding to host cells, enabling intracellular colonization. The S protein is primed by host cell proteases and recognized by cellular receptors. The human serine protease TMPRSS2 is responsible for priming the S protein of both SARS-CoV and SARS-CoV-2, and angiotensin-converting enzyme 2 (ACE2) acts as a receptor for entry for both viruses. In the case of MERS-CoV, it specifically binds to another receptor, dipeptidyl peptidase 4 (DPP4).

[0029] The varied pathogenesis and epidemiology of SARS-CoV-2 are partly related to interindividual and interpopulation differences in ACE2 expression levels and possibly also to structural differences in ACE-2 among individuals. Children and young adults generally express lower levels of ACE-2, which may contribute to their relative resistance to severe COVID-19 disease. Although several ACE-2 gene variants have been identified in human populations, which may further affect viral infection and pathogenesis, no heterogeneity has been found in ACE-2 residues involved in SARS-CoV-2 viral S protein binding. This indicates that the virus likely utilizes a highly conserved attachment / entry site, which correlates with the rapid spread of SARS-CoV-2 across continents and different human populations (Cao et al., 2020). In light of these reports, ACE-2 has been widely considered a potential target for intervention in the development of anti-COVID-19 prevention and treatment tools. However, significant obstacles remain to achieve success along this discovery path.

[0030] Another challenging path for innovation toward COVID-19 management involves the use of anti-inflammatory cytokines and related inhibitors and immunomodulators to treat virus-mediated ARDS and CSS in COVID-19 patients. These goals are complicated by concerns about inhibiting beneficial antiviral immune and inflammatory functions. Numerous targets have been considered for cytokine modulation as a means to combat SARS-CoV infection and pathogenesis. Recent studies have confirmed that severe COVID-19 patients exhibit elevated erythematosus sedimentation rates (ESRs) and persistently elevated levels of C-reactive protein (CRP), IL-6, TNFα, IL-1b, IL-8, and IL2R (Tu et al.). These and other proinflammatory markers are clinically associated with ARDS, hypercoagulability, and disseminated intravascular coagulation (DIC), which manifests as thrombosis, thrombocytopenia, and limb gangrene in severe cases. These and other sequelae of CSS can exacerbate lung injury and lead to a systemic CSS-related illness called "extrapulmonary systemic hyperinflammation syndrome" (ESHS), which can be a fatal complication in pregnant subjects.

[0031] The question of when and how to block CSS to mediate anti-inflammatory therapy is crucial for reducing mortality in COVID-19 subjects. In this regard, it is important to consider the underlying immune impairment mediated by SARS-CoV. Lymphopenia is one of the most prominent diagnostic markers of COVID-19. Both T cells and NK cells are substantially reduced in patients with COVID-19, while white blood cell counts are elevated. In critically ill patients, NK cells are extremely low or even undetectable, and memory helper T cells and regulatory T cells are severely reduced (Hui et al., 2019). Remarkable autopsy findings of COVID-19 reveal that secondary lymphoid tissues are also destroyed in severe cases, which is a striking difference from typical CSS disease. Splenic atrophy is commonly observed and correlates with splenic lymphocytosis, significant cytopathic changes, focal hemorrhagic necrosis, macrophage proliferation, and macrophage phagocytosis. Similarly, lymph node atrophy and reduction in lymph node numbers, as well as decreased CD4+ and CD8+ T cell counts in the spleen and lymph nodes, are also observed [Hui et al., 2019]. Additionally, in lungs with characteristic diffuse alveolar damage (DAD), the predominant infiltrating cells were monocytes, macrophages, and moderately multinucleated giant cells, with very few lymphocytes. The majority of infiltrating lymphocytes were CD4+ T cells. Importantly, even when PCR tests were negative in blood or throat swabs, viral inclusion bodies were still detected in type II alveolar epithelium and macrophages (Zu et al., 2020; De Wit et al., 2016; Chan et al., 2015B). This finding is consistent with a so-called "primary cytokine storm" induced by viral infection and primarily triggered by alveolar macrophages, epithelial cells, and endothelial cells, rather than a "secondary cytokine storm" induced by different subsets of activated T lymphocytes in the later stages of viral infection or as a complication of T cell-engaging therapy (Chan et al., 2012; Kanne et al., 2020).

[0032] Yet another complication of severe COVID-19 disease, generally correlated with ARDS severity, is the phenomenon of neutrophil infiltration into pulmonary capillaries, accompanied by the formation of neutrophil extracellular traps (NETs), which has been identified in several recently published COVID-19 autopsies. Neutrophils are the most common type of white blood cell (WBC) in the bloodstream and are phagocytes that migrate from the blood to sites of injury or infection during the acute phase of inflammation. Neutrophils migrate freely from the source of infection into and through vascular and interstitial compartments by chemotaxis, attracted by cytokines (e.g., interleukin-8 (IL-8), C5a, fMLP, leukotriene B4, and H2O2) expressed by activated endothelium, mast cells, and macrophages. Once localized, neutrophils themselves express and release cytokines that act to amplify the inflammatory response by recruiting and activating other cell types. Neutrophils significantly outnumber monocyte / macrophage phagocytes and are generally considered a hallmark of early acute inflammation.

[0033] In addition to recruiting and activating other cells of the immune system, neutrophils play a critical role in the first line of defense against pathogens. Neutrophils have three ways of directly attacking viral and bacterial pathogens: phagocytosis (ingestion), degranulation (release of soluble antimicrobial factors), and generation of neutrophil extracellular traps (NETs). In ARDS in general, and SARS-CoV infection more specifically, it appears that at least some of these normally beneficial immune / inflammatory functions of neutrophils may be destructively overactivated or overactivated.

[0034] Neutrophil degranulation typically releases an assortment of proteins from three distinct types of granules. Azurophilic granules (or "primary granules") release myeloperoxidase, bactericidal / permeability-increasing protein (BPI), defensins, and the serine proteases neutrophil elastase and cathepsin G. Specialized granules (or "secondary granules") release alkaline phosphatase, lysozyme, NADPH oxidase, collagenase, lactoferrin, histaminase, and cathelicidin. Tertiary granules release cathepsins, gelatinases, and collagenases. When abnormally overexpressed or overactivated, these various enzymes and antimicrobial agents can have profound deleterious effects on tissues and extracellular components, including through the destruction of essential "barrier" components present in blood vessels, lungs, and kidneys that preserve their structural integrity, pathogen resistance, and function.

[0035] A more significant pathogenic consequence of excessive neutrophil numbers, infiltration, and activation in COVID-19 disease appears to involve the generation of neutrophil extracellular traps (NETs). NETs are webs of chromatin, microbicidal proteins, oxidative enzymes, and cytokines that are normally released by neutrophils to suppress infection. However, if not properly controlled, NETs appear to spread inflammation and cause microvascular thrombosis, possibly contributing to pulmonary failure / ARDS in COVID-19 patients with CSS and PMIS, and playing a similar role in vasculitis and secondary organ failure. Various other diseases known to be caused by NETs also present with similar symptoms, including thick mucus secretions and blood clots in the airways, as seen in ARDS / SARS.

[0036] Elevated blood neutrophil levels worsen outcomes in COVID-19, and the role of NETs appears to be paramount. In a recent study, Zuo and colleagues (2020) reported that increased neutrophil infiltration into pulmonary capillaries and overexpression of NETs strongly correlated with the severity of viral pneumonia / ARDS in COVID-19 patients. Serum from severe COVID-19 patients exhibited elevated levels of cell-free DNA, myeloperoxidase (MPO)-DNA, and citrullinated histone H3 (Cit-H3), which indicate elevated NET levels. Cell-free DNA levels also strongly correlated with acute inflammatory phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase. MPO-DNA correlated with both cell-free DNA and absolute neutrophil counts, while Cit-H3 correlated with platelet levels and the observed prothrombic (clot-forming) effect of NETs. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation compared with those able to breathe unaided. Finally, serum from individuals with COVID-19 elicited NET release from control neutrophils in vitro. These data reveal that high levels of neutrophils and NETs in severe COVID-19 patients are likely an important contributor to CSS, ARDS, vasculitis, and thrombus formation in these patients. Other studies have more broadly and specifically linked elevated NETs to ARDS, comparable to these findings for severe SARS-CoV infection.

[0037] It is important to note that neutrophils can be beneficial during periods of viral infection or disease and then destructive during other periods when they become overwhelmed or overactivated. In diseases with alpha-1 antitrypsin deficiency, the key neutrophil enzyme elastase, which is normally beneficial in managing pathogenesis, is not properly inhibited by alpha-1 antitrypsin, causing excessive tissue damage during a normally adaptive inflammatory response (as occurs in emphysema). In other situations, including apparently in the context of COVID-19 disease, excessive neutrophil activation releases elastase and other destructive enzymes into the extracellular compartment, contributing to lung barrier disruption and acute lung injury. This duality of regulated and unregulated function reflects an adaptive process gone awry and arguably indicates that the process is being hijacked or overwhelmed by the pathogen in question or due to the novel and fluctuating pathogenesis of the progressive disease process.

[0038] There are numerous examples of host-targeting molecular strategies pursued by viruses and bacteria to subvert, misdirect, or even diverge host immune and inflammatory processes to the pathogen's advantage. In the key example of neutrophils, molecular interactions between these key effector cells and their target pathogens can profoundly alter neutrophil proliferation and lifespan. Both viral and bacterial pathogens have been shown to extend neutrophil lifespan or accelerate neutrophil lysis after phagocytosis. Chlamydia pneumoniae and Neisseria gonorrhoeae have both been shown to inhibit neutrophil apoptosis. Other pathogens are known to extend neutrophil lifespan by both interfering with spontaneous apoptosis and inhibiting phagocytosis-induced cell death (PICD). At the other end of the spectrum, some pathogens can directly alter the fate of neutrophils after phagocytosis by accelerating cell lysis and / or apoptosis (a type of neutrophil "overactivation" that leads to tissue necrosis).

[0039] Viruses and bacteria can also directly attack and destroy elements of the host immune system or inflammatory control mechanisms. As mentioned above, severe COVID-19 disease is characterized by a significant reduction in lymphocyte numbers, and it is quite possible that NK cells and other crucial lymphocytes lost in this disease could be directly invaded and destroyed by the SARS-CoV virus. Alternatively, the destruction of these cells may be indirect, as a concomitant consequence of CSS. Because the COVID-19 virus appears to infect target cells primarily through angiotensin-converting enzyme 2 (ACE2), and ACE2 expression is absent on lymphocytes, these specific cells likely fall victim to the CSS destruction mechanism.

[0040] In relation to these mechanisms, vasculitis and thrombosis with endothelial injury are prominent features in severe COVID-19 patients. Many critically ill COVID-19 patients have vasculitis-like manifestations, often accompanied by gangrene in the extremities. Autopsies reveal that pulmonary vessels (those associated with the alveolar septa) are congested and edematous, with intravascular and perivascular infiltration of monocytes and their macrophage progeny. Small vessels exhibit hyperplasia, vessel wall thickening, luminal narrowing, obstruction, and focal hemorrhage. Microvascular hyaline thrombi are found in severe cases (Zu et al., 2020; Hui et al., 2019; Chan et al., 2015B). The underlying mechanisms of vascular injury may involve direct endothelial cell injury by the virus or downstream CSS effects leading to DIC, antiphospholipid syndrome (APS), and vasculitis mimicry. Pathological "autoimmune" responses induced by antiviral immunity may also be involved.

[0041] COVID-19 patients with a hypercoagulable state include those with PIMS. Common sequelae of these complications include prolonged prothrombin time, elevated D-dimer and fibrinogen levels, and near-normal activated partial thromboplastin time. A minority of patients progress to overt disseminated intravascular coagulation (DIC). Tang et al. (2020) reported that 71.4% of nonsurvivors and 0.6% of survivors of COVID-19 showed evidence of overt DIC. Furthermore, many nonsurvivors exhibited subclinical DIC (indicated by fibrin clot formation) characterized by a hypercoagulable state upon postmortem examination. A high proportion of patients with deteriorating COVID-19 presented with acroischemia, which indicates a hypercoagulable state prior to the onset of overt DIC.

[0042] Several factors may contribute to coagulation disorders in COVID-19 patients. The persistent inflammatory state in severe and critically ill COVID-19 patients acts as an important trigger of the coagulation cascade. Certain cytokines, including IL-6, can activate the coagulation system and suppress the fibrinolytic system. In the context of COVID-19, pulmonary and peripheral endothelial injury due to direct viral attack may be an important inducer of hypercoagulation. Endothelial cell injury strongly activates the coagulation system through tissue factor exposure and other pathways. Aggressive immune and inflammatory responses, in turn, are exacerbated by dysfunctional coagulation, and these two processes may act in a kind of feedback loop toward an uninhibited endpoint. Finally, because anticardiolipin and anti-β2GP1 antibodies have been detected in COVID-19 patients, the emergence of antiphospholipid antibodies in COVID-19 patients may exacerbate their coagulation abnormalities (Zhang et al., 2020).

[0043] While antiviral and supportive treatments are important components of COVID-19 disease management, blocking CSS using anti-inflammatory therapy represents a critical unrealized goal for the treatment and management of COVID-19 disease. Various anti-inflammatory drugs are known in the art, including nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and chloroquine / hydroxychloroquine, which may be useful in the treatment of COVID-19. To reduce pulmonary and systemic inflammation, immunosuppressants, proinflammatory cytokine antagonists (e.g., IL-6R monoclonal antibodies, TNF inhibitors, IL-1 antagonists, Janus kinase (JAK) inhibitors, etc.), and other immunomodulatory agents are also widely considered.

[0044] Despite this abundance of available anti-inflammatory agents, none of them are clear regarding their efficacy and potential adverse side effects in the context of ARDS and COVID-19 disease. Anti-inflammatory treatment of patients with COVID-19 disease raises fundamental risk / benefit concerns regarding whether and when to treat subjects with an anti-inflammatory regimen. These fundamental issues remain hotly debated and no consensus has emerged. A major concern is that anti-inflammatory drugs, such as corticosteroids, may delay or impair beneficial antiviral defenses and / or simultaneously increase the risk of secondary infections, particularly in subjects facing pre-existing immune system disorders or disorders mediated by the virus itself. Another issue arises with biological agents that target proinflammatory cytokines; they may only inhibit one specific inflammatory factor and therefore not be able to halt CSS in general. Alternatively, as with steroids, anti-inflammatory drugs may impair beneficial immune functions. For example, JAK inhibitors exert potent anti-inflammatory effects but simultaneously impair important immune mechanisms mediated by INF-α. Yet another fundamental and complex issue concerns the optimal time window for anti-inflammatory treatment, which appears to be crucial in the case of COVID-19 disease. Severely ill patients typically exhibit prolonged initial moderate symptoms followed by a rapid exacerbation 1–2 weeks after onset, and subsequent anti-inflammatory treatment may fail to achieve a favorable therapeutic response.

[0045] Further examining the pathogenesis of the SARS virus, SARS-CoV-2 exhibits tropism for and actively replicates in upper respiratory tract tissues. Like SARS-CoV, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2), which is widely expressed in vascular endothelium, respiratory epithelium, alveolar monocytes, and macrophages, as its primary receptor for cellular entry (Lu et al., 2020). The primary route of transmission is via direct or indirect respiratory tract exposure (demonstrated by live virus isolation from pharyngeal swabs and detection of subgenomic messenger RNA (sgRNA) in ependymal cells) (Wolfel et al., 2020). Tropism for upper respiratory tract tissues facilitates higher contact transmission of SARS-CoV-2 through continuous pharyngeal shedding of virus, even when symptoms are minimal and restricted to the upper respiratory tract. Late in the disease course, SARS-CoV-2 resembles SARS-CoV in that viral replication progresses to the lower respiratory tract and, in severe cases, then launches a widespread attack on the lungs and other ACE2-expressing target organs, including the heart, kidneys, gastrointestinal tract, and distal vasculature. The extent and duration of this viral spread correlates with clinical deterioration, which occurs primarily during the second week after disease onset. However, the worsening disease throughout the later stages in severe cases cannot be attributed solely to direct viral damage but also involves SARS-CoV-2-induced immune-mediated injury. Two distinguishing features of severe and critically ill patients at this stage of COVID-19 disease are a progressive increase in inflammation and an abnormal tendency toward hypercoagulation.

[0046] As was also the case with SARS-CoV, immune-mediated inflammation undoubtedly plays a key role in this late stage of severe COVID-19 pathogenesis. The progression of COVID-19 involves a continuing decline in lymphocyte counts, along with a significant increase, infiltration, and hyperactivation of neutrophils accompanied by a broad increase in inflammatory markers (including C-reactive protein, ferritin, interleukin (IL)-6, IP-10, MCP1, MIP1A, and TNFα). Various studies have reported that reduced lymphocyte counts and elevated levels of ferritin, IL-6, and D-dimer are directly associated with increased mortality in COVID-19 patients. While the mechanisms underlying the progressive lymphopenia in severe and critically ill COVID-19 patients remain unclear, declines in B cells, T cells, and natural killer (NK) cells are all more pronounced in severe cases. Other studies have reported increased levels of CD8+ T cell activation (measured by the ratio of CD38 expression to HLA-DR expression) despite a decrease in CD8+ T cell counts. Lymphopenia is also a key feature of SARS-CoV disease progression, with declines in both CD4+ and CD8+ T lymphocytes often correlating with pathogenic radiographic changes. Although some studies have demonstrated direct infection of macrophages and lymphocytes by SARS-CoV, the rapid decline in lymphocyte numbers in SARS-CoV-1 has been attributed to two additional mechanisms: redistribution of circulating lymphocytes or lymphocyte depletion through apoptosis or pyroptosis. Viral gene expression was not observed in peripheral blood mononuclear cells (PBMCs) from COVID-19 patients, and the normal viral transporter ACE2 is not expressed on lymphocytes. However, Wang et al. (2020) suggested that T lymphocytes may be more permissive to SARS-CoV-2 than SARS-CoV due to spike protein-triggered endocytosis.Other reports have shown that there is upregulation of apoptosis, autophagy, and p53 pathways in PBMCs from COVID-19 patients (Xiong et al., 2020), and other researchers have reported that NK cells and CD8+ T cells are functionally exhausted in COVID-19 patients due to overexpression of NKG2A (Zheng et al., 2020).

[0047] The above collective findings indicate that immune disruption begins early in COVID-19 disease as a result of a combination of direct and bystander effects. While the gradual pathogenic changes of COVID-19 may be reversible with timely intervention, especially in mild and moderate cases, the complexity and intractability of this disease pose many obstacles. The clinical course of SARS-CoV-2 infection can conceptually be divided into three phases: the viremic phase, the acute (pneumonic) phase, and the severe or convalescent phase. Patients with competent immune function and no significant risk factors (e.g., old age, significant comorbidities) may generate effective and adequate immune responses to suppress the virus in the first or second phase without manifesting the hyperimmune / hyperinflammatory symptoms of CSS, ARDS, or PIMS. In contrast, patients with impaired immune function may fail in the early stages and be at higher risk of developing severe or critical illness with increased morbidity and mortality. Therefore, treatment of COVID-19 should potentially involve meticulous triaging and patient staging, relying on observation of clinical deterioration (e.g., by detecting rapid hyperinflammation, threshold lymphopenia, hypercoagulable states, etc.) to enable treatment of high-risk subjects between stages 1 and 2.

[0048] In light of the above, there is an urgent need in the art for more effective tools and methods to combat acute respiratory distress syndrome (ARDS), including coronavirus-mediated severe acute respiratory syndrome (SARS), cytokine storm syndrome (CSS), and other hyperinflammatory diseases such as pediatric inflammatory multisystem syndrome (PIMS), Kawasaki disease, extrapulmonary systemic hyperinflammatory syndrome (ESHS), and their associated vascular congestion and thrombotic conditions such as disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and gangrene, and the widespread cellular, tissue, and organ injury associated with these conditions.

[0049] The associated need to combat the devastating destruction mediated by COVID-19 and the related SARS coronavirus, which has emerged as a novel pathogen three times in the past two decades and will likely re-emerge. The unmet needs for treating and managing COVID-19 disease are numerous and complex, spanning the vast range of pathogenic, immunological, and inflammatory insults mediated by COVID-19, including ARDS / SARS, CSS, PIMS, ESHS, and DIC in severe cases.

[0050] Addressing these needs and overcoming the associated obstacles will require the discovery and development of powerful, innovative tools, including multi-active and multi-targeted drugs and therapies that can impair viral function and control hyperimmune and hyperinflammatory responses while enhancing, or at least sparing, the host's beneficial immune and inflammatory capabilities and responses. Additionally, these objectives will be most beneficially met by developing novel combinations of complementary drugs and treatments that simultaneously reach multiple targets and induce multiple (e.g., antiviral, pro-immune, and anti-inflammatory) effects. Successful approaches to combating COVID-19 and its diverse pathogenic sequelae will be further optimized for individual patients by timing and delivering treatment forms, dosages, and modalities tailored to the disparate manifestations of each patient's unique disease pathology. Summary of the Invention

[0051] SUMMARY OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION The present invention achieves the above-mentioned goals and fulfills additional objectives and advantages through the novel and potent use of "TPA" compounds and related compositions and methods to treat and / or prevent acute respiratory distress syndrome (ARDS) in mammalian subjects, including COVID-19 and other SARS-coronavirus-mediated severe acute respiratory syndrome (SARS) in humans. Related aspects of the invention utilize clinically effective TPA compounds, compositions, and methods to prevent and / or treat hyperimmune and hyperinflammatory conditions, including "cytokine storm syndrome" (CSS) generally, and numerous other hyperinflammatory diseases, such as, but not limited to, pediatric inflammatory multisystem syndrome (PIMS) associated with COVID-19 disease, a related pediatric condition known as Kawasaki disease, extrapulmonary systemic hyperinflammatory syndrome (ESHS), and hyperinflammation-induced vascular congestion and thrombotic conditions, such as disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and gangrene.

[0052] A further aspect of the present invention utilizes TPA compounds, compositions and methods to effectively limit or treat the wide range of cellular, tissue and organ injury associated with ARDS, SARS, CSS, PIMS, ESHS, DIC, and other immune and inflammatory injuries or disease states that may accompany these conditions.

[0053] Further aspects of the present invention are directed to compositions and methods utilizing TPA compounds for treating and / or preventing COVID-19 disease and associated adverse conditions and symptoms in humans caused by SARS coronaviruses (e.g., SARS-CoV, SARS-CoV-2 (COVID-19), and Middle East Respiratory Syndrome coronavirus (MERS-CoV)). In exemplary embodiments, the present invention provides clinically effective TPA compounds, compositions, and methods for preventing and / or treating ARDS / SARS in subjects with severe COVID-19 disease. In another exemplary embodiment, the compositions and methods of the present invention utilize TPA compounds or compositions to effectively treat and / or prevent the hyperimmune and hyperinflammatory states associated with severe COVID-19 disease and associated cellular, tissue, and organ injury and dysfunction, such as CSS, PIMS, ESHS, DIC, and other immune and inflammatory injuries or disease states that may accompany these conditions, such as vascular congestion and thrombotic conditions, including, but not limited to, disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and gangrene.

[0054] TPA compounds useful in the methods and compositions of the present invention include those represented by Formula I: phorbol esters and derivatives according to TIFF2023528804000001.tif51128, wherein: R1 and R2 are hydrogen; hydroxyl; The alkyl group contains 1 to 15 carbon atoms TIFF2023528804000002.tif9128; The lower alkenyl group contains 1 to 7 carbon atoms TIFF2023528804000003.tif11128; TIFF2023528804000004.tif39128, R3 is hydrogen or It may be TIFF2023528804000005.tif10128.

[0055] In some embodiments, at least one of R1 and R2 is other than hydrogen and R3 is hydrogen or TIFF2023528804000006.tif9128 and substituted derivatives thereof. In another embodiment, either R1 or R2 is TIFF2023528804000007.tif9128, and the remaining R1 or R2 TIFF2023528804000008.tif9128, where the lower alkyl is 1 to 7 carbons and R3 is hydrogen.

[0056] The alkyl, alkenyl, phenyl and benzyl groups herein may be unsubstituted or substituted with halogen, preferably chlorine, fluorine or bromine, nitro, amino and / or similar type groups.

[0057] In another aspect, the present invention provides a compound of formula II: Utilizing the exemplary phorbol esters in TIFF2023528804000009.tif58128.

[0058] Phorbol esters for use in the compositions and methods of the present invention will be understood to include pharmaceutically acceptable active analogs and derivatives of the compounds of Formula I and Formula II, e.g., rationally designed chemical analogs and derivatives in which selected substitutions, deletions, or additions of functional groups are applied to the parent compound structure within Formula I or Formula II, as well as salts, isomers, enantiomers, polymorphs, solvates, hydrates, and / or prodrugs of the compounds. DETAILED DESCRIPTION OF THE INVENTION

[0059] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION The following description is provided to illustrate the underlying means and principles of the present invention through the description of exemplary embodiments. The present invention is not intended to be limited by this description. Those skilled in the art will understand that any modifications, changes, substitutions, improvements, and further applications of the objects, materials, and principles described herein are within the scope of the presently disclosed invention and the scope of the appended claims.

[0060] The present invention provides "TPA compounds" for use as pharmaceutical agents and in drug combinations and methods to mediate novel, diverse, and often multifunctional therapeutic effects. The TPA compounds of the present invention and related compositions and methods are effective in treating and preventing a diverse range of intractable disease states mediated by abnormal, hyperimmune and hyperinflammatory responses in humans and other mammals. The TPA compounds, compositions, and methods of the present invention potently exert one or more effective activities, including antiviral, anti-inflammatory, and immunoregulatory activities, to ameliorate immune dysfunction and hyperinflammatory diseases and conditions caused by viral and bacterial infections, burns, trauma, and other insults that result in harmful impairment, misdirection, or escalation of normally beneficial immune and inflammatory mechanisms.

[0061] Among the novel and powerful uses of the TPA compounds described herein, the present invention provides effective clinical tools and methods for preventing and treating a previously intractable condition known as acute respiratory distress syndrome (ARDS). ARDS is a complex disease of immune dysfunction and hyperinflammatory disease mediated by complex pathogenic factors and mechanisms. ARDS can be caused by causes such as thermal or chemical lung injury, traumatic lung injury, or severe lung infection due to viral or bacterial agents. The unifying features of ARDS include intense inflammation of the lung parenchyma, infiltration of excessive numbers of destructive neutrophils and macrophages into the lung tissue and alveolar spaces, oxidative stress, disruption of the endothelial and epithelial barriers, damage to the lung epithelial lining, and pulmonary fibrosis. The impact of these pathogenic events on patients leads to restricted respiratory effort, hypoxemia, and eventual pulmonary failure, long-term fibrosis and other pathological damage, and death in severe cases. In a related aspect of the invention, the TPA method and compositions are utilized to treat ARDS mediated by severe acute respiratory syndrome (SARS), a SARS coronavirus, including the COVID-19 virus.

[0062] In another aspect of the present invention, effective TPA compounds, compositions and methods are used to prevent and treat hyperinflammatory conditions including "cytokine storm syndrome" (CSS) generally, pediatric inflammatory multisystem syndrome (PIMS) associated with COVID-19 disease, Kawasaki disease, extrapulmonary systemic hyperinflammatory syndrome (ESHS) generally, and vascular congestion and thrombotic conditions caused by hyperinflammation, such as disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and gangrene.

[0063] A further aspect of the present invention utilizes TPA compounds and methods to directly prevent or inhibit productive infection by coronaviruses, particularly SARS coronaviruses. The TPA compounds of the present invention are effective in treating and preventing SARS-CoV-2 infection and disease in human subjects, as evidenced by TPA's ability to induce anti-SARS-CoV-2 immunoregulatory, immunopotentiating, and / or anti-inflammatory responses in subjects with COVID-19 disease. The multiple antiviral activities of the TPA compounds described herein are effective in preventing or reducing SARS-CoV-2 infection by reducing viral load in the upper and lower respiratory tract; reducing viral titers in non-respiratory ACE-2-positive cells and tissues; preventing or reducing viral attachment to and entry into the lungs and other ACE-2-positive cells and tissues; preventing or reducing viral replication in the lungs and other ACE-2-positive cells and tissues; and / or preventing or reducing viral shedding from the upper respiratory tract of infected subjects. These antiviral effects may be direct or indirect, but the antiviral outcome is specific to preventing or limiting viral infection, replication, virulence, transmissibility, and associated COVID-19 disease severity in treated subjects.

[0064] More detailed aspects of the present invention are directed to compositions and methods utilizing TPA compounds, compositions, or methods for treating and / or preventing COVID-19 disease and / or associated adverse conditions and symptoms caused by SARS coronavirus (SARS-CoV, SARS-CoV-2 (COVID-19), or Middle East Respiratory Syndrome coronavirus (MERS-CoV)).

[0065] In exemplary embodiments, the present invention provides clinically effective TPA compounds that function as "antiviral" agents. Antiviral activity, as used herein, includes, for example, reducing viral load / titer in targeted cells, tissues, or compartments, or impairing or otherwise limiting viral function (e.g., viral attachment, viral entry into cells, viral replication, viral egress, viral defenses against host antiviral mechanisms, the impact of viral pathogenicity on host cellular integrity, physiology, gene expression, immune activity, inflammatory activity, life cycle / longevity, etc.). These and related antiviral methods and compositions will prevent and / or treat COVID-19 infection and / or associated disease symptoms in at-risk, virus-exposed subjects. In another exemplary embodiment, the compositions and methods of the present invention address the hyperimmune and hyperinflammatory states associated with severe COVID-19 disease and associated cell, tissue, and organ injury and dysfunction, e.g., CSS, PIMS, ESHS, DIC, and other immune and inflammatory injuries or disease states that may accompany these conditions, e.g., vascular congestion and thrombotic events. The TPA compounds or compositions are utilized to effectively treat and / or prevent conditions such as, but not limited to, disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and gangrene.

[0066] In certain embodiments, the present invention utilizes one or more TPA or TPA-like compounds, either the parent TPA compound (12-O-tetradecanoylphorbol-13-acetate (TPA); also known as phorbol-12-myristate-13-acetate (PMA)), or structurally related functional analogs, derivatives, salts, or other derivative forms of this parent TPA compound. The TPA compounds utilized in the present invention are useful in compositions and methods administered to subjects to mediate antiviral, anti-ARDs, anti-CSS, anti-inflammatory, anti-cytopathic, immunostimulatory, and apoptosis-regulating effects, as well as other clinically relevant activities. These activities are effective, individually or cooperatively, in mediating the prevention and / or treatment of a range of ARDS and SARS virus (e.g., COVID-19) diseases, conditions, and symptoms, as well as associated immunological, cellular, tissue, and organ injury and dysfunction, in disparate disease contexts.

[0067] TPA compounds for use in the present invention include one or more phorbol ester compounds of Formula I and / or Formula II that are effectively administered to prevent and / or treat targeted viral infections, diseases, conditions, or symptoms as described herein. In exemplary embodiments, for illustrative purposes, the parent or prototype TPA compound, phorbol 12-myristate-13-acetate ("PMA," also referred to herein as 12-O-tetradecanoyl-phorbol-13-acetate, "TPA"), is utilized as the clinically effective agent in the pharmaceutical compositions and methods of the present invention. However, it will be appreciated that the present disclosure provides numerous other pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, complexes, analogs, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of compounds conforming to the structural basis described below for compounds of Formula I and Formula II.

[0068] Phorbol is a natural plant-derived polycyclic alcohol of the tigliane family of diterpenes. It was first isolated in 1934 as a hydrolysis product of Croton tiglium oil from the seeds of the lotus flower. It is highly soluble in most polar organic solvents and water. Esters of phorbol have the following formula I: TIFF2023528804000010.tif54128, wherein: R1 and R2 are hydrogen; The alkyl group contains 1 to 15 carbon atoms TIFF2023528804000011.tif9128; TIFF2023528804000012.tif10128 and substituted derivatives thereof; R3 is hydrogen, TIFF2023528804000013.tif9128 or a substituted derivative thereof.

[0069] As used herein, the term "lower alkyl" or "lower alkenyl" refers to a moiety containing 1 to 7 carbon atoms. In compounds of Formula I, the alkyl or alkenyl group can be straight or branched. In some embodiments, either R or R or both are long-chain carbon moieties (i.e., Formula I is decanoate or myristate).

[0070] The alkyl, alkenyl, phenyl and benzyl groups of the formulae herein can be unsubstituted or substituted with, for example, halogen (eg, chlorine, fluorine or bromine), nitro, amino and other functional groups.

[0071] Organic and synthetic forms of phorbol esters, including any preparations or extracts from herbal sources such as rosehip, are contemplated as useful compositions comprising phorbol esters (or phorbol ester analogs, related compounds, and / or derivatives) for use in embodiments herein. Useful phorbol esters and / or related compounds for use in the present invention will typically have a structure according to Formula I. However, those skilled in the art will recognize that functionally equivalent analogs, complexes, conjugates, and derivatives of such compounds are also within the scope of the present invention.

[0072] In more particular embodiments, exemplary structural modifications according to Formula I above will be selected to yield useful candidate compounds for treating and / or preventing COVID-19 infection or disease, ARDS generally, SARS, CSS, PIMS, and other targeted infections, diseases, conditions, and symptoms described herein. Among the various modifications of compounds of Formula I contemplated herein are those in which at least one of R1 and R2 is not hydrogen, R3 is hydrogen, Exemplary analogs and derivatives selected from the group consisting of TIFF2023528804000014.tif9128 and its substituted derivatives can be constructed and tested by routine methods. In another embodiment, either R1 or R2 is TIFF2023528804000015.tif10128, and the remaining R1 or R2 TIFF2023528804000016.tif9128, and R3 is hydrogen.

[0073] Alternatively, certain rationally designed analogs and derivatives according to Formula II: R1 and R2 are hydrogen; hydroxyl; The alkyl group contains 1 to 15 carbon atoms TIFF2023528804000017.tif9128; The lower alkenyl group contains 1 to 7 carbon atoms TIFF2023528804000018.tif9128; TIFF2023528804000019.tif47128 and substituted derivatives thereof; R3 is hydrogen or TIFF2023528804000020.tif9128, analogs and derivatives can be constructed.

[0074] Other exemplary compositions and methods of the invention illustrated herein include those of formula II: Utilizing phorbol 12-myristate-13-acetate (TPA) according to TIFF2023528804000021.tif61128, including its pharmaceutically acceptable active salts, active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and / or prodrugs.

[0075] Further exemplary phorbol esters for use in the present compositions include phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate; phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol 12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate; phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol 12-myristate; phorbol 13-myristate; phorbol 12,13,20-triacetate; 12-deoxyphorbol Examples include, but are not limited to, 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol 13-isobutyrate; 12-deoxyphorbol 13-isobutyrate-20-acetate; 12-deoxyphorbol 13-phenylacetate; 12-deoxyphorbol 13-phenylacetate 20-acetate; 12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate 13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; and phorbol 13-acetate.

[0076] Additional TPA compounds may be engineered for use in the antiviral, anti-ARDS, anti-CSS, and other primary therapeutic compositions and methods of the invention by structural modifications accomplished according to rational design chemistry to improve relevant pharmacological properties such as solubility, lipophilicity, bioavailability, in vivo half-life, resistance or susceptibility to endogenous enzymes, amenability to formulation for particular delivery forms, modalities, and delivery targets / compartments, shelf-life stability, etc. Additional phorbol esters and related compounds and derivatives useful in the formulations and methods of the invention include, but are not limited to, other pharmaceutically acceptable active salts of the compounds, as well as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and / or prodrugs of the compounds. Further exemplary forms of phorbol esters for use in the compositions and methods of the present invention include phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate; phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol 12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate; phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol 12-myristate; phorbol 13-myristate; phorbol 12,13,20-triacetate; 12-deoxy These include, but are not limited to, cyphorbol 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol 13-isobutyrate; 12-deoxyphorbol 13-isobutyrate-20-acetate; 12-deoxyphorbol 13-phenylacetate; 12-deoxyphorbol 13-phenylacetate 20-acetate; 12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate 13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; and phorbol 13-acetate. The structures shown in Table 1 exemplify these diverse targets for rationally designed chemical modification of parent phorbol ester compounds.

[0077] Table 1. Exemplary phorbol esters TIFF2023528804000022.tif78128TIFF2023528804000023.tif20096TIFF20235288040 00024.tif201123TIFF2023528804000025.tif21496TIFF2023528804000026.tif242109

[0078] Antiviral Compositions and Methods The present invention provides novel tools and methods for the treatment and prevention of viral infections and associated diseases, including the treatment and prevention of SARS-CoV-2 infection and associated COVID-19 disease. In exemplary embodiments, subjects at risk of SARS-CoV-2 infection, and infected subjects who test positive for the virus, are administered an antivirally effective amount of a TPA compound sufficient to elicit an antiviral response to SARS-CoV-2 and / or prevent or reduce one or more clinical symptoms of COVID-19 disease. In certain embodiments, the TPA compound is administered in an amount and dosage form effective to reduce or eliminate one or more signs of the severity of a viral infection selected from: (1) viral load / titer in cells, tissues, or samples of the subject's upper or lower respiratory tract; (2) viral load / titer in ACE-2-positive cells, tissues, or samples of the subject's non-respiratory tract, or in the subject's plasma; (3) viral attachment and / or entry into the lungs or other tissues / cells; (4) viral replication in the lungs or other ACE-2-positive cells, tissues, or organs of the subject; and / or (5) viral shedding from upper respiratory tract tissues or samples of the infected subject (each indicator / value is measured and determined in the treated subject in comparison to the same indicator / value measured and determined in a similar placebo-treated control subject).

[0079] Demonstrating the antiviral efficacy of TPAs ​​through clinical evaluation of treated and control subjects to determine the viral "load" in the subjects involves comparing the number or estimated "titer" of SARS-CoV-2 virions, DNA, or other quantitative measures of SARS-CoV-2 levels in test and control biological samples (e.g., nasopharyngeal swab samples). A variety of assay methods and materials are published, widely known, and routinely performed for this purpose. According to the teachings herein, patients treated with the antiviral TPA compositions and methods of the present invention will exhibit at least a 20% reduction, often a 25-50% reduction, and often a 75-95% or greater reduction, and up to 100% elimination, of detectable viral load / titer compared to placebo-treated control subjects similarly at risk or infected with SARS-CoV-2. Comparable clinical efficacy is similarly provided for other SARS coronaviruses (SARS-CoV and MERS) and other viruses and pathogens that cause or contribute to ARDS, CSS, or PIMS in human subjects.

[0080] In related compositions and methods of the invention directed to antiviral treatment of SARS-CoV-2-positive patients, therapeutic TPA compositions can clear or eliminate virus from at least the upper respiratory tract within one to two weeks after treatment. In particular, DNA or other quantitative measures of SARS-CoV-2 levels in nasopharyngeal swab samples from subjects who screened positive for SARS-CoV-2 infection before TPA treatment will be reduced by 100% in at least 25-50%, 50-75%, up to 90% or more of TPA-treated subjects within two weeks after TPA treatment (corresponding to complete elimination of detectable SARS-CoV-2 virus in the upper respiratory tract and indicating a non-contagious state).

[0081] In related embodiments, the anti-COVID-19 therapy is administered before (or as soon as possible after) the subject develops one or more indexes of severe COVID-19 disease selected from: (1) fever lasting two or more days; (2) severe lower respiratory symptoms, including pulmonary congestion, chest tightness, shortness of breath, and / or hypoxemia; and / or (3) acute respiratory distress syndrome (ARDS), cytokine storm syndrome (CSS); pediatric inflammatory multisystem syndrome (PIMS); extrapulmonary systemic hyperinflammatory syndrome (ESHS), and / or any other condition or symptom associated with an exaggerated immune hyperinflammatory response in the subject, such as any symptom of vascular congestion and thrombotic conditions, disseminated intravascular coagulation (DIC), thrombosis, stroke, and / or thrombocytopenia. Patients who test positive for SARS-CoV-2 or have known elevated risk factors for COVID-19 disease should be closely monitored (e.g., with blood tests for cytokines, pulmonary function and hypoxemia testing, and lung scanning for pathogenic lesions) to ensure TPA treatment is initiated.

[0082] Without wishing to be bound by theory, the TPA compounds of the present invention exert multiple antiviral activities, both directly and indirectly, in treated subjects that are effective in mediating both prophylactic (i.e., preventing the occurrence or reducing the extent of actual infection) and therapeutic (reducing or eliminating viral load, thereby reducing disease severity) effects and responses in treated subjects.

[0083] Certain antiviral effects of TPA compounds involve their immunostimulatory and anti-inflammatory interactions with protein kinases. Protein kinases are key components of biological signal-response pathways that "control" immune and inflammatory responses to viruses and other pathogens. In one aspect of the present invention, the interaction between TPA compounds and one or more protein kinases involved in immune regulation and inflammation mediates potent antiviral effects for preventing and treating SARS-CoV-2 infection.

[0084] Protein kinase C (PKC) is a key kinase for regulating immune function and inflammation in humans. As used herein, "regulate" refers to beneficially managing specific immune or inflammatory signals or triggers (e.g., cytokine or chemoattractant signals), cells, mechanisms, or pathways by activating, inhibiting, and / or selecting them. In the context of PKC, the TPA compounds of the present invention bind to and activate PKC, and this binding and activation in SARS-CoV-2 patients mediates the transcriptional activation of interferon-stimulated genes (ISGs), which are involved in immune-stimulating signaling and downstream activation of immune effector functions. In exemplary embodiments, TPA-activated PKC stimulates ISGs that encode products that can interfere with viral replication and spread, such as by slowing cellular metabolism and activating adaptive immunity. Other ISGs that can be activated by TPA include pattern recognition receptors (PRRs) that further sensitize cells to pathogens, proteins that reduce membrane fluidity and permeability (hindering membrane fusion and impairing viral entry and release), and other antiviral factors that inhibit specific targets involved in the viral cell entry, replication, and release / extrusion cycles (Schneider et al., 2014; Totura et al.).

[0085] Human interferons comprise a large group of proteins that help regulate the activity of the immune system. Generally, type 1 interferon (IFN-I) exerts potent antiviral effects. In COVID-19 infection, IFN-I is produced by immune cells, epithelial cells, and endothelial cells as an early response to viral infection. The potential use of type 1 interferon (IFN-I) for COVID-19 treatment was recently reviewed by Sallard et al. (2020). Following recognition of viral components by pattern recognition receptors (PRRs), IFN-I is among the first immune effector molecules produced during most viral infections. IFN-I is recognized by the IFNAR receptor, which is present on the plasma membrane in a variety of cell types. Binding of IFN-I to IFNAR induces phosphorylation of transcription factors such as STAT1 and STAT2, directing their localization to the nucleus, where they activate interferon-stimulated genes (ISGs).

[0086] ISGs encode a series of key molecules responsible for regulating various immune responses, including inflammation, cytokine signaling, and immunoregulation. Certain ISGs interfere with viral replication and spread through various mechanisms (Schneider et al., 2014; Totura et al.). At the same time, IFN-I stimulates downstream proinflammatory cytokines, which raises some concerns regarding the possibility that IFNs may induce or contribute to cytokine storm syndrome (CSS).

[0087] The present invention controls IFN-I activation (through TPA activation of PKC) and provides a safe and discrete antiviral effect. Specifically, the present TPA compositions and methods enhance IFN-mediated antiviral defenses without increasing inflammation or suppressing other beneficial immune functions.

[0088] IFN-I treatment has been investigated against MERS-CoV and SARS-CoV, both alone and in combination with lopinavir / ritonavir (Chan et al., 2015; Shealan et al., 2020), ribavirin (Chen et al., 2004; Morgenstern et al., 2005; Omrani et al., 2014), remdesivir, corticosteroids (Loutfy et al., 2003), or IFN-γ (Sainz et al., 2004; Scagnolari et al., 2004) (reviewed in Stockman et al., 2006). IFN-α and IFN-β have reportedly demonstrated antiviral activity in vitro and in certain animal models (Chan et al., 2015), but failed to significantly ameliorate disease in humans (Stockman et al., 2006). The combination of IFNβ and lopinavir / ritonavir against MERS-CoV improved lung function but did not significantly reduce either viral replication or lung pathology (Shealan et al., 2020). The combination of IFNα2a and ribavirin delayed death but did not significantly reduce final mortality (Omrani et al., 2014). The combination of IFNα2b and ribavirin produced positive results in rhesus monkeys (Falzarano et al., 2013), but results in humans were inconclusive (Arabi et al., 2017).

[0089] The failure of these previous studies to demonstrate substantial disease improvement with IFN-I treatment may be related to viral inhibition of the IFN signaling pathway by MERS-CoV and SARS-CoV, the limited number of subjects used, or the difficulty of discerning the individual effects of IFN-I when used in combination with other drugs (Sallard et al., 2020). It is also possible that IFNs are more effective in patients without comorbidities (Al-Tawfiq et al., 2014; Shalhoub et al., 2015). The diversity of IFN subtypes may be another explanation for the discrepancies observed among these studies. IFN-β has repeatedly been shown to be more potent than IFN-α as an inhibitor of coronaviruses (Scagnolari et al., 2004; Stockman et al., 2006). This subtype dominance may be related to the protective activity of IFNβ1 in the lung, where it upregulates cluster of differentiation 73 (CD73) in pulmonary endothelial cells, promotes the secretion of anti-inflammatory adenosine, and supports endothelial barrier maintenance. These beneficial effects correlate with the reduced vascular leakage observed in ARDS patients treated with IFNβ1a (Bellingan et al., 2014). However, this effect alone was insufficient to reduce ARDS mortality (Ranieri et al., 2020).

[0090] Recent studies suggest that the timing of IFN-I administration during SARS-CoV disease may play a critical role in determining treatment outcomes. One study observed positive effects when IFN-I was administered shortly after infection, but when administered later, IFN-I failed to inhibit viral replication and induced negative side effects (Channappanavar et al., 2019).

[0091] Our pilot study demonstrates that early administration of TPA provides clinically beneficial antiviral effects against SARS-CoV-2. Typically, TPA treatment would be initiated as soon as a patient is diagnosed and referred for treatment. However, if the diagnosis and referral are made very early, it may be advisable to withhold treatment until significant symptoms develop. For example, if a patient is virus-positive in the upper respiratory tract but does not have signs of severe infection (e.g., fever, labored breathing / hypoxemia), treatment may be delayed until such signs first appear (usually around days 7–8 after symptom onset). This delay may be beneficial for optimizing treatment timing to counteract disease escalation without prematurely exhausting the immune system, particularly the lymphocyte reserve.

[0092] The use of the TPA compounds described herein to stimulate PKC and IFN-I is particularly promising because this mechanism should circumvent the SARS virus's counter-immune strategies. SARS-CoV has been reported to be able to disrupt certain immune signaling pathways in its host. For example, the SARS-CoV Orf6 protein disrupts karyopherin transport, thereby inhibiting the nuclear import of transcription factors, including STAT1, and thereby impairing the STAT-1-mediated interferon response. Similarly, the SARS-CoV Orf3b protein inhibits the phosphorylation of IRF3, another factor involved in IFN activation (Frieman et al., 2007; Kopecky-Bromberg et al., 2007). However, the SARS-CoV-2 Orf6 and Orf3b proteins are truncated, which may explain why SARS-CoV-2 exhibits considerable sensitivity to IFNα in vitro (Lokugamage et al., 2020). SARS-CoV-2 is significantly more sensitive to IFN-I than SARS-CoV, suggesting that IFN-I treatment may be more effective against COVID-19 disease. Supporting this hypothesis, it has been reported that IFNα2b spray can reduce the infectivity of SARS-CoV-2 (Shen and Yang, 2020). This report of IFN-I prophylaxis against SARS-CoV-2 is consistent with data reported on the in vitro efficacy of interferon pretreatment against SARS-CoV-2 virus (Lokugamage et al., 2020).

[0093] In favor of early TPA treatment of SARS-CoV-2, the inventors emphasize that COVID-19 pathology is accompanied by extensive lung lesions in severe cases. Some researchers have compared these lesions to a form of "interferonopathy" associated with excessive use of IFN drug therapy (e.g., in multiple sclerosis patients). Furthermore, it is possible that the SARS-CoV-2 virus induces an excessive IFN response within a more general "cytokine storm" response, thereby contributing to CSS and lung injury in severe cases (Siddiqi and Mehra, 2020; Zhang et al., 2020). Therefore, TPA treatment of the present invention is likely to activate IFN-I and ISGs without excessively stimulating cytokines to levels associated with CSS, ARDS, and PIMS. Therefore, TPA treatment would likely be directed toward the early stages of SARS-CoV-2 infection. Typically, TPA treatment would be initiated within 7–10 days of diagnosis. In certain instances where the risk of or symptoms of ARDS and CSS are elevated, TPA treatment may be terminated simultaneously or shortly thereafter (e.g., upon the observation of a substantial increase in proinflammatory cytokine levels and / or the observation of substantial ARDS symptoms). In another aspect of the invention, the TPA compound is administered at a selected time point during the first stage of SARS-CoV-2 infection (e.g., 3-7 days after the onset of upper respiratory tract symptoms and confirmed by a positive nasopharyngeal SARS-CoV-2 test), maintained for 1-5 days, and then combined with an anti-interferon drug 10-12 days after the onset of symptoms (or upon the observation of a substantial increase in proinflammatory cytokine levels and / or the observation of substantial ARDS symptoms).

[0094] The novel TPA compositions and methods of the present invention also mediate indirect antiviral effects through immune-stimulating activation and lymphocyte rescue to reduce the dangerous lymphopenia mediated by SARS-CoV-2 and other SARS coronaviruses. Perhaps the most detrimental effect observed in subjects with COVID-19 disease is the sudden crash of lymphocytes, including T cells, B cells, and NK cells. In the most severely ill COVID-19 patients, NK cells are minimal or absent, and memory helper T cells and regulatory T cells are severely reduced (Hui et al., 2019). This correlates with autopsy evidence of secondary lymphoid destruction and splenic atrophy in fatal COVID-19 cases. In addition to these manifestations, severely ill COVID-19 patients exhibit lymphocyte crashes, along with evidence of hemorrhagic necrosis, macrophage proliferation, and macrophage phagocytosis in the spleen. Similar pathogenic changes are also seen in the lungs of severe COVID-19 patients with ARDS, characterized by very few lymphocytes in the lung parenchyma, prominent macrophage and neutrophil infiltration, disruption of the epithelial and endothelial barriers, and diffuse alveolar damage (DAD). This occurs while viral inclusions remain detectable in the lung epithelium, even when PCR tests are negative on blood or throat swabs (Zu et al., 2020; De Wit et al., 2016; Chan et al., 2015B). These pathogenic indicia are also correlated with elevated cytokine levels, collectively representing a condition known as "cytokine storm syndrome" (CSS).

[0095] In addition to minimizing the hyperinflammatory activity of macrophages and neutrophils, the TPA compositions and methods of the present invention mediate lymphocyte protection (extension of lifespan) and directly stimulate lymphocyte proliferation and differentiation. Our pilot studies have demonstrated a direct, clinically effective mitogenic effect of TPA on peripheral blood lymphocytes (primarily T cells). Because SARS-CoV-2 appears to overwhelm and exhaust T, B, and NK cells, this evidence indicates that TPA compounds and methods may effectively reduce or prevent lymphopenia in COVID-19 patients. This activity may protect lymphocytes and increase lymphocyte numbers, which in turn may mediate a potent antiviral effect.

[0096] In view of the above, the present invention provides novel TPA compositions and methods that mediate clinical antiviral benefits in subjects at risk for or exhibiting SARS-CoV-2 and other viruses. The compositions and methods will substantially reduce total virus numbers and reduce associated adverse effects in treated subjects by reducing viral load / titer in the upper or lower respiratory tract; reduce viral load / titer in non-respiratory tissues and organs (e.g., in subjects exhibiting CSS or PIMS affecting multiple organs); reduce or prevent viral attachment and entry into cells; impair viral replication in infected cells; and / or 5) inhibit or block viral shedding from the respiratory tract.

[0097] Anti-ARDS Compositions and Methods In another aspect of the invention, an anti-ARDS effective amount of a TPA compound is administered to a mammalian subject to induce an anti-ARDS response in a subject exhibiting acute respiratory distress syndrome (ARDS), including a subject exhibiting severe acute respiratory syndrome (SARS) mediated by the human SARS (hSARS) coronavirus. In an exemplary embodiment, a subject suitable for TPA anti-ARDS treatment will exhibit a positive SARS-CoV-2 nasopharyngeal or other test result indicating active SARS-CoV-2 infection, and treatment will be initiated within the first 1-8 days after early COVID-19 disease symptoms (upper respiratory symptoms such as cough, sneezing, sore or itchy throat, and / or fever) and before the onset of severe lower respiratory COVID-19 disease. The compositions and methods are effective in reducing or eliminating one or more symptoms of ARDS in treated subjects compared to control subjects, selected from: (1) dyspnea (shortness of breath); (2) hypoxemia; (3) elevated levels of one or more proinflammatory cytokines or other inflammatory factors in the lung parenchyma (the lung parenchyma is the portion of the lung involved in gas transfer—the alveoli, alveolar ducts, and respiratory bronchiolar tissue), or (2) increased infiltration and / or elevated levels of monocytes, macrophages, and / or neutrophils in the lung parenchyma and / or alveolar airspaces; (3) disruption of the endothelial and / or epithelial barrier of the lung; (4) pathogenic fibrosis in the lung and / or other histopathological signs of immunogenic or hyperinflammatory disease injury; or (5) elevated signs of oxidative stress in lung tissue or other tissues, organs, or biological samples from the subject (each indicator / value being measured and determined in treated subjects compared to the same indicator / value measured and determined in similar placebo-treated control subjects).

[0098] In certain embodiments of the present invention, the TPA compounds and methods herein exert antiviral and other preventive and therapeutic activities against COVID-19, ARDS, and CSS through activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). NFκB is a protein complex that controls DNA transcription and is a critical regulator of immune and inflammatory function. The NFκB pathway mediates important cellular processes, such as stress response, inflammation, and adaptive and innate immunity. Aberrant transcriptional regulation of NFκB has been linked to cancer and autoimmune disease. It is now clear that proper regulation of NFκB is essential for overall healthy and balanced immune and inflammatory function, including by determining the balanced activation of key immune and inflammatory effector cells and cellular decisions, such as the timing and progression of immune cell differentiation, cell fate, cellular programming, signal response capabilities, e.g., receptor expression and function, cell lifespan, and apoptosis.

[0099] A latent, inactive form of NFκB is constantly present in the cytosol of most cells. This allows NFκB to be the primary transcription factor in response to viruses and other stimuli and a key component in the inflammasome cascade (Oeckinghaus et al., 2009). Upon activation, NFκB primes the NLRP3-inflammasome for activation by inducing IL-1beta and NLRP3 expression. The signaling adaptor p62 / SQSTM1, mediated by NFκB activation, has been identified as a positive mediator of Ras-induced inflammation and tumorigenesis (Duran et al., 2008), making it an attractive target for cancer therapy (Zhang et al., 2016). Researchers are now also discovering that NFκB guards against excessive inflammation through the p62 / SQSTM1-mediated mitophagy process. Specifically, NFκB limits its own pro-inflammatory activity in macrophages by promoting p62 / SQSTM1-mediated removal of damaged mitochondria (Zhong et al., 2016), which likely serves as a negative feedback loop to manage the inflammatory response and prevent tissue damage.

[0100] According to the teachings herein, TPA compounds function as activators of NFκB in a novel manner that downregulates hyperinflammatory effects while maintaining downstream antiviral effects in subjects treated with COVID-19, ARDS, and CSS. While NFκB initially functions as an early primer for inflammation, timely intervention with TPA prior to the irreversible stage of hyperinflammation (i.e., before the onset of CSS) "activates" NFκB, thereby downregulating hyperinflammation in macrophages and neutrophils through a novel mitophagy pathway. In this capacity, NFκB acts to feedback-limit its own pro-inflammatory activity in macrophages by promoting p62 / SQSTM1-mediated removal of damaged mitochondria (Zhong et al., 2016). Our pilot study demonstrates that when TPA-mediated NFκB activation is applied at the appropriate stage in COVID-19 and ARDS (e.g., when macrophages and neutrophils are activated but not yet hyperactivated, and before macrophage- and neutrophil-mediated COVID-19- and ARDS-associated tissue damage occurs), this control may clinically reduce or block excessive inflammation, including CSS, and associated tissue damage.

[0101] Another important aspect of TPA efficacy in treating human SARS virus infection, ARDS, CSS, PIMS, and other virus-induced hyperinflammatory and pathogenic conditions (e.g., in COVID-19 subjects) relates to TPA's effects on the activation and signaling of kinases that control immunity and inflammation, as well as downstream inflammation and pathogenesis. ACE2 is downregulated upon SARS-CoV-2 attachment, leading to hyperactivation of NFκB and mitogen-activated protein kinase (MAPK) signaling pathways, resulting in lung inflammation and injury. These and other interactions between the virus and the host correlate with MAPK activation and dysregulation of PKC, another regulator of the MAPK proinflammatory signaling cascade. In general terms, TPA can interact with and activate PKC, resulting in substrate phosphorylation to propagate signals involved in the MAPK inflammatory cascade.

[0102] The more complex effects of TPA on the MAPK pathway involve TPA regulation of immune cell differentiation and apoptosis. Our studies show that the exemplary TPA compound 12-O-tetradecanoylphorbol-13-acetate not only activates PKC but also stabilizes it, achieving a clinically beneficial reduction in MAPK overstimulation (reduced MAPK activation and phosphorylation activity, which attenuates downstream MAPK-mediated proinflammatory signaling).

[0103] In certain aspects of the present invention, the pro-apoptotic effects of TPA compounds mediate increased apoptosis in MAPK-stimulated target cells (including macrophages and neutrophils), further limiting MAPK-mediated hyper-inflammatory activity of these target cells.

[0104] In a related embodiment, the pro-apoptotic effects of TPA compounds mediate increased apoptosis in pathogenic inflammatory cells overstimulated and reprogrammed by viral dysregulation of transforming growth factor beta (TGF-β). TGF-β is a multifunctional cytokine belonging to the transforming growth factor superfamily, which includes three mammalian isoforms (TGF-β1-3, HGNC symbols TGFB1, TGFB2, and TGFB3) and many other signaling proteins. TGF-β proteins are produced by all leukocyte lineages. In the presence of viral infection, activated TGF-β forms a serine / threonine kinase complex with other immune / inflammatory cofactors, which then binds to the TGF-β receptor. The TGF-β receptor is composed of both type 1 and type 2 receptor subunits. After TGF-β receptor binding, type 2 receptor kinase phosphorylates and activates type 1 receptor kinase, activating the immune / inflammatory signaling cascade. This enhances the synthesis and / or activation of a variety of target genes and gene products that function in immune and inflammatory cell differentiation, chemotaxis, proliferation, activation, programming, and apoptosis.

[0105] TGF-β is secreted in a latent form by many cell types, including macrophages, in a complex with two other polypeptides: latent TGF-beta binding protein (LTBP) and latency-associated peptide (LAP). Serum proteinases, such as plasmin, catalyze the release of active TGF-β from this complex. This occurs on the surface of macrophages, where the latent TGF-β complex binds to CD36 via its ligand, thrombospondin-1 (TSP-1). Inflammatory stimuli, such as viral infection, that activate macrophages enhance the release of active TGF-β by promoting plasmin activation. Macrophages also endocytose IgG-bound latent TGF-β complexes secreted by plasma cells, which then release active TGF-β extracellularly.

[0106] Therefore, the primary function of TGF-β is as a proinflammatory cytokine. However, TGF-β also plays distinct roles in the specification and differentiation of immune and inflammatory cells. Currently, only a few TGF-β activation pathways are known, and the broader mechanisms underlying TGF-β activation remain poorly understood. Known activators of TGF-β include proteases, integrins, pH, and reactive oxygen species (ROS).

[0107] It is well known that perturbations leading to dysregulated TGF-β activation and signaling can mediate hyperinflammation, autoimmune disorders, fibrosis, and cancer. This is well demonstrated by the intense hyperinflammation in TGF-β null transgenic mice. As described above, COVID-19 disease and other SARS virus infections cause intense pulmonary inflammation, fibrosis, and diffuse alveolar damage (DAD) in severe cases. Macrophages and neutrophils infiltrate the lung parenchyma and alveolar spaces in large numbers, correlating with ARDS in severe COVID-19 patients. This lesion often includes neutrophil infiltration into pulmonary capillaries, associated with pulmonary fibrosis, thrombosis, and vasculitis, and extensive deposition of neutrophil extracellular traps (NETs). Similar mechanisms of hyperinflammation manifesting as CSS are evident in PIMS and late-stage COVID-19, where CSS hyperinflammation and pathogenesis can lead to multiple organ involvement and failure.

[0108] Zhao et al. (2008) studied the original SARS-CoV coronavirus and reported that the SARS-CoV nucleocapsid (N) protein actually enhances TGF-β, mediating hyperinflammation through certain pathways, while simultaneously abolishing critical TGF-β pathways / activities involved in inducing apoptosis in mature immune / inflammatory cells. The SARS N protein specifically induces the expression of plasminogen activator inhibitor-1 and simultaneously attenuates Smad3 / Smad4-mediated apoptosis in human peripheral lung epithelial (HPL) cells. The hyperactivating effect of the SARS N protein on the TGF-β inflammatory cascade is Smad3-specific. The N protein associates with Smad3 and promotes Smad3-p300 complex formation, while inhibiting the complex formation between Smad3 and Smad4 (Zhao et al., 2008). These findings imply a surprising mechanism by which the SARS virus N protein disrupts and hijacks one specific pathway of TGF-β activation and signaling (while simultaneously activating other mechanisms of TGF-β-mediated hyperinflammation), detrimentally blocking normal apoptosis and extending the lifespan of pathogenic inflammatory host cells infected with SARS-CoV.

[0109] An important goal of the present invention is to prevent and shorten pulmonary fibrosis and other damage in COVID-19 subjects, which is mediated by the massive influx of activated macrophages and neutrophils into the lung parenchyma and alveolar spaces in severe cases. The TPA compositions and methods of the present invention achieve this clinical objective, in part, by reversing SARS N protein blockade of TGF-β-mediated apoptosis. This viral "reprogramming" mechanism is thought to explain the extremely elevated numbers of activated macrophages and neutrophils in COVID-19 subjects with severe CSS and ARDS symptoms. Although these inflammatory / immune cells normally undergo apoptosis after performing their respective antiviral functions (phagocytosis and degranulation), COVID-19 recruits these cells by extending their lifespan (through N protein suppression of TGF-β-induced apoptosis), thereby misregulating continued hyperinflammation and enhanced viral replication and spread. This recruitment and protection of pathogenic macrophages and neutrophils by SARS coronavirus is clearly part of the virus's pathogenic and evolutionary strategies. Although circulating lymphocytes (T, B, and NK cells) apparently lack ACE-2 receptors, ACE2-expressing CD68+CD169+ macrophages have been detected in COVID-19 patients, particularly in the splenic marginal zone and marginal sinus of lymph nodes. These macrophages contain SARS-CoV-2 nucleoprotein antigens and show upregulated IL-6 (Feng et al., 2020). This suggests that macrophages may be infected by COVID-19 in a "Trojan horse" manner, transport the virus to vulnerable tissues and organs, and fundamentally contribute to the excessive inflammation and activation-induced lymphocyte cell death during SARS-CoV-2 infection.

[0110] While it is unclear whether neutrophils express the ACE-2 receptor and can be directly infected by the hSARS virus, the role of ACE-2 in SARS-CoV-2 hyperinflammatory activation and neutrophil recruitment is clear. Research by Li et al. (2020) demonstrates that ACE2 is not only a receptor that facilitates SARS-CoV-2 cell entry and replication, but also contributes to virus-mediated dysregulation of the host immune response, cytokine expression, and inflammation after infection. ACE-2 levels in infected cells and tissues correlate with the severity of SARS-CoV-2-induced ARDS and are directly associated with the excessive elevation of proinflammatory cytokines that mediate CSS and ARDS. More specifically, Li and colleagues report that high ACE2 expression is associated with the strength of innate and adaptive immune responses, B cell regulation, and cytokine secretion, as well as hyperinflammatory responses induced by IL-1, IL-10, IL-6, and IL-8. In other words, CSS-mediated immune system dysfunction is broadly associated with high ACE2 expression. Additionally, high ACE2 expression correlates with increased expression of genes involved in viral replication and transcriptome changes in SARS-CoV-2-infected epithelial cells that enhance viral entry, replication, and assembly. T cell activation and T cell-mediated inflammatory responses are also induced by SARS-CoV-2 transcriptome changes in infected cells. Increased levels of IL-1β, IFN-γ, IP10, and MCP1 in patients infected with SARS-CoV-2 appear to be associated with hyperactivation of T-helper 1 (Th1) cell responses. ACE2 also mediates the activation of neutrophils, NK cells, Th17 cells, Th2 cells, Th1 cells, dendritic cells, and TNFα-secreting cells, leading to severe and exaggerated inflammatory responses. High ACE2 expression in lung tissue specifically promotes macrophage cytotoxic activation, neutrophil inflammation, and a Th2-dominant immune response (Li et al., 2020). Interestingly, ACE2 expression changes in a time-dependent manner after SARS-CoV infection.

[0111] As a consequence of the "recruitment and reprogramming" of host macrophages and neutrophils by the SARS-CoV-2 virus, hijacked and "immortalized" inflammatory effector cells congeste and impair the host circulation, degrade and congeste the interstitial and alveolar compartments (e.g., through pulmonary fibrosis, NET deposition, epithelial and endothelial barrier disruption, vasculitis, and thrombosis), and radically limit the host's ability to successfully mount an immune response (e.g., by preventing immune effector cells and secreted antibodies from reaching viral nursery sites in the lungs and other organs). The culmination of these hijacked and dysregulated processes is ARDS / SARS, with extreme pulmonary inflammatory pathogenesis, blockage of circulation and gas exchange, hypoxemia, and eventual pulmonary / heart failure. These are the characteristic effects of a novel (e.g., zoonotic) universal virus, rather than the fine-tuned, attenuated effects of a specialized virus that has long coevolved with its host.

[0112] By using the TPA compositions and methods of the present invention, the infection and pathogenic mechanisms of SARS-CoV-2 are effectively blocked or substantially reduced. In exemplary embodiments, anti-inflammatory methods utilizing TPA compounds reduce SARS-CoV-2-induced hyperinflammation, CSS, ARDS, PIMS, and the associated tissue and organ damage associated with these pathogenic conditions. In certain embodiments, the TPA compounds of the present invention prevent or reduce SARS-CoV-2-induced activation and lifespan extension (N protein-mediated disruption of normal apoptosis) in host macrophages and neutrophils, thereby limiting the pathogenic effects of CSS, ARDS, DAD, PIMS, and ESHS, and consequently limiting viral replication, intrahost viral dissemination, viral shedding, and viral transmission. Without wishing to be bound by theory, these anti-inflammatory and "pro-apoptotic" effects of the TPA compounds and methods of the present invention may be directly or indirectly related to, or entirely independent of, PKC, MAPK, NFκB, and / or TGF-β targets, mechanisms, and pathways.

[0113] In accordance with the teachings herein, patients treated with the anti-ARDS compositions and methods of the present invention may experience symptoms such as, for example, dyspnea, elevated levels of one or more pro-inflammatory cytokines in the lungs, elevated levels of monocytes, macrophages, and / or neutrophils in the lung parenchyma and / or alveolar air spaces, disruption of the endothelial and / or epithelial barrier of the lungs, elevated signs of oxidative stress in lung tissue (e.g., elevated levels of reactive oxygen species (ROS) in the lungs), and / or one or more pathogenic symptoms of lung injury. The claimed compositions and methods will demonstrate at least a 20% reduction, often a 25-50% reduction, and often a 75-95% or greater reduction, up to 100% elimination of one or more indices, conditions, or symptoms correlated with ARDS severity selected from the group consisting of: hyperinflammation, fibrosis, diffuse alveolar damage (DAD), macrophage and / or neutrophil infiltration into the lung parenchyma, macrophage and / or neutrophil infiltration into pulmonary capillaries, widespread neutrophil extracellular trap (NET) deposition, pulmonary interstitial, pulmonary, and / or coronary vascular thrombosis, and vasculitis, among other pathological signs associated with ARDS injury. Efficacy of the claimed compositions and methods is determined by measuring and determining, for each of the aforementioned indices of ARDS, the clinical diagnostic value of that in a treated subject compared to the same indices / values ​​measured and determined in similar placebo-treated control subjects.

[0114] The anti-ARDS composition methods and compositions described herein are effective in treating or preventing one or more ARDS symptoms in a range of treated ARDS subjects, including ARDS caused by viral pathogens (e.g., SARS), bacterial pathogens, caustics, lung injury or trauma, burns, and other causes.

[0115] Anti-CSS Compositions and Methods In a further exemplary embodiment, an anti-CSS effective amount of a TPA compound is administered to a mammalian subject to induce an anti-CSS response in a subject infected with the SARS virus, a subject exhibiting COVID-19 disease, or other subject exhibiting symptoms of cytokine storm syndrome (CSS). The rationale, strategies, and mechanisms of action for using TPA compounds to treat and prevent CSS in COVID-19 disease and other hyperinflammatory conditions generally follow those described above for anti-ARDS compositions and methods.

[0116] In accordance with these aspects of the invention, patients treated with the anti-CSS compositions and methods of the invention may exhibit one or more indexes, conditions, or symptoms that correlate with the occurrence and / or severity of CSS, such as excessively elevated activation, expression, and / or levels of pro-inflammatory cytokines in cells or tissues affected by CSS; increased infiltration and / or elevated numbers of macrophages and / or neutrophils in the lung parenchyma, alveolar air spaces, or another tissue or organ affected by CSS; lymphopenia (a dramatic decrease in the number of lymphocytes (T, B, and NK cells)); elevated oxidative stress markers; elevated levels of markers of oxidative stress in the lung or other tissue or organ affected by CSS; and / or oxidative stress symptoms (each indicator / value being measured and determined in treated subjects in comparison to the same indicator / value measured and determined in similar placebo-treated control subjects).

[0117] In more detailed aspects, the anti-CSS methods and compositions of the present invention are surprisingly effective in reducing or eliminating a major causative factor in CSS: the dysregulation and excessive elevation of pro-inflammatory cytokines above levels normally associated with a beneficial inflammatory response. According to the teachings herein, patients treated with the anti-CSS compositions and methods of the present invention will exhibit at least a 20% reduction, often a 25-50% reduction, and often a 75-95% or greater reduction, and up to 100% elimination, of excessively elevated levels of one or more pro-inflammatory cytokines associated with CSS.

[0118] In exemplary embodiments, subjects treated with TPA will exhibit substantially reduced levels of IL-6, a key pro-inflammatory cytokine associated with COVID-19-induced CSS. In exemplary examples, IL-6 levels in TPA-treated subjects who, at the onset of treatment, exhibit COVID-19 disease with elevated pro-inflammatory cytokines and other CSS symptoms described herein will exhibit therapeutic downregulation of IL-6, corresponding to at least a 20%, 25-50%, and up to a 75-95% reduction in IL-6 levels in a target tissue or sample (e.g., a circulating blood sample, or lung tissue from a biopsy or autopsy) compared to placebo-treated control subjects with comparable cytokine levels and CSS symptoms. To demonstrate preventive efficacy, patients in the test and control groups would be selected to have comparable CSS risk factors (e.g., all have early-diagnosed COVID-19 disease), and after completion of treatment, fewer patients in the TPA-treated group would develop CSS symptoms, including elevated IL-6 levels, and those who developed CSS symptoms would have less severe symptoms, including substantially lower IL-6 levels, compared with the incidence and severity of CSS and overstimulated IL-6 levels in CSS-positive placebo-treated control subjects.

[0119] Equivalent efficacy in preventing and reducing excessive elevations in proinflammatory cytokine levels associated with CSS may also be achieved using TPA treatment to mediate reductions in the expression or levels of not only IL-6 but also a wide range of proinflammatory cytokine targets, including, but not limited to, (IL)-1B; IL-7; IL-8; IL-9; IL-10; fibroblast growth factor (FGF); granulocyte-macrophage colony-stimulating factor (GM-CSF); IFNγ; granulocyte colony-stimulating factor (G-CSF); interferon-γ-inducible protein (IP10); monocyte chemoattractant protein (MCP1); macrophage inflammatory protein 1 alpha (MIP1A); platelet-derived growth factor (PDGF); tumor necrosis factor (TNFα); and vascular endothelial growth factor (VEGF) in target plasma, cells, or tissues. Each of these proinflammatory cytokine targets for TPA-mediated attenuation was reported by Huang et al. (2020) to be abnormally elevated in patients with severe COVID-19 disease. Huang and colleagues reported that IL-2, IL-7, IL-10, G-CSF, IP10, MCP1, MIP1A, and TNFα were higher in critically ill ICU patients than in non-ICU patients (Huang et al., 2020; Conti et al., 2020). In exemplary embodiments of the present invention, the target of TPA treatment is directed at reducing the expression, activation, half-life and / or levels of one or more CSS-associated proinflammatory cytokines selected from IL-10 (which inhibits proinflammatory cytokine production by monocytes / macrophages and neutrophils and inhibits TH1-type lymphocyte responses); IL-11 (which inhibits proinflammatory cytokine responses by monocytes / macrophages and promotes Th2 lymphocyte responses); IL-13 (which attenuates monocyte / macrophage function), IL-37, and other proinflammatory cytokines and chemokines associated with CSS.

[0120] Without wishing to be bound by theory, the TPA compositions and methods of the present invention potently attenuate the hyperactivation and overexpression of proinflammatory cytokines indirectly associated with CSS at the cellular level (e.g., by reducing activation-induced lymphopenia, which results in the attenuation of normal T, B, and NK cell function), and also through the above-mentioned signal response pathways involving PKC, MAPK, NFκB, TGF-β, and other targets, mechanisms, and pathways associated with normal immune and inflammatory control.

[0121] In another exemplary embodiment, subjects receiving TPA treatment will exhibit a substantial increase in anti-inflammatory cytokine levels. This novel effect, in turn, mediates a further reduction in CSS symptoms and pathology, such as in subjects with COVID-19 disease. In an exemplary embodiment, the levels of anti-inflammatory cytokines in TPA-treated controls who, at the onset of treatment, exhibit COVID-19 disease and associated elevated pro-inflammatory cytokines and other CSS symptoms described herein will exhibit therapeutic upregulation of one or more anti-inflammatory cytokines, corresponding to a 20%, 25-50%, or even a 75-95% increase in cytokine levels in a target tissue or sample (e.g., a circulating blood sample, or lung tissue from a biopsy or autopsy) compared to placebo-treated control subjects with comparable pro-inflammatory cytokine levels and CSS symptoms. To demonstrate preventive efficacy, if patients in the test and control groups are selected to have comparable CSS risk factors (e.g., all have early-diagnosed COVID-19 disease), then fewer patients in the TPA-treated group will develop CSS symptoms, including elevated pro-inflammatory cytokine levels, and those who do develop CSS symptoms will have less severe symptoms, correlated with substantially elevated anti-inflammatory cytokine levels and substantially reduced pro-inflammatory cytokine levels, compared with the incidence and severity of CSS and anti- and pro-inflammatory cytokine profile values ​​in placebo-treated control subjects.

[0122] Equal efficacy in upregulating anti-inflammatory cytokines to prevent and reduce CSS symptoms, including excessive elevation of pro-inflammatory cytokine levels, is expected to be achieved by targeting a broad range of anti-inflammatory cytokine targets, including, but not limited to, IL-1RA (pro-immune soluble TNF receptors (sTNFRs), including sTNFR1 (the circulating counterpart of membrane-bound TNFR1 (mTNFR1) that binds to TNF trimers in the circulation and prevents membrane-bound TNF receptor-TNF ligand interaction) and sTNFR2 (the counterpart of mTNFR2 that binds to TNF trimers in the circulation and prevents membrane-bound TNF receptor-TNF ligand interaction); soluble IL-1 receptor type 2 (sIL-1RII) (which binds to circulating IL-1 ligand in plasma and prevents IL-1β from binding to IL-1 receptor type 1); and membrane-bound IL-1 receptor type 2 (mIL-1RII) I) (which lacks intracellular signaling function but functions as a "decoy" receptor, competing with type 1 IL-1R for IL-1 ligand binding at the cell membrane); IL-10 (which inhibits Th1 cytokines including IL-2 and IFN-γ, inactivates monocyte / macrophage proinflammatory cytokine synthesis, and downregulates or inhibits monocyte / macrophage-derived TNF-α, IL-1, IL-6, IL-8, IL-12, granulocyte colony-stimulating factor, MIP-1α, MIP-2α, and IL-18BP); IL-11 (which blocks the nuclear translocation of NF-κB, thereby impairing the transcriptional activation of proinflammatory cytokines, attenuates IL-1 and TNF synthesis in macrophages by upregulating inhibitory NF-κB, and inhibits CD41 inhibits the synthesis of IFN-g and IL-2 by T cells, functions as a Th2-type cytokine, and inhibits cytokine expression by Th1 lymphocytes; IL-13 (downregulates the production of TNF, IL-1, IL-8, and MIP-1α by monocyte / macrophage cells);and TGF-β (which has both pro- and anti-inflammatory effects, serves as a biological switch to antagonize, enhance, or modify the actions of other cytokines and growth factors, can convert active sites of inflammation into sites dominated by resolution and repair, can function as an immunopotentiator locally and an immunosuppressant in the systemic circulation, suppresses T-cell and B-cell proliferation and differentiation; downregulates IL-2, IFN-γ, and TNF; serves as a monocyte / macrophage inactivator, similar to IL-10, and induces apoptosis in mature immune / inflammatory cells) may also be achieved with TPA treatment.

[0123] In related aspects of the invention directed to the treatment and prevention of CSS, the TPA compositions and methods described herein effectively block or reduce the infiltration and increased numbers of macrophages and / or neutrophils in the lung parenchyma, alveolar air spaces, or another tissue or organ affected by CSS. These effects of TPA compounds may involve a variety of mechanisms, including blocking or reducing pro-inflammatory cytokine and / or chemokine signaling in macrophages and / or neutrophils (blocking or reducing their activation, migration / infiltration; blocking or reducing macrophage and / or neutrophil pro-inflammatory cytokine expression, cytotoxicity, destruction of epithelial and endothelial cells and structural components; blocking or reducing NET deposition by activated neutrophils; and blocking or reducing macrophage and / or neutrophil pro-thrombotic activity and associated pathogenic effects. In addition, the TPA compositions and methods of the invention mediate an apoptosis-inducing effect on macrophages and neutrophils that positively regulates their normal inflammatory activity and timely apoptosis (and, in the case of COVID-19 disease, disrupts SARS-CoV-2 recruitment and reprogramming in these cells, mediated by the effect of the SARS N protein on TGF-β, which pathogenically impairs normal apoptosis, thereby prolonging the lifespan and hyper-inflammatory effects of these cells).

[0124] In a further aspect of the invention relating to the treatment and prevention of CSS, the TPA compounds and methods of the invention are also clinically effective for treating or preventing T lymphopenia, a significant reduction in lymphocyte (T, B, and NK cell) numbers in the blood, spleen, and / or lymph nodes observed in severe CSS cases (i.e., cases progressing to tissue / organ pathology, injury, and dysfunction), mediated at least in part by activation-induced lymphocyte cell death.

[0125] In other embodiments, the TPA compositions and methods of the present invention are effective in treating or preventing oxidative stress associated with CSS, as determined, for example, by a reduction in oxidative stress markers (e.g., elevated / ROS levels) in lung tissue or other tissues or organs affected by CSS.

[0126] Other treatment targets of the present invention related to CSS include preventing or reducing inflammatory damage to the endothelial and / or epithelial barrier in the lung or other tissues or organs affected by CSS. Diagnostic indicia of efficacy in these examples include known histological and histochemical assays evaluating biopsy and / or autopsy results between treated and control groups of CSS subjects.

[0127] The related TPA treatment methods and compositions of the present invention are directed to the prevention and treatment of pathogenic fibrosis and / or other histopathological signs of immunogenic or hyperinflammatory disease injury in the lung or other tissue or organ / organs affected by CSS, lymph node loss and / or atrophy, and splenic inflammation and / or atrophy, all of which are assessed for efficacy using conventional pathological examination, histological and histochemical methods.

[0128] Further aspects of the present invention are directed to the prevention and treatment of CSS, involving the reduction or blocking of sepsis, toxic shock syndrome (TSS), and / or oxidative stress symptoms associated with CSS. Again, conventional assay methods for measuring diagnostic parameters for the evaluation of treatment / prophylactic efficacy, symptoms, are widely known in the art.

[0129] Anti-inflammatory and immunoregulatory compositions and methods A further aspect of the present invention is a TPA composition and method for preventing or treating hyperimmune or hyperinflammatory conditions, such as, but not limited to, pediatric inflammatory multisystem syndrome (PIMS), Kawasaki disease, extrapulmonary systemic hyperinflammatory syndrome (ESHS), which are associated with COVID-19 disease, or vascular congestion or thrombotic conditions caused by hyperinflammation, which may include, but are not limited to, disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and / or gangrene. When these compositions and methods are administered to a human subject at increased risk for or exhibiting a hyperimmune or hyperinflammatory condition or disorder, clinical efficacy can be demonstrated by: (1) elevated levels of one or more proinflammatory cytokines or other inflammatory factors in the subject's affected tissue, organ, or plasma; (2) increased infiltration and / or elevated levels of monocytes / macrophages and / or neutrophils in the subject's affected tissue, organ, or compartment (e.g., the alveoli, kidney, or vascular lumen); (3) disruption of the endothelial and / or epithelial barrier in the subject's affected tissue or organ; (4) pathogenic fibrosis and / or other histopathological signs of immunogenic or hyperinflammatory disease injury in the subject's affected tissue or organ; (5) toxic shock syndrome (TSS) symptoms; (6) elevated signs of oxidative stress in biological samples from affected tissues, organs, or subjects; and / or (7) a substantial reduction (i.e., at least a 20% reduction, a 25-50% reduction, a 75-95% reduction, or up to 100% prevention / elimination) of one or more targeted hyperimmune or hyperinflammatory conditions or symptoms selected from one or more Pediatric Inflammatory Multisystem Syndrome (PIMS)-associated symptoms selected from sudden fever, rash, bloodshot eyes, dry or cracked mouth, redness in the palms and soles, enlarged glands, enlarged blood vessels, and / or coronary artery aneurysms (each indicator / value measured and determined in treated subjects compared to the same indicator / value measured and determined in similar placebo-treated control subjects).

[0130] In a related aspect of the invention, TPA is administered to a human subject to prevent or treat hyperimmune or hyperinflammatory conditions, such as, but not limited to, pediatric inflammatory multisystem syndrome (PIMS), Kawasaki disease, extrapulmonary systemic hyperinflammatory syndrome (ESHS), or vascular congestion or thrombotic conditions caused by hyperinflammation, which may include, but are not limited to, disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and / or gangrene. According to these aspects, a human subject at increased risk for or exhibiting a hyperimmune or hyperinflammatory condition or disorder may exhibit: (1) elevated levels of one or more proinflammatory cytokines or other inflammatory factors in the subject's affected tissue, organ, or plasma; (2) increased infiltration and / or elevated levels of monocytes / macrophages and / or neutrophils in the subject's affected tissue, organ, or compartment (e.g., alveoli, kidney, or vascular lumen); (3) disruption of the endothelial and / or epithelial barrier in the subject's affected tissue or organ; (4) pathogenic fibrosis and / or other histopathological signs of immunogenic or hyperinflammatory disease injury in the subject's affected tissue or organ; (5) toxic shock syndrome (TSS) symptoms; (6) elevated signs of oxidative stress in a biological sample from the affected tissue, organ, or subject; and / or (7) one or more pediatric inflammatory multisystem syndrome (PIMS)-associated symptoms selected from rash, bloodshot eyes, dry or cracked mouth, redness in the palms and soles, enlarged glands, enlarged blood vessels, and / or coronary artery aneurysms, wherein each indicator / value is measured and determined in the treated subject in comparison to the same indicator / value measured and determined in a similar placebo-treated control subject.

[0131] Combination drug therapy and coordinate treatment methods In a further aspect of the present invention, combinatorial formulations and coordinated treatment methods are provided that utilize effective amounts of a TPA compound and one or more "secondary agents." The secondary agents may be "secondary therapeutic agents" or "secondary preventative agents" that can be co-formulated with or coordinately administered with the TPA compound to provide a combined formulation, combination drug therapy, or coordinated treatment or prevention method effective to treat or prevent a targeted COVID-19 infection or disease, ARDS generally, SARS, CSS, PIMS, or other targeted infection, disease, condition, and / or symptom described herein. For example, an exemplary combination formulation and coordinated treatment / prevention method for ARDS includes a TPA compound with anti-ARDS effects combined with one or more secondary or adjunctive treatment or prevention agents effective to treat and / or prevent ARDS, or a targeted co-morbidity, condition, or symptom. In alternative aspects, the secondary agent can have the same, similar, or different pharmacological activity as the TPA compound for treating and / or preventing a targeted or coexisting condition.

[0132] In related embodiments, any TPA compound disclosed herein can be used in a drug combination or combined therapy with one or more secondary therapeutic or prophylactic agents to treat and / or prevent any infection, disease, condition, and / or symptom described herein. In an exemplary embodiment, an antiviral TPA compound is combined or coordinated with a conventional antiviral drug. In another exemplary embodiment, an anti-ARDS, anti-CSS, anti-PIMS, anti-ESHS, anti-DAD, anti-inflammatory, immunostimulatory, anti-cytopathic, and / or pro-apoptotic TPA compound is combined or coordinated with a secondary agent that mediates a significant clinical benefit in preventing or treating any disease, condition, or symptom associated with or coexisting with viral infection, hyperinflammation, ARDs, CSS, PIMS, ESHS, DAD, increased cytopathic activity, immunosuppression, impairment of normal apoptotic activity of immune and / or inflammatory cells, and / or any other target symptom, condition, or index described herein in a selected patient.

[0133] The TPA compounds of the present invention can be administered simultaneously or sequentially with a secondary agent, which may act additively, synergistically, or independently to treat and / or prevent the same or a different disease, condition, or symptom as the disease, condition, or symptom for which the TPA compound is administered. The TPA compound and secondary agent may be combined in a single formulation or administered separately at the same time or at different times. Thus, administration of the TPA compound and secondary agent(s) can be simultaneous or sequential in either order, and the treatment interval may include time during which either or both (or all) of the TPA compound and secondary therapeutic agent(s) individually and / or collectively exert their therapeutic effects. A distinctive aspect of all such coordinated treatment methods is that the selected TPA compound exerts at least some detectable therapeutic activity toward alleviating or preventing the targeted disease, condition, or symptom described herein, eliciting a favorable clinical response, which may or may not be accompanied by a separate or enhanced clinical response mediated by the secondary therapeutic agent. In another exemplary embodiment, coordinated administration of a TPA compound and a secondary therapeutic agent will typically result in a stronger therapeutic response and reduced side effects compared to the clinical response observed after administration of the same dosage of either the TPA compound or the secondary agent alone. For example, coordinated treatment using an antivirally active TPA compound in combination with a conventional antiviral agent may result in a substantial therapeutic effect and avoid or reduce associated side effects (compared to those observed when a therapeutic-equivalent dose of the TPA compound or the conventional antiviral agent is used alone), even when both the TPA compound and the conventional antiviral agent are individually administered at subtherapeutic doses. In this way, the TPA compound and the conventional antiviral agent are "potentiating" each other, eliciting combined therapeutic efficacy at dosages where neither drug alone provides a detectable therapeutic benefit.The surprising combined efficacy of TPA with a complementary or augmenting secondary therapeutic agent at doses that would normally (i.e., when administered alone) be sub-therapeutic has significant advantages in that adverse side effects that may accompany the separate administration of TPA or the secondary therapeutic agent at fully therapeutic doses are eliminated or substantially reduced. While these coordinate dosing regimens utilized herein will vary depending, for example, on well-known clinical and patient-specific parameters, the terms "therapeutic dose" and "sub-therapeutic dose" have their ordinary and clear meanings to those skilled in the art (and are herein applicable to any one or combination of symptoms associated with the targeted diseases, conditions, and symptoms).

[0134] Combination of antiviral drugs and TPA In coordinated treatments of the present invention in which the secondary agent is an antiviral agent, the secondary agent can be a conventional antiviral agent, for example, abacavir, acyclovir, adefovir, amantadine, Ampligen, amprenavir (Agenerase), arbidol, atazanavir, atripla, Balavir, baloxavir marboxil (Xofluza), bictarvy, boceprevir (Victrelis), cidofovir, cobicistat (Tybost), combivir, daclatasvir (Daklinza), darunavir, delavirdine, descovy, didanosine, docosanol, dolutegravir, or doravirine. (Pifeltro), Ecoliever, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine (Intelence), famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir (Cytovene), ibacitabine, ibalizumab (Trogarzo), idoxuridine, imiquimod, Imunovir, indinavir, inosine, integrase inhibitors Factors, interferon type I, interferon type II, interferon type III, lamivudine, letermovir (Prevymis), lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir (Nexavir), nitazoxanide, norvir, nucleoside analogues, oseltamivir (Tamiflu), peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir (Rapivab), pleconaril, podophyllotoxin Pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant), rimantadine, ritonavir, saquinavir, simeprevir (Olysio), sofosbuvir, stavudine, telaprevir, telbivudine (Tyzeka), tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir (Valtrex), valganciclovir, vicriviroc,Antiviral agents may be selected from any one or combination of vidarabine, viramidine, zalcitabine, zanamivir (Relenza), and / or zidovudine, as well as many other safe and effective antiviral drugs known and readily available for clinical use. Antiviral agents may also be selected from monoclonal antibodies and other biologics that immunospecifically bind to or otherwise neutralize target viruses, and steroids and corticosteroids, such as prednisone, cortisone, fluticasone, and glucocorticoids.

[0135] In exemplary combination compositions and methods of the present invention, a TPA compound with anti-SARS-CoV-2 efficacy is co-formulated with remdesivir and delivered together (e.g., via a multidrug IV infusion), or co-administered simultaneously or sequentially with remdesivir. Remdesivir (GS-5734) is currently a promising anti-COVID-19 drug that exhibits broad-spectrum antiviral activity against RNA viruses. It is a prodrug with a structure similar to adenosine. Remdesivir is incorporated into nascent viral RNA and also inhibits RNA-dependent RNA polymerase. This leads to premature termination of the viral RNA chain, resulting in the cessation of viral genome replication. Remdesivir was originally developed by Gilead Sciences (USA) for the Ebola virus and underwent clinical trials during the recent Ebola outbreak in the Democratic Republic of the Congo. Although it was not found to be effective against Ebola during this study, its safety in humans was established, allowing its entry into ongoing clinical trials to determine remdesivir's efficacy against COVID-19 disease. Importantly, remdesivir has previously been shown to exhibit in vitro and in vivo antiviral activity against different coronaviruses, including SARS-CoV and MERS-CoV. A recent in vitro study also reported that remdesivir inhibits SARS-CoV-2 (Wang et al., 2020). Remdesivir is currently being tested in multiple trials in different countries, including two randomized Phase III trials in China (NCT04252664 and NCT04257656).

[0136] In other exemplary combination compositions and methods of the present invention, anti-SARS-CoV-2 TPA compounds are co-formulated and delivered together with favipiravir, or co-administered simultaneously or sequentially with favipiravir. Like remdesivir, favipiravir inhibits viral RNA-dependent RNA polymerase by structurally mimicking endogenous guanine. Competitive inhibition can substantially impair the efficacy of viral replication. While favipiravir has been approved for the treatment of influenza, it has less preclinical support than remdesivir for the treatment of SARS-CoV-2. Clinical studies in China evaluated the efficacy of favipiravir plus interferon-α to treat SARS-CoV-2 (ChiCTR2000029600), and in March 2020, favipiravir was approved by the National Medical Products Administration of China as the first safe and effective anti-COVID-19 drug.

[0137] In other exemplary combination compositions and methods of the present invention, the anti-SARS-CoV-2 TPA compound is co-formulated and delivered together with ivermectin, or co-administered simultaneously or sequentially with ivermectin. Ivermectin is an FDA-approved antiparasitic drug that has been shown to exert antiviral activity against both human immunodeficiency virus (HIV) and dengue virus. Ivermectin can dissociate preformed IMRα / β1 heterodimers, which are responsible for the nuclear transport of viral protein cargo. Because nuclear transport of viral proteins is essential for the replication cycle and inhibition of host antiviral responses, targeting the nuclear transport process may be a viable therapeutic approach against RNA viruses. Recently, in vivo studies have demonstrated the ability of ivermectin to reduce viral RNA by up to 5,000-fold 48 hours after infection with SARS-CoV-2 (Caly et al., 2020). Now that the safety profile for antiparasitic use has been established, the next step in proving the efficacy of ivermectin for treating COVID-19 will involve trials to determine optimal dosing.

[0138] In other exemplary combination compositions and methods of the present invention, anti-SARS-CoV-2 TPA compounds are co-formulated and delivered together with lopinavir and ritonavir, or co-administered simultaneously or sequentially with lopinavir and ritonavir. The human immunodeficiency virus (HIV) protease inhibitors lopinavir and ritonavir are promising candidates in the TPA methods and compositions of the present invention for antiviral treatment of COVID-19 subjects. Aspartyl protease is an enzyme encoded by the HIV pol gene that cleaves precursor polypeptides in HIV and therefore plays an essential role in its replication cycle. Although coronaviruses encode proteases of different enzyme classes (cysteine ​​proteases), evidence suggests that lopinavir and ritonavir may also inhibit the coronavirus 3CL1 pro protease. Several clinical, animal, and in vitro model studies conducted on SARS and MERS have confirmed that the protease inhibitor lopinavir / ritonavir combination works against each of these viruses. Lopinavir / ritonavir is currently undergoing clinical trials for use in patients with COVID-19 (NCT04252885; ChiCTR2000029308), but no clear benefit over standard treatment has yet been reported.

[0139] Combination of anti-ACE2 drugs and TPA In other exemplary combination compositions and methods of the present invention, the anti-SARS-CoV-2 TPA compound is co-formulated and delivered together with recombinant human angiotensin-converting enzyme 2 (APN01), or co-administered simultaneously or sequentially with recombinant human angiotensin-converting enzyme 2 (APN01). Soluble recombinant human angiotensin-converting enzyme 2 (rhACE2) is expected to block SARS-CoV-2 entry by blocking the S protein from interacting with cellular ACE2. Indeed, a recent study reported that rhACE2 was able to inhibit SARS-CoV-2 replication by 1,000- to 5,000-fold in cells and embryonic stem cell-derived organoids (Monteil et al., 2020). rhACE2 likely reduces serum angiotensin II by redirecting substrates away from the related enzyme ACE. This may prevent further activation of the ACE2 receptor, thereby preserving pulmonary vascular integrity and preventing ARDS. APN01, originally developed by Apeiron Biologics, is already undergoing Phase II trials for ARDS. A small pilot study in China (NCT04287686) is currently evaluating the biological and physiological roles of rhACE2 in COVID-19 pneumonia, specifically as a treatment for ARDS. Apeiron Biologics has also initiated a placebo-controlled, double-blind, dose-escalation study to evaluate the safety and tolerability of intravenous APN01. Measuring serum levels of angiotensin II and angiotensin I-7 will further elucidate the bioproducts blocked by this drug and the biological and physiological roles of rhACE2 in COVID-19 pneumonia.

[0140] Viral entry inhibitor In other exemplary combination compositions and methods of the present invention, a TPA compound with anti-SARS-CoV-2 efficacy is co-formulated and delivered together with arbidol, or co-administered simultaneously or sequentially with arbidol. Arbidol is a viral entry inhibitor developed for influenza and arboviruses. This drug targets the major glycoprotein hemagglutinin (HA) on the surface of influenza viruses. Arbidol prevents fusion of the viral membrane with endosomes after endocytosis. It is currently being tested as a single agent against SARS-CoV-2 (NCT04260594, NCT04255017). Another clinical trial is comparing arbidol with favipiravir against SARS-CoV-2 (ChiCTR2000030254).

[0141] In other exemplary combination compositions and methods of the present invention, the anti-SARS-CoV-2 TPA compound is co-formulated with interferon and delivered together, or co-administered simultaneously or sequentially with interferon. Clinical trials have recently been registered to evaluate the combination of lopinavir / ritonavir with IFNα2b (ChiCTR2000029387) or the combination of lopinavir / ritonavir with ribavirin and subcutaneously administered IFNβ1b (NCT04276688) for the treatment of COVID-19. IFN administration by vapor inhalation is now part of the standard of care for COVID-19 in China, with the advantage of specifically targeting the airways. Intravenous and subcutaneous modes of IFN administration have been well described and have been found to be safe in several clinical trials. Combining IFN-I with lopinavir / ritonavir, ribavirin, or remdesivir may improve efficacy based on the enhanced efficacy of these combinations reported for other coronaviruses (Sheal et al., 2020). Type III IFNs are also candidates for the treatment of COVID-19 due to their known protective effects in the respiratory tract (Lokugamage et al., 2020). Subcutaneous IFNβ1a combined with lopinavir / ritonavir is being compared with lopinavir / ritonavir alone, hydroxychloroquine, and remdesivir in the DisCoVeRy trial (NCT04315948), the first clinical trial of the WHO Solidarity consortium of clinical trials.

[0142] Combination of anti-inflammatory drugs and TPA In addition to combinations and coordinated treatment methods with antiviral drugs, the TPA compounds may be beneficially combined with secondary therapeutic and / or prophylactic agents to treat and / or prevent any of the other diseases, conditions, and symptoms described herein as targets for therapeutic intervention. For example, "anti-inflammatory" drugs and biologics may be beneficially combined with the TPA compounds in anti-ARDS, anti-CSS, anti-PIMS, anti-ESHS, anti-DAD, anti-inflammatory, immunostimulatory, anti-cytopathic and / or pro-apoptotic drug mixtures, coordinated administration protocols, and complementary multi-drug treatment methods, provided that the combination is effective (e.g., complementary, additive, synergistic, potentiating) to coordinately treat one or more diseases, conditions, or symptoms associated with or coexisting with viral infection, hyperinflammation, ARDs, CSS, PIMS, ESHS, DAD, increased cytopathic activity, immunosuppression, impairment of normal apoptotic activity of immune and / or inflammatory cells, and / or any other target symptom, condition, or index described herein in the treated patient.

[0143] A wide variety of anti-inflammatory agents may be useful in various aspects of the present invention, including, but not limited to, anti-inflammatory cytokines, steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), antioxidants, prostaglandins, and antibiotics, among other anti-inflammatory agents.

[0144] In exemplary combination compositions and methods of the present invention, the anti-inflammatory TPA compounds are co-formulated and delivered together with a nonsteroidal anti-inflammatory drug (NSAID), or are co-administered simultaneously or sequentially with a nonsteroidal anti-inflammatory drug (NSAID). Exemplary NSAID candidates for use in these embodiments include aspirin, celecoxib (Celebrex), diclofenac (Cambia, Cataflam, Voltaren-XR, Zipsor, Zorvolex), diflunisal, etodolac, ibuprofen (Motrin, Advil), indomethacin (Indocin), celecoxib (Celebrex), piroxicam (Feldene), indomethacin (Indocin), meloxicam (Mobic), and the like. Non-limiting examples of NSAID anti-inflammatory drugs include, but are not limited to, Vivlodex, ketoprofen (Orudis, ketoprofen ER, Oruvail, Actron), sulindac (Clinoril), diflunisal (Dolobid), nabumetone (Relafen), oxaprozin (Daypro), tolmetin (tolmetin sodium, Tolectin), salsalate (Disalcid), fenoprofen (Nalfon), flurbiprofen (Ansaid), ketorolac (Toradol), meclofenamate, mefenamic acid (Ponstel), and other safe and effective NSAID anti-inflammatory drugs that are widely known and available for clinical use.

[0145] Combination of cytokine inhibitors and TPA In other exemplary combination compositions and methods of the invention, the anti-inflammatory TPA compounds are co-formulated and delivered together with, or co-administered simultaneously or sequentially with, anti-inflammatory drugs or agents that directly or indirectly inhibit / reduce the induction, synthesis, activation and / or circulating levels of one or more pro-inflammatory cytokine targets that are excessively elevated in association with SARS-CoV-2 infection, COVID-19 disease, ARDS, SARS, CSS, PIMS, ESHS, DAD, or any other hyperinflammatory state or symptom described herein. In exemplary embodiments, the anti-inflammatory TPA compound will be co-formulated with or coordinately administered with one or more secondary therapeutic agents that inhibit / reduce the induction, synthesis, activation and / or circulating levels of one or more pro-inflammatory cytokine targets selected from (IL)-1B; IL-2; IL-6, IL-7; IL-8; IL-9; IL-10; fibroblast growth factor (FGF); granulocyte-macrophage colony-stimulating factor (GM-CSF); IFNγ; granulocyte colony-stimulating factor (G-CSF); interferon-γ-inducible protein (IP10); monocyte chemoattractant protein (MCP1); macrophage inflammatory protein 1 alpha (MIP1A); platelet-derived growth factor (PDGF); tumor necrosis factor (TNFα); and vascular endothelial growth factor (VEGF). In these aspects of the invention, the TPA compound and the secondary therapeutic agent, in combination, are effective in reducing the expression, activation, half-life and / or levels of said one or more targeted pro-inflammatory cytokines, often with fewer side effects than administration of equally therapeutically effective doses of the individual drugs.

[0146] Exemplary anti-inflammatory drug candidates for use as secondary therapeutic agents in these embodiments include the anti-inflammatory cytokines described above, as well as various drugs and biological agents that specifically, directly or indirectly, target, bind, block, inactivate, and / or inhibit the synthesis or activity of one or more pro-inflammatory cytokines.

[0147] Anti-IL-6 biologics and drug-TPA combinations Illustrative of these aspects of the present invention, TPA compounds, when used in combination with anti-IL-6 drugs or biologics, are effective in combination to substantially reduce the induction, synthesis, activation, and / or circulating levels of IL-6. IL-6 has long been considered a keystone proinflammatory cytokine, along with TNF-α and IL-1, involved in the proinflammatory cascade. In the case of IL-6, this cytokine is considered a marker of systemic activation of inflammatory effectors. Like many other cytokines, IL-6 possesses both pro- and anti-inflammatory properties. Of particular importance, IL-6 is a potent inducer of the acute phase inflammatory response, and elevated IL-6 levels strongly correlate with poor prognosis in COVID-19 patients with ARDS (excessive elevation of IL-6 is strongly associated with the need for mechanical ventilation). The classical pathway of IL-6 signaling occurs via the IL-6 receptor, which is expressed on neutrophils, monocytes, macrophages, and other leukocyte populations. In addition to binding to the membrane-bound IL-6 receptor (mIL-6R, CD126), IL-6 can also bind to soluble forms of the IL-6 receptor, which are generated by proteolytic cleavage of mIL-6R or alternative splicing of mRNA. Increased levels of circulating IL-6 are associated with a more rapid decline in lung elasticity and more severe bronchoalveolar inflammation. Therefore, specific blockade of IL-6-regulated signaling pathways represents a promising approach for attenuating the acute inflammation associated with lung injury in COVID-19 disease subjects.

[0148] In one exemplary embodiment of targeting IL-6 with a multi-drug TPA strategy, a TPA compound is coordinately administered with an anti-IL inhibitory, binding, or inactivating agent, such as a specific IL-6 monoclonal antibody or Fab fragment or a soluble IL-6 receptor or receptor analog, and related biologics containing their cognate anti-IL-6 binding or inactivating domains.

[0149] In certain embodiments, the anti-IL-6 monoclonal antibody siltuximab, manufactured by EUSA Pharma, is utilized in a coordinated anti-inflammatory treatment method with the TPA compound. Siltuximab is currently under clinical investigation for use in treating COVID-19 patients with ARDS.

[0150] In another embodiment, the IL-6 receptor antagonist sarilumab (Kevzara) is utilized in conjunction with anti-inflammatory TPA compounds to neutralize or impair IL-6 activity, thereby mediating clinical benefit in subjects with COVID-19, ARDS, CSS, and other hyperinflammatory conditions. Kevzara has shown efficacy in treating arthritis. Regeneron Pharmaceuticals and Sanofi are currently conducting Phase II and III trials of Kevzara in severe and critically ill COVID-19 patients (NCT04315298).

[0151] In another embodiment, the recombinant human anti-IL-6 monoclonal antibody tocilizumab (TCZ) is utilized in conjunction with an anti-inflammatory TPA compound to neutralize or impair IL-6 activity to achieve clinical benefit in subjects with COVID-19, ARDS, CSS, and other hyperinflammatory conditions. TCZ specifically binds to soluble and membrane-bound IL-6 receptors (IL-6R), thereby blocking IL-6 signaling and IL-6-mediated inflammatory responses. TCZ has been widely used in rheumatic diseases such as rheumatoid arthritis. In 2017, TCZ was approved in the United States for life-threatening severe CSS caused by chimeric antigen receptor T cell (CART) immunotherapy. Recent studies have evaluated the efficacy of TCZ in treating severely and critically ill COVID-19 patients. Preliminary reports indicate that TCZ reduces fever and other ARDS symptoms, and 75.0% of treated subjects showed improved oxygenation. Opaque lung lesions on CT scans were remitted in 90.5% of patients. In addition, peripheral lymphocyte levels returned to normal in 52.6% of patients. To evaluate the safety and efficacy of tocilizumab in the treatment of severe COVID-19 pneumonia in hospitalized adults, several clinical trials are currently being enrolled, including a multicenter randomized controlled trial on the efficacy and safety of tocilizumab in patients with novel coronavirus pneumonia (NCP) (ChiCTR2000029765), a single-arm open multicenter study on tocilizumab (ChiCTR2000030796), and studies on tocilizumab combined with antiviral drugs (ChiCTR2000030442 and ChiCTR2000030894).

[0152] In a related embodiment, the anti-inflammatory TPA compound is co-formulated and delivered together with, or co-administered simultaneously or sequentially with, an anti-IL-6 drug that directly blocks or inhibits IL-6 or indirectly inhibits, reduces, or alters the pro-inflammatory activity of IL-6. One such drug is andrographolide, originally identified in the plant Andrographis paniculata. Andrographolide is a diterpenoid labdane compound and is the primary bioactive component found in the traditional medicinal plant Andrographis paniculata, widely used in India and China to treat infections, inflammation, colds, fever, pneumonia, and other conditions, as well as diarrhea. Andrographolide has been documented to exhibit a wide range of therapeutically important biological activities, including antibacterial, anti-inflammatory, antimalarial, and anticancer activity. Andrographolide exhibits potent anti-inflammatory activity, including through inhibition of NF-κB signaling. Andrographolide also suppresses the production of inducible nitric oxide synthase and reactive oxygen species, which are associated with excessive inflammation. Andrographolide can also induce cell cycle arrest and apoptosis via the caspase-8-dependent pathway by increasing p27 expression and decreasing cyclin-dependent kinase expression. In mouse peritoneal macrophages, andrographolide inhibits the production of TNF-α and interleukin-12 through the suppression of ERK1 / 2 signaling.

[0153] Andrographolide drugs combined with TPA With regard to IL-6-mediated inflammation, andrographolide has also been shown to have potent anti-inflammatory activity, affecting IL-6-mediated hyperinflammatory mechanisms and pathways. Andrographolide dose-dependently inhibits IL-6 production and suppresses IL-6 hyperinflammatory signaling (including within the Stat3, Akt, and ERK1 / 2 pathways) both in vitro and in vivo (Chun, 2010). According to an exemplary embodiment of the present invention, coordinated administration of an anti-inflammatory TPA compound and andrographolide is effective in combination to block or inhibit IL-6 activity, often via multiple pathways and / or mechanisms, directly or indirectly inhibiting, reducing, or altering the proinflammatory activity of IL-6 or correcting the hyperinflammatory or immunological dysregulatory effects of IL-6 (e.g., dysregulation of differentiation, proliferation, activation, inflammatory cytokine synthesis, and / or apoptotic activity of immune and / or inflammatory effector cells, such as lymphocytes, monocytes / macrophages, and neutrophils).

[0154] Combination of kinase modulators and TPA In other exemplary combination compositions and methods of the invention, the anti-inflammatory TPA compound is co-formulated with, delivered together with, or co-administered simultaneously or sequentially with, a kinase-modulating drug or agent that directly or indirectly inhibits, reduces, activates, or alters the immune or inflammatory activity of one or more kinases involved in mediating or suppressing an excessive inflammatory response or regulating the differentiation, proliferation, activation, inflammatory cytokine synthesis, and / or apoptotic activity of immune and / or inflammatory effector cells (e.g., lymphocytes, monocytes / macrophages, and neutrophils). Exemplary target kinases in these aspects of the invention include mitogen-activated protein kinases (MAPKs) and Janus kinases (JAKs). In exemplary embodiments, coordinated treatment of a subject with COVID-19 disease with TPA and a JAK inhibitor clinically reduces one or more disease states or symptoms associated with ARDS, SARS, CSS, PIMS, ESHS, DAD, or other hyperinflammatory states or symptoms described herein. In some embodiments, the anti-inflammatory TPA compound is co-formulated with or coordinately administered with the JAK inhibitor jakotinib hydrochloride or another JAK inhibitor baricitinib.

[0155] The JAK inhibitor anti-COVID-19 treatment strategy described herein is based on the role of ACE2 in SARS-CoV-2 pathogenesis, as detailed above. ACE2 is a cell surface protein widely expressed in the heart, kidney, blood vessels, and especially in the alveolar epithelium. SARS-CoV-2 apparently binds to ACE2 and enters cells via ACE2-mediated endocytosis. One known regulator of endocytosis is AP2-associated protein kinase 1 (AAK1). AAK1 inhibitors can impair viral infection and replication by blocking viral passage into cells. Baricitinib, a JAK inhibitor and AAK1 inhibitor, has already been shown to be safe in COVID-19 subjects. Therapeutic dosing of either 2 mg or 4 mg of baricitinib per day was sufficient to induce measurable SARS-CoV-2 inhibition. Concerns regarding the use of JAK inhibitors are based on reports that JAKs inhibit various cytokines, such as INF-α, that mediate antiviral immune responses.The JAK inhibitor jacotinib hydrochloride is currently undergoing clinical trials (Study for safety and efficacy of Jakotinib hydrochloride tablets in the treatment severe and acute exacerbation patients of novel coronavirus pneumonia (COVID-19) (ChiCTR2000030170) and Severe novel coronavirus pneumonia (COVID-19) patients treated with ruxolitinib in combination with mesenchymal stem cells: a prospective, single-blind, randomized controlled clinical trial (ChiCTR2000029580)).

[0156] Combination of anti-SARS-CoV-2 vaccine and TPA In other exemplary combination compositions and methods of the invention, antiviral or anti-inflammatory TPA compounds are co-formulated and delivered together with anti-SARS-CoV-2 vaccine agents, or co-administered simultaneously or sequentially with anti-SARS-CoV-2 vaccine agents, to prevent or reduce viral infection and thereby prevent or reduce COVID-19 disease states and symptoms. Exemplary candidate vaccines for use in these coordinated treatment methods are currently numerous as the world races for effective protection against SARS-CoV-2 and other past and potential future hSARS viruses. In certain embodiments, secondary therapeutic agents in the form of anti-hSARS virus vaccines can include live-attenuated recombinant or recombinant chimeric hSARS viruses, inactivated or killed hSARS viruses, subunit vaccines composed of all or part of an immunogenic subunit of the hSARS virus, such as the hSARS spike (S) protein, and many other diverse anti-hSARS vaccine tools are currently being investigated. Vaccines are particularly important in the combined TPA treatment methods and compositions of the invention, in part because there is currently insufficient evidence that any existing antiviral drugs (administered as monotherapy) effectively treat COVID-19 pneumonia.

[0157] Vaccine development is a critical long-term strategy to prevent future COVID-19 outbreaks. Sequencing of the SARS-CoV-2 genome has led to the proposal of multiple nucleic acid-based vaccine candidates, many of which are based on the S protein coding sequence.

[0158] mRNA-1273 vaccine The SARS-CoV-2 genome was sequenced in early January 2020, shortly after the COVID-19 pneumonia outbreak. Moderna's mRNA-1273 vaccine candidate is a synthetic strand of mRNA encoding a perfusion-stabilized viral spike (S) protein. It is expected to elicit an antiviral immune response specifically directed against the SARS-CoV-2 spike protein after intramuscular injection in humans. Unlike conventional vaccines made from inactivated or killed pathogens, live-attenuated viruses, or small immunogenic viral subunits, Moderna's lipid nanoparticle-encapsulated mRNA vaccine does not require any use, handling, or patient exposure to the SARS-CoV-2 virus. Therefore, it is relatively safe and readily available for testing. If mRNA-1273 proves safe in humans and passes Phase 1 trials, subsequent evaluation of its efficacy will be immediately conducted (NCT04283461). This vaccine candidate is a potential useful secondary agent in combination formulations and coordinated treatments with the TPA described herein.

[0159] INO-4800 INO-4800 is a DNA vaccine candidate developed by Inovio Pharmaceuticals. Similar to Moderna's mRNA-1273, INO-4800 is a genetic vaccine that can be delivered to human cells, translated into protein, and elicit an immune response. Compared to conventional vaccines, genetic vaccines are less costly to produce, easier to produce, and safer to administer. Because nucleic acids have a simple structure, they eliminate the risk of improper folding that can occur with recombinant protein-based vaccines. However, the amount of plasmid delivered and the appropriate administration interval and route are uncertainties that can affect the immunogenicity of genetic vaccines. This vaccine candidate is a potential secondary agent useful in combination formulations and coordinated treatments with the TPA described herein.

[0160] ChAdOx1 nCoV-19 The ChAdOx1 nCoV-19 vaccine, developed by the University of Oxford, is composed of a non-replicating adenovirus vector and the genetic sequence of the SARS-CoV-2 S protein and is currently undergoing Phase I / II clinical trials (NCT04324606). Because adenoviruses are non-replicating within the host, they are relatively safe for children and individuals with underlying medical conditions. Adenovirus-based vectors are characterized by broad tissue tropism, covering both respiratory and gastrointestinal epithelia, the two major sites expressing the SARS-CoV-2 ACE-2 receptor. This vaccine candidate is a potential secondary agent for future use in combination formulations and coordinated treatments with the TPA described herein.

[0161] stabilized subunit vaccines Enveloped viruses require fusion of the viral membrane with the host cell membrane for infection. This process involves a conformational change in viral glycoproteins from pre-fusion to post-fusion forms. Although pre-fusion glycoproteins are relatively unstable, they are still capable of eliciting strong immune responses. The University of Queensland is developing a stabilized subunit vaccine against SARS-CoV-2 based on molecular clamp technology, which will allow recombinant viral proteins to remain stable in their pre-fusion form. Molecular clamp vaccines have previously been applied to influenza and Ebola viruses, demonstrating their ability to induce neutralizing antibody production. Efficacy after two weeks at 37°C has also been reported. This vaccine candidate is a potentially useful secondary agent in combination formulations and coordinated treatments with the TPA described herein.

[0162] Nanoparticle-based vaccines Nanoparticle-based vaccines are an alternative strategy to incorporate antigens for vaccination of at-risk subjects. By encapsulation or covalent functionalization, nanoparticles can be conjugated with antigenic epitopes, mimicking viruses and inducing antigen-specific lymphocyte proliferation and cytokine production. In addition, mucosal vaccination via intranasal or oral spray can not only stimulate immune responses at mucosal surfaces but also induce systemic responses. This demonstrates the ability of nanoparticle-based vaccines to protect humans from respiratory viruses that cause systemic symptoms. Novavax, Inc. is producing a nanoparticle-based anti-SARS-CoV-2 vaccine using an antigen derived from the virus's S protein. This protein is stably expressed in a baculovirus system, and the product is expected to enter Phase I trials this summer. This vaccine candidate is a potential secondary agent useful in combination formulations and coordinated treatments with the TPA described herein.

[0163] Pathogen-specific artificial antigen-presenting cells Based on the knowledge that antigen-specific T cells can eradicate cancer cells and viral infections, generating large numbers of T cells with viral antigen specificity may enhance resistance to SARS-CoV-2 infection. An efficient method for generating large numbers of T cells involves the use of appropriate antigen-presenting cells capable of activating effector T cells, as well as the differentiation and proliferation of corresponding effector cytotoxic T cells. Genetically modified artificial antigen-presenting cells (aAPCs) expressing conserved domains of viral structural proteins delivered by lentiviral vectors can induce the differentiation and proliferation of naive T cells. Several studies are underway to evaluate the safety and immunogenicity of aAPCs alone and in combination with antigen-specific cytotoxic T cells (NCT04299724, NCT04276896). aAPCs may be useful secondary agents in future combination formulations and coordinated treatments with the TPAs ​​described herein.

[0164] Natural killer cells The highest mortality rate from COVID-19 is observed in elderly patients, which may be due, at least in part, to a weakened immune system caused by aging. Approaches aimed at boosting the innate antiviral immune response against SARS-CoV-2 hold great promise. Natural killer (NK) cells constitute a key component of the innate immune system, mediating rapid responses to viral infections. Previous studies have shown that pulmonary migration of NK cells and macrophages plays a crucial role in the elimination of SARS-CoV (Chen et al., 2009). The innate response itself can suppress SARS-CoV infection by increasing cytokine and chemokine production without the assistance of CD8+ T cells or antibodies. Whether the addition of NK cells can mediate viral clearance in COVID-19 pneumonia is currently under phase I clinical trial in China (NCT04280224), which is expected to be completed by the end of 2020. Several companies are aiming to repurpose their NK-based anticancer products for the treatment of COVID-19. Among these is a product being co-developed by South Korea's Green Cross LabCell and the U.S.-based Kleo Pharmaceuticals. Also, the U.S.-based company Celularity has developed placental hematopoietic stem cell-derived NK cells, CYNK-001. NK cell-boosting methods and compositions may be useful secondary agents in future combination formulations and coordinated treatments with the TPA described herein.

[0165] Recombinant interferon Type I interferons are secreted by virus-infected cells. When used alone or in combination with other drugs, they exert broad-spectrum antiviral effects against HCV, respiratory syncytial virus, SARS-CoV, and MERS-CoV. Studies are currently focused on their safety and efficacy in treating COVID-19 pneumonia (NCT04293887). Type I interferons may be useful secondary agents in future combination formulations and coordinated treatments with TPAs ​​described herein.

[0166] Mesenchymal stem cells Mesenchymal stem cells (MSCs) have been shown to exert anti-inflammatory functions by reducing proinflammatory cytokines and producing paracrine factors for tissue repair. Preclinical evidence has shown that MSCs can not only restore endothelial permeability but also reduce inflammatory infiltrates. The immunomodulatory effects of MSCs have been demonstrated with avian influenza viruses, and their role in treating COVID-19 pneumonia is promising. Currently, MSCs from umbilical cord and dental pulp are being clinically tested in COVID-19 research (NCT04293692, NCT04269525, NCT04288102, NCT04302519). MSCs may be useful secondary agents in future combination formulations and coordinated treatments with TPAs ​​described herein.

[0167] Intravenous immune globulin Intravenous immunoglobulin (IVIG) has been widely applied in the fields of neurology, dermatology, and rheumatology. IVIG exerts diverse, dose-dependent effects on the immune system. Low doses (0.2–0.4 g / kg) of IVIG are useful as replacement therapy for antibody deficiencies. Higher doses (up to 2 g / kg) of IVIG exhibit potent immunomodulatory functions by suppressing inflammatory cell proliferation, inhibiting phagocytosis, and interfering with antibody-dependent cellular cytotoxicity (Jolles et al., 2005). Recent trials are evaluating the safety and efficacy of IVIG in COVID-19 subjects (NCT04261426). IVIG compositions may be useful secondary agents in future combination formulations and coordinated treatments with TPAs ​​described herein.

[0168] SARS-CoV-2 specific neutralizing antibody Antibody-mediated humoral immune responses are crucial for preventing viral infections. The development of specific neutralizing antibodies targeting viral surface epitopes is a promising approach for targeting COVID-19. AbCellera (Canada) and Eli Lilly and Company (USA) are collaborating to develop functional antibodies capable of neutralizing SARS-CoV-2 in infected patients. To this end, they screened over 5 million immune cells from one of the first US patients to recover from COVID-19 and identified over 500 promising anti-SARS-CoV-2 antibody sequences, which are currently undergoing screening to identify the most effective ones. This approach has been successfully applied to produce functional specific antibodies against West Nile virus. Vir Biotechnology, Inc., ImmunoPrecise, Mount Sinai Health System, and Harbour BioMed (HBM) are conducting screening to find monoclonal antibodies that will bind to and neutralize SARS-CoV-2. These and other SARS-CoV-2-specific neutralizing antibodies may be useful secondary agents in future combination formulations and coordinated treatments with the TPAs ​​described herein.

[0169] Anti-C5a monoclonal antibody Complement activation correlates with acute lung injury, and the cytokine C5a (a bioactive molecule cleaved from C5) has been implicated as a key effector mediating tissue injury. The role of C5a includes recruiting neutrophils and T lymphocytes and increasing pulmonary vascular permeability. Anti-C5a treatment has been shown to reduce lung injury by reducing vascular leakage and neutrophil influx into the lung interstitium and alveolar space (Guo et al., 2005). Examples of anti-C5a agents for use as secondary agents in combination formulations and coordinated treatment methods with TPA include BDB-1, marketed by Beijing Defengrei Biotechnology Co., and IFX-1, produced by Beijing Staidson Biopharma and InflaRx.

[0170] thalidomide Recently, thalidomide has reemerged as an antiangiogenic, anti-inflammatory, and anti-fibrotic pharmaceutical agent for diverse therapeutic applications. By reducing the synthesis of TNF-alpha, thalidomide has been utilized as a treatment for various hyperinflammatory diseases, such as Crohn's disease and Behçet's disease. Thalidomide is effective in treating H1N1-infected mice by reducing inflammatory cell infiltration and inhibiting or blocking the production of proinflammatory cytokines (Zhu et al., 2014). Studies are currently underway to investigate the immunomodulatory effects of thalidomide to reduce lung injury caused by an excessive immune / inflammatory response to SARS-CoV-2 (NCT04273529, NCT04273581). Thalidomide is a potentially useful secondary agent in combination formulations and coordinated treatments with TPA, as described herein.

[0171] Fingolimod Fingolimod is an oral immunomodulatory agent primarily used to treat refractory multiple sclerosis. It is structurally similar to the lipid sphingosine-1-phosphate (S1P) and can act as a highly potent antagonist of the S1P1 receptor in lymph node T cells. Effective binding leads to internalization of the S1P1 receptor, followed by sequestration of lymph node T cells. Reducing pulmonary influx of T lymphocytes is another approach to attenuating uncontrolled immunopathogenesis and is currently being tested in clinical trials (NCT04280588). Fingolimod is a potentially useful secondary agent in certain combination formulations and coordinated treatments with TPA described herein.

[0172] Combination of antiangiogenic drugs and TPA Elevated levels of vascular endothelial growth factor (VEGF) are observed in patients with acute respiratory distress syndrome. VEGF functions as an inflammatory mediator that can induce endothelial injury and increase vascular permeability (Thickett et al., 2001). Various anti-VEGF drugs and biologics are being developed for clinical use. Among these, bevacizumab is a recombinant humanized monoclonal antibody that can bind to VEGF and neutralize its function, e.g., block angiogenesis. It is currently approved and widely used in the United States to treat multiple types of cancer. Ongoing trials are currently evaluating the efficacy of bevacizumab for treating SARS-CoV-2 infection (NCT04275414). Antiangiogenic drugs and biologics, including anti-VEGF drugs and biologics, may be useful secondary agents in future combination formulations and coordinated treatments with the TPAs ​​described herein.

[0173] Hydroxychloroquine In other exemplary combination compositions and methods of the present invention, anti-SARS-CoV-2 TPA compounds are co-formulated and delivered together with chloroquine or hydroxychloroquine, or co-administered simultaneously or sequentially with chloroquine or hydroxychloroquine. Hydroxychloroquine, which has long been used as an antimalarial and anti-autoimmune drug, also appears to limit viral infectivity by increasing the endosomal pH required for membrane fusion between the virus and host cells. One study reported that hydroxychloroquine specifically inhibits SARS-CoV replication by interfering with the glycosylation of its cellular receptor, ACE2. Recent in vitro studies suggest that hydroxychloroquine effectively reduces SARS-CoV-2 viral load / titer in COVID-19 subjects (Lan et al., 2020). Several clinical trials were rapidly conducted in China, and hydroxychloroquine was reported to be, to varying degrees, effective in treating COVID-19-associated pneumonia. A small, open-label, nonrandomized clinical trial in France reported that hydroxychloroquine, in combination with azithromycin, had a positive effect. The U.S. FDA issued an emergency use authorization for hydroxychloroquine to treat COVID-19 in the United States, and the drug has since been widely used off-label for this purpose in the country. More recent studies have found limited evidence of substantial clinical benefit for hydroxychloroquine in treating COVID-19 disease, and concerns about the drug's cardiac safety have led to the early termination of at least one large clinical trial.

[0174] Glucocorticoids Numerous clinical trials have reported the efficacy of glucocorticoids for the treatment of coronavirus and influenza pneumonia. During the 2003 SARS epidemic, glucocorticoids were the primary immunomodulatory therapy. The timely use of glucocorticoids can improve early fever and reduce the severity of hypoxia associated with pneumonia. However, some studies have not found any beneficial effects of glucocorticoids, and some reports have been associated with immunosuppression, delayed viral clearance, and adverse reactions. According to international guidelines for the management of sepsis and septic shock, when glucocorticoids are used, they should be administered at low doses for a short period of time only in patients in whom adequate fluid and vasopressor therapy do not restore hemodynamic stability.

[0175] Systemic glucocorticoid administration has been used in severe complications of COVID-19, including ARDS, acute cardiac injury, acute renal complications, and to suppress CSS manifestations in patients with elevated D-dimer levels, which are associated with thrombus formation. Without further evidence, WHO interim guidelines do not support the use of systemic corticosteroids to treat viral pneumonia and ARDS associated with COVID-19 disease.

[0176] Although the benefit of systemic glucocorticoids in treating SARS-CoV-2 is currently questionable (based on their potential immunosuppressive effects that prolong viral clearance), these drugs may nonetheless have future therapeutic use in treating certain aspects of COVID-19 disease. The underlying pathogenesis of COVID-19 pneumonia consists of both direct damage caused by the virus and substantial pathogenic effects caused by the host's hyperimmune and hyperinflammatory responses. Methylprednisolone administration is an exemplary glucocorticoid to help suppress hyperimmune and inflammatory responses, and studies are underway to explore its efficacy and safety in COVID-19 subjects (NCT04273321, NCT04263402). In certain COVID-19 and ARDS treatments, as well as in targeted and staged treatment protocols, glucocorticoids may be useful secondary agents in future combination formulations and coordinated treatments with the TPAs ​​described herein.

[0177] Pharmaceutical Compositions, Administration, Delivery and Formulations With respect to each of the exemplary aspects of the invention described above, certain embodiments will utilize one or more TPA or TPA-like compounds, such as the parent TPA compounds of Formula I or Formula II above, e.g., 12-O-tetradecanoylphorbol-13-acetate (formally "TPA"; also known as phorbol-12-myristate-13-acetate (PMA)), or structurally related functional analogs, conjugates, prodrugs, salts, or other modified or derivative forms of the parent TPA compounds. The TPA compounds utilized in the invention are useful in compositions and methods administered to subjects to mediate antiviral (e.g., anti-SARS-CoV-2), anti-inflammatory, anti-ARDs, anti-CSS, anti-PIMS, anti-ESHS, anti-DAD, anti-cytopathic, immunostimulatory, and / or pro-apoptotic effects, as well as other clinically relevant activities. The potent and diverse clinical effects of these compositions and methods are effective, individually and collectively, in treating and / or preventing the diverse array of conditions, symptoms, and accompanying immunological, cellular, tissue, and organ injury and dysfunction of ARDS and COVID-19 disease.

[0178] Generally, a clinically effective amount or clinically effective dose of a TPA compound of Formula I or Formula II is administered to a subject suitable for TPA treatment to effectively induce an antiviral, anti-inflammatory, anti-ARDs, anti-CSS, anti-PIMS, anti-ESHS, anti-DAD, anti-cytopathic, immunostimulatory, and / or pro-apoptotic response in the treated subject. As described above, clinical efficacy is demonstrated by comparing the therapeutic indices before and after treatment in test subjects and placebo-treated control subjects. In exemplary embodiments, an effective amount of an active TPA compound will provide a quantitatively or qualitatively significant therapeutic benefit in one or more unit dosage forms, at a certain administration frequency, and over a selected therapeutic intervention period to alleviate one or more symptoms of viral infection, excessive inflammation, ARDs, CSS, PIMS, ESHS, DAD, increased cytopathic activity, immunosuppression, dysfunction of the normal apoptotic activity of immune and / or inflammatory cells, and / or any other target symptom, condition, or index described herein in the treated subject.

[0179] The compositions of the invention typically comprise an effective amount or unit dosage of a TPA compound of Formula I or Formula II formulated with one or more pharmaceutically acceptable carriers, excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers and / or other additives that may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics and / or pharmacodynamics, reduce adverse side effects, or provide other benefits for pharmaceutical use. Effective dosages will be readily determined by the clinician depending on the target condition and clinical and patient-specific factors. For administration to mammalian subjects, including humans, suitable effective unit dosages of the active TPA compound may range from about 10 to about 1500 μg, about 20 to about 1000 μg, about 25 to about 750 μg, about 50 to about 500 μg, about 150 to about 500 μg, about 125 μg to about 500 μg, about 180 to about 500 μg, about 190 to about 500 μg, about 220 to about 500 μg, about 240 to about 500 μg, about 260 to about 500 μg, and about 290 to about 500 μg. In certain embodiments, disease-treating effective dosages of phorbol ester compounds of Formula I or related or derivative compounds can be selected within narrower ranges, such as 10-25 μg, 30-50 μg, 75-100 μg, 100-300 μg, or 150-500 μg. These and other effective unit dosages can be administered in a single dose or in the form of multiple daily, weekly, or monthly doses, e.g., in a dosing regimen comprising 1-5 or 2-3 doses per day, week, or month. In an exemplary embodiment, dosages of 10-30 μg, 30-50 μg, 50-100 μg, 100-300 μg, or 300-500 μg are administered once, twice, three times, four, or five times per day. In more particular embodiments, 50-100 μg, 100-300 μg, 300-400 μg, or 400-600 μg are administered once or twice daily. In a further embodiment, a dosage of 50-100 μg, 100-300 μg, 300-400 μg, or 400-600 μg is administered every other day.In alternative embodiments, dosage is calculated based on body weight, e.g., from about 0.5 μg / m2 to about 300 μg / m2 per day, from about 1 μg / m2 to about 200 μg / m2 per day, from about 1 μg / m2 to about 187.5 μg / m2, from about 1 μg / m2 per day to about 175 μg / m2 per day, from about 1 μg / m2 per day to about 157 μg / m2 per day, μg / m² per day, about 1 μg / m² to about 125 μg / m² per day, about 1 μg / m² to about 75 μg / m² per day, 1 μg / m² to about 50 μg / m² per day, 2 μg / m² to about 50 μg / m² per day, 2 μg / m² to about 30 μg / m² per day, or 3 μg / m² to about 30 μg / m² per day.

[0180] In another embodiment, the dosage is administered even less frequently, e.g., from 0.5 μg / m to about 300 μg / m every other day, from about 1 μg / m to about 200 μg / m every other day, from about 1 μg / m to about 187.5 μg / m, from about 1 μg / m to about 175 μg / m every other day, or from about 1 μg / m to about 157 μg / m every other day. 2, about 1 μg / m to about 125 μg / m every other day, about 1 μg / m to about 75 μg / m every other day, 1 μg / m to about 50 μg / m every other day, 2 μg / m to about 50 μg / m every other day, 2 μg / m to about 30 μg / m per day, or 3 μg / m to about 30 μg / m per day. In further embodiments, dosages can be administered 3 times per week, 4 times per week, 5 times per week, weekdays only, only when concomitant with other treatment regimens, daily, or any other suitable dosing regimen depending on clinical and patient-specific factors.

[0181] The amount, timing and delivery mode of the therapeutic composition of the present invention will be adjusted individually and routinely according to factors such as body weight, age, sex and individual condition, the acuteness and severity of the target disease or condition, whether the administration is prophylactic or therapeutic, and other factors known to achieve drug delivery, absorption, pharmacokinetics and efficacy. Effective dosage and administration protocol will often include repeated administration over several days, one week or more, one month or more, or even one year or more. Effective treatment regimens can also involve prophylactic administration administered daily or multiple times per day for several days, weeks, months or years.

[0182] The pharmaceutical composition of the present invention can be administered by any clinically acceptable route and means that achieves the intended therapeutic or prophylactic purpose.Suitable administration routes include all conventional delivery routes, devices and methods.Currently used delivery methods include injectable methods such as intravenous injection and infusion, intramuscular, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial and subcutaneous injection.Oral and mucosal solid and liquid dosage forms, as well as intranasal and intrapulmonary aerosol delivery, are also contemplated.

[0183] Effective dosage forms of the present invention will often contain excipients recognized in the art of pharmaceutical compounds as suitable for preparing dosage units. Such excipients include, but are not limited to, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavors, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other conventional excipients and additives. The therapeutic compositions of the present invention can also be administered in sustained-release, delayed-release, or other controlled-release forms, for example, by using slow-release carriers or excipients or sustained-, delayed-, or controlled-release polymers.

[0184] Certain TPA compositions of the present invention are advantageously delivered by parenteral administration, such as intravenous, intramuscular, subcutaneous, or intraperitoneal. These dosage forms will typically be provided in the form of aqueous or non-aqueous sterile injectable solutions, optionally containing additives such as antioxidants, buffers, bacteriostats, and / or solutes that render the formulation isotonic with the blood of a mammalian subject. Aqueous and non-aqueous sterile suspensions may include suspending agents and / or thickening agents. The formulations may be presented in unit-dose or multi-dose containers. Additional compositions and formulations of the present invention may include polymers, liposomes, micelles, conjugates, and other agents that improve bioavailability in specific targets (e.g., the lungs) and / or prolong release following parenteral administration. Useful parenteral preparations may be solutions, dispersions, or emulsions suitable for such administration. Extemporaneous injection and infusion solutions, emulsions, and suspensions may be prepared according to conventional practices from sterile powders, granules, and tablets or other starting forms. Useful unit dosage forms will contain a daily dose or units, a sub-daily dose, or a therapeutic dose effective over multiple days.

[0185] In certain embodiments, the compositions of the present invention may comprise the TPA compounds of Formula I or Formula II encapsulated for delivery in microcapsules, microparticles, or microspheres, such as hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared, for example, by coacervation techniques or interfacial polymerization, encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or encapsulated in macroemulsions. Microencapsulation processes are well known and can be routinely performed to prepare microparticles containing the active ampakines described herein.

[0186] As mentioned above, in certain embodiments, the methods and compositions of the present invention utilize pharmaceutically acceptable salts, such as acid addition salts or base salts of the TPA compounds described above, to enhance solubility, bioavailability, or other performance criteria. Examples of pharmaceutically acceptable addition salts include inorganic and organic acid addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts, such as hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate, and hydrogen phosphate salts. Additional pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium salts, potassium salts, and cesium salts; alkaline earth metal salts such as calcium salts and magnesium salts; organic amine salts such as triethylamine salts, pyridine salts, picoline salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, and N,N'-dibenzylethylenediamine salts; organic acid salts such as acetate, citrate, lactate, succinate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, and formate; sulfonate salts such as benzenesulfonate and p-toluenesulfonate; and amino acid salts such as alginate, aspartate, glutamate, tartrate, and gluconate. Suitable base salts are formed from bases that form non-toxic salts, such as aluminum salts, calcium salts, lithium salts, magnesium salts, potassium salts, sodium salts, zinc salts, and diethanolamine salts.

[0187] In another embodiment, the methods and compositions of the present invention utilize prodrugs of phorbol esters of Formula I or Formula II. A prodrug is considered to be any covalently attached carrier that releases the active parent drug in vivo. Examples of prodrugs useful in the present invention include esters or amides with hydroxyalkyl or aminoalkyl substituents, which can be prepared by reacting such compounds with anhydrides such as succinic anhydride. It will also be understood that a related aspect of the present invention includes methods and compositions comprising phorbol esters of Formula I or Formula II with in vivo metabolic products of the compounds (either generated in vivo after administration of the precursor compound or administered directly in the form of the metabolite itself). Such products can result from, for example, oxidation, reduction, hydrolysis, amidation, esterification, etc., of the precursor drug or administered compound, primarily by enzymatic processes. Thus, the present invention includes methods and compositions for making and using TPA compounds, derivatives, and metabolites produced by a process comprising contacting a phorbol ester compound of Formula I or Formula II with a mammalian body fluid, cell, tissue sample, or subject for a period of time sufficient to yield a metabolic product of the TPA compound (e.g., starting with a radiolabeled compound administered parenterally at a detectable dose, allowing sufficient time for metabolic conversion products of the labeled compound to appear and be isolated from urine, blood, or other biological sample).

[0188] It will also be understood that the invention disclosed herein encompasses diagnostic compositions for diagnosing or otherwise managing the risk level, presence, severity, or treatment indications of diseases in a mammalian subject, such as, but not limited to, viral infection, hyperinflammation, ARDs, CSS, PIMS, ESHS, DAD, increased cytopathic activity, immunosuppression, impairment of normal apoptotic activity of immune and / or inflammatory cells, and / or any other target symptom, condition, or index described herein. An exemplary diagnostic method involves contacting a labeled (e.g., isotopically, fluorescently, or otherwise labeled to permit detection of the labeled compound using conventional methods) TPA compound of Formula I or Formula II with a mammalian subject (e.g., cell, tissue, plasma, organ, or individual) at risk or exhibiting one or more target symptoms, and then detecting the presence, location, metabolism, and / or binding state of the labeled TPA compound using any of a wide range of known assays and labeling / detection methods. In certain embodiments, the TPA compound is isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively. The isotopically labeled compounds are then administered to an individual or other subject and then detected as described above to provide useful diagnostic and / or therapeutic management data according to conventional techniques.

[0189] As will be understood by those skilled in the art, the present invention is not limited to the specific compounds, formulations, process steps, and materials disclosed herein above, which are provided for illustrative purposes only. In accordance with the overall discovery and teachings of the present invention, these compounds, formulations, process steps, and materials can be modified, extended, or substituted in equivalent form and purpose without undue experimentation. Furthermore, the terminology used herein is merely exemplary to describe exemplary embodiments and is not intended to limit the scope of the present invention. The following examples are also provided for illustrative purposes, but are not limiting. [Example]

[0190] Example I TPA compounds are effective in preventing and treating acute respiratory distress syndrome (ARDS), including sudden acute respiratory syndrome (SARS), associated with severe COVID-19 viral disease The inventors have detailed the disparate cellular, molecular, gene regulatory, biochemical, physiological, and pathogenic effectors, functions, targets, pathways, and responses affected and achieved by the active TPA compounds of the present invention to mediate novel and profound antiviral, immunoregulatory, and anti-hyperinflammatory clinical benefits in patients suffering from ARDS, COVID-19 disease, and the other hyperinflammatory and immunodysregulatory conditions detailed above. Attempting to illustrate all targets and activities of TPA within this broad range of clinical utility would exceed the practical needs and expectations of those skilled in the art. For the sake of a streamlined and clear description, the inventors have incorporated by reference all manner and forms of cellular, molecular, gene regulatory, biochemical, physiological, and pathological research, as well as diagnostic assay techniques, tools, and methods, relevant to the practical implementation of the full range of activity and clinical mechanisms of the TPA compounds and methods disclosed herein.

[0191] Certain embodiments of the present invention relating to ARDS exemplify the broad range of activity and clinical benefits of TPA compounds in general. In the specific example of ARDS caused by the human SARS coronavirus SARS-CoV-2 (COVID-19), TPA-related compositions and methods for treating and preventing ARDS utilize and elicit a wide range of such activities and benefits. For example, the inventors' pilot studies investigating the clinical optimization of TPA compounds and methods for treating ARDS have revealed a number of crucial immunostimulatory and anti-inflammatory targets, mechanisms, and pathways, whereby TPA compounds target, eliminate, or reduce abnormal effector cells, molecules, and biochemical effectors, and downstream effects, that cause immune dysfunction and hyperinflammation. In another aspect, the TPA compounds and methods of the present invention interact with, induce, or promote beneficial immune regulatory cells and their associated molecules and biochemical effectors, thereby beneficially modifying their downstream effects. Similarly, the TPA compounds inhibit, prevent, promote, and / or modify other immune and inflammatory targets known to mediate associated immune dysfunction and hyperinflammatory conditions, such as cytokine storm syndrome (CSS), pediatric inflammatory multisystem syndrome (PIMS), extrapulmonary systemic hyperinflammatory syndrome (ESHS), and the vascular congestion and thrombotic conditions associated with severe COVID-19-associated ARDS (e.g., disseminated intravascular coagulation (DIC), thrombosis, stroke, thrombocytopenia, and gangrene, as well as other cellular, tissue, and organ injury associated with these conditions).

[0192] This disclosure, along with the inventors' ongoing pilot studies, illustrates the broad-spectrum immunostimulatory and anti-inflammatory effects of TPA, which may jointly mediate the reduction and / or prevention of ARDS conditions and symptoms, as well as other pathogenic conditions and symptoms associated with severe COVID-19 disease. In this context, TPA compounds possess multiple demonstrated immunostimulatory and anti-inflammatory activities mediated by key immune and inflammatory regulatory cells, genetic targets, and molecular and biochemical targets, such as cytokines, chemokines, and kinases. Exemplary pilot studies have demonstrated that TPA compounds effectively treat and prevent lymphopenia by promoting a full range of immunostimulatory effects related to lymphocyte activation, proliferation, and survival. Further teachings suggest that TPA compounds administered to SARS-CoV-2 subjects exhibiting ARDS clinically support the normal health, function, and survival of lymphocytes, including T cells, B cells, and NK cells, which are known to be critically impaired and reduced in number in severe COVID-19 / ARDS cases. In a related context, TPA was shown to exert a proapoptotic effect through interactions with immune regulatory kinases such as MAPK and JAK, which may potentially correct the aberrant programming and activation of neutrophils implicated as key effectors in the pathogenesis associated with severe COVID-19 disease or in ARDS, CSS, PIMS, ESHS, and other conditions. Neutrophils are apparently "hijacked" and reprogrammed by SARS-CoV-2 proteins (and other emerging proinflammatory factors that emerge during the course of COVID-19 disease development), leading to profound dysregulation of neutrophil immune and inflammatory activity.This includes an array of excessive inflammatory activity of neutrophils and their upstream regulator cells and signals, such as elevated proinflammatory cytokines and chemokines; increased neutrophil and macrophage migration (extravasation) into the lung and alveolar compartments (facilitated by disruption of vascular and endothelial barriers); excessive pathogenic neutrophil and macrophage activity, including tissue- and extracellular matrix-destructive degranulation, increased cellular and tissue oxidative stress (e.g., due to excessive release of peroxides and other ROS), excessive phagocytosis, and alveolar-associated neutrophil extracellular traps (NETs), as well as vascular congestion and thrombosis; and dysregulation of normal neutrophil and macrophage apoptosis (perhaps in part due to disruption of spontaneous apoptosis mediated by the SARS-CoV-2 N protein and inhibition of phagocytosis-induced cell death (PICD)), which contributes significantly to COVID-19 tissue and organ pathogenesis by abnormally extending neutrophil and macrophage lifespan and extending and otherwise dysregulating inflammatory activity.

[0193] Yet another activity of TPA, confirmed by our pilot study, demonstrates its remarkable ability to block and reduce excessive proinflammatory cytokine activation, which is critically associated with CSS and ARDS associated with severe COVID-19 disease. TPA compounds block or impair the over-induction, over-synthesis, and over-activity of proinflammatory cytokines directly linked to CSS and ARDS via various targets, mechanisms, and pathways, as described. Due to these activities, our evidence indicates that the anti-inflammatory TPA compounds and methods of the present invention will also block and impair the hyperinflammatory sequelae of SARS-CoV-2 infection, such as lymphopenia (e.g., by blocking or inhibiting proinflammatory hyperactivation of T, B, and NK lymphocytes, thereby reducing activation-induced lymphocyte cell death), and the excessive infiltration, overactivation, and elevated numbers of destructive macrophages and neutrophils in the lung parenchyma, alveolar air spaces, and other tissues and organs affected by CSS. These anti-inflammatory effects of TPA, which limit the expression and activity of pro-inflammatory cytokines, will consequently mediate a reduction in COVID-19 / CSS / ARDS-associated pathogenic states, including oxidative stress; disruption of endothelial and epithelial barriers; fibrosis and other inflammatory injuries in the lungs and other tissues and organs affected by CSS; inflammatory injury, loss, and atrophy of lymph nodes; inflammatory injury and atrophy of the spleen; sepsis; toxic shock syndrome (TSS) and other lesions.

[0194] In keeping with the inventors' goal of being unreliable, the inventors have provided representative scientific examples, explanations, and analyses throughout this specification, along with citations that introduce the reader to publications that provide additional technical descriptions of known and available research and diagnostic assay tools and methods, including all in vitro cell-based biochemical molecular and gene regulation assays, as well as in vivo research and diagnostic clinical assays. These research assays and clinical diagnostic tools and methods are followed by detailed descriptions herein and cross-reference citations to supporting scientific articles. Each of the publications cited herein is incorporated by reference for all purposes, including as supplementary references to research and clinical protocols and technical materials and methods directed to the purpose, which are known and readily implemented in the art.

[0195] To further clarify the understanding and practice of the invention herein, the inventors are currently extending their pilot studies toward preclinical and clinical trials in animal and human subjects focused on the clinical use of antiviral and anti-inflammatory TPA compounds to reduce or prevent ARDS and CSS associated with SARS-CoV-2 infection.

[0196] Preclinical studies of TPA compounds for treating and preventing ARDS The efficacy of the TPA compounds and methods of the present invention in mediating multiple broad immunostimulatory and anti-inflammatory effects for treating and preventing ARDS will be further demonstrated by studies using a well-known endotoxin-induced mouse model of ARDS. Using this model, TPA compounds will be shown to reduce pulmonary histopathological changes, including neutrophil extravasation, thrombosis characterized by extravasated and clotted red blood cells in the lung parenchyma and alveolar spaces, and alveolar wall thickening. TPA compounds will also inhibit the endotoxin-induced increase in protein content found in bronchoalveolar lavage (BALF) samples from study subjects, confirming TPA's protective function against endothelial and epithelial barrier disruption. Endotoxin-induced release of proinflammatory cytokines will also be reduced in study subjects treated with TPA, as evidenced by, for example, the documented endotoxin-induced hyperstimulation of tumor necrosis factor-alpha (TNFα). These studies also show that neutrophils washed out from TPA-treated subjects produce reduced levels of reactive oxygen species (ROS), further demonstrating the anti-inflammatory, immunostimulatory (including lymphocyte protection), and barrier-protective effects of TPA compounds mediated by neutrophil-regulatory and other immune-effecting signaling cascade targets and pathways described herein. Related assays, following the extensive references provided herein, will confirm the protective effects of TPA compounds on the ability of in vitro endothelial monolayers to resist peroxide-induced barrier dysfunction. These data confirm that TPA broadly regulates pro-immune functions and inhibits or "reprograms" hyper-inflammatory functions, particularly those through direct effects on upstream signals (cytokines and chemokines) and molecular / gene regulatory and "switch" effectors (e.g., MAPK and JAK kinases) that program and drive immune and inflammatory effector cells (T, B, and NK lymphocytes, monocytes / macrophages, and neutrophils) that are implicated as playing key roles in severe COVID-19 / ARDS pathogenesis, as well as their differentiation, activation, cytokine and receptor synthesis / response activity, migration, and longevity / apoptosis.

[0197] In a mouse model of endotoxin-induced ARDS, the TPA composition is injected intravenously into the tail vein of mice 4 hours after endotoxin instillation into the lungs. At this time, the mice exhibit hypothermic shock, and the lungs already show signs of neutrophil infiltration (inflammation). This model therefore demonstrates the effect of TPA on the ongoing pathogenesis of ARDS. Mice administered the TPA compound will exhibit significantly reduced lung histopathological changes at both 24 and 48 hours after injection. The immunostimulatory and anti-inflammatory effects of TPA will be further demonstrated by reduced protein extravasation in BALF samples at 48 hours, including reduced TNFα content in these samples. Neutrophils washed from the bronchoalveolar compartment of TPA-administered study mice will exhibit significantly reduced ROS generation after 48 hours, and endothelial monolayers preincubated with TPA will also exhibit increased resistance to peroxide-induced barrier dysfunction.

[0198] Study Design: To evaluate the effects of TPA on endotoxin-induced lung injury, LPS-endotoxin or saline was delivered to 20–25 g isoflurane-anesthetized C57Bl / 6 mice as previously described (Zhang et al., 2013). To assess dose-dependent safety and efficacy, nested TPA dosage ranges were selected as described above for different study groups and injected into the tail vein 4 h after LPS administration. Animal temperatures were determined every 2 h. Animals were sacrificed for analysis 24–48 h later.

[0199] To assess vascular leakage and pulmonary inflammation, Evans Blue Dye (EBD)-albumin conjugate (0.5% EBD in 4% BSA solution) was administered (30 mg / kg) into the tail vein 1 h before the end of the experiment. In anesthetized animals, the chest cavity was opened and blood was collected by cardiac puncture to determine the level of circulating EBD. The lungs were lavaged for gross pathology, histopathology, and collection of lavage samples.

[0200] This study design allows for tracking of TPA efficacy when therapeutic intervention is initiated after the first signs of ARDS. To further correlate the study parameters with human ARDS status and responses, animal body temperature and neutrophil infiltration in BALF are closely monitored. Mice undergo a hypothermic response that peaks 4–6 h after LPS administration. Total white blood cell (WBC) and neutrophil cell counts are measured in BALF after saline or LPS administration. Within 4 h of LPS administration, the presence of neutrophils in BALF from TPA-treated controls will already be significantly reduced compared to saline-injected control subjects.

[0201] To further evaluate ARDS pathogenesis in this model, hematoxylin and eosin staining of lung sections will also demonstrate TPA inhibition of hyperinflammatory lung pathogenesis. As LPS-induced lung inflammation progresses to a more severe stage at 48 hours, TPA-treated subjects will show a significant reduction in neutrophils and RBCs entering the lung interstitial and alveolar air spaces, as well as reduced alveolar wall swelling.

[0202] The effects of TPA on lung permeability and neutrophil infiltration would also correlate with reduced levels of extravasated proteins and cytokines in treated subjects, as indicated by TPA inhibition of LPS-induced EBD extravasation, and reduced monocyte / macrophage and neutrophil counts in BALF and histological samples of lung interstitium and alveolar air spaces. The balance between macrophages, lymphocytes, and neutrophils in BALF from TPA-treated mice would also indicate significant protective activity of TPA on lymphocytes (to ameliorate the lymphopenia seen in ARDS) and suppression of overstimulation and proapoptotic control of macrophage and neutrophil populations (which would reduce the total number of these cells and their ratio to WBCs).

[0203] To further characterize the efficacy of TPA on ARDS, the ROS-producing capacity of BALF WBCs from TPA and control mice will be measured. WBC ROS production will be significantly lower in TPA-treated mice.

[0204] This model will also help elucidate the effects of TPA on proinflammatory, immunostimulatory, anti-inflammatory, and other cytokine effects associated with ARDS disease development and protection. Endotoxin-induced ARDS subjects in this model exhibit a profound hyperinflammatory increase in proinflammatory cytokines, including IL-10. TPA will block or reduce the excessive elevation of IL-6, IL-10, TNFα, MIP2, and other proinflammatory cytokines measured in BALF and histological samples from these subjects.

[0205] To assess the protective effects of TPA on endothelial and epithelial barrier integrity and protection from oxidative stress, the barrier function of endothelial and epithelial monolayers is directly assessed. In one protocol, human pulmonary artery endothelial cells (HPAECs) are grown as monolayers on collagen-treated polyester inserts in the presence of TPA in the lower chamber. Prior to analysis, the inserts are mirrored onto fresh wells to avoid direct ROS capture. The upper chamber is loaded with FITC-dextran, and the monolayers are stimulated with 250 mM H2O2, an edema-forming product of neutrophil oxidative burst. Significant HPAEC barrier dysfunction (as evidenced by transendothelial electrical resistance (TER) studies) is observed in control subjects in response to H2O2, which is significantly attenuated or blocked by TPA treatment of ASCs.

[0206] The following exemplary protocols and materials are currently being implemented to advance exemplary aspects of the present invention.

[0207] Scope of work in-life: TPA / ARDS studies in mice 1. Purpose 1.1. To determine the efficacy of the inflammatory control drug 12-O-tetradecanoylphorbol-13-acetate (TPA) to prevent pulmonary hyperinflammation in a mouse model of adult respiratory distress syndrome (ARDS). 2. Materials and Methods 2.1. Test and Control Articles / Cells in Ready-to-Administer or Stock Concentrations 2.2. Cell Product Thawing and Dilution Instructions; Storage and Stability Information 2.3. The research protocol must be approved by the sponsor prior to the start of the study. Target Data / Submissions 3.1. In-Life Draft Report with all study data Individually tabulated and QC'd raw animal observation data, in-life observations, dose group assignment, body weights and adverse events. Autopsy findings and macroscopic evaluation 3.2. Histopathology report TIFF2023528804000027.tif192167TIFF2023528804000028.tif165166TIFF2023528804000029.tif121166TIFF2023528804000030.tif108166

[0208] Demonstration that TPA treatment regulates and attenuates immune / inflammatory responses, including by biasing these responses toward Th1 activation relative to Th2 activation. TPA may minimize the likelihood of severe Covid-19 disease by controlling and attenuating virus-induced hyperimmune and hyperinflammatory activation, including CSS (by blocking or reducing the overexpression of proinflammatory cytokines associated with the overactivation of inflammatory signaling and cellular responses). In certain embodiments, TPA mediates attenuated, immunoselective blunting of Th2 T cell responses associated with hyperimmune and hyperinflammatory activity, while mediating neutral or enhancing effects on beneficial Th1-biased T helper cell differentiation and marker expression. Various assays may be useful to demonstrate that administration of TPA to animal or human subjects mediates a controlled / attenuated immune response, typically one that is biased toward a Th1-specific cytokine / chemokine / growth factor expression pattern compared to a Th2-specific cytokine / chemokine / growth factor expression pattern (compared to patterns determined among untreated control subjects). In one exemplary assay, peripheral blood mononuclear cells (including cells and / or tissues) from subjects (i.e., those) induced with ARDS, infected with SARS-CoV-2, or exposed to hCoV pseudoviruses, with or without TPA treatment (i.e., treatment before, during, or after ARDS induction or virus or pseudovirus exposure / activation), are analyzed to assess marker expression, histology, histochemistry, and immune cell differentiation in TPA-treated and untreated control samples. In certain assays, PBMC samples are isolated from animal subjects after ARDS induction (e.g., using endotoxin induction) in the absence of virus, while another useful assay would utilize samples from subjects exposed to hCoV-2 or from animal or human subjects exposed to CoV pseudoviruses to assess Th1-biased versus Th2-biased T cell differentiation or marker expression. In an exemplary study, cellular immune responses in TPA-treated test subjects and untreated control model subjects can be assessed using a multicolor T cell ELISpot (such as that provided by CTL Laboratories).CD4+ Th1 responses can be identified, for example, by measuring IFN-γ, while CD4+ Th2 responses can be identified by measuring IL-5, among other useful activation / differentiation markers. TPA mediates a controlled and attenuated immune / inflammatory response biased toward Th1 T cell activation / differentiation and characterized by blunted Th2 activation / differentiation compared to controls, which will reduce the hyperimmune and hyperinflammatory activation associated with CSS and ARDS.

[0209] The enzyme-linked immune absorbent spot (ELISpot) is a highly sensitive and specific assay that quantitatively measures the frequency of cytokine or immunoglobulin secretion by single cells. ELISpot has been widely applied to investigate specific immune responses in infections, cancer, allergies, and autoimmune diseases. With a detection level as low as 1 cell per 100,000, ELISpot is one of the most sensitive cellular assays currently available. The FluoroSpot assay is a variant of the ELISpot assay that uses fluorescence to analyze multiple cytokines in a single well. ELISpot assays are performed in 96-well plates and analyzed using an automated ELISpot reader. Therefore, this assay is robust, easy to perform, and suitable for large-scale testing. T cell ELISpot has been widely applied in studying specific immune responses in infectious diseases, cancer, allergies, and autoimmune diseases. In the present invention, the T cell ELISpot assay is particularly useful for guiding the development and monitoring the efficacy and safety of TPA and related compounds that mediate healthy, balanced, and attenuated immune and inflammatory responses to SARS-CoV-2 and other respiratory viral infections to reduce the risk and incidence of ARDS and other related conditions described herein (e.g., pneumonia, vasculitis, thrombosis, etc.).

[0210] CSS assay Additional assays will elucidate the potent activity of TPA in preventing CSS and the associated harmful hyperimmune and / or hyperinflammatory responses that contribute to ARDS in patients with COVID-19. Because the complex and uncertain immune and inflammatory interactions associated with SARS-CoV-2 infection and COVID-19 disease remain to be fully understood, the target of these assays will be fundamental, focusing on the potential of Ii-Key-SARS-CoV-2 peptides to induce the expression of CSS and associated pro-inflammatory cytokines.

[0211] An exemplary cytokine assay utilizes a modified cytometric bead array (CBA) screen that uses a flow cytometry system adapted to simultaneously quantify multiple cytokines in, for example, cell culture supernatants (SN) or biological fluids such as serum or plasma. The CBA system uses the wide dynamic range of fluorescence detection provided by flow cytometry along with antibody-coated beads for efficient analyte capture. Because each bead in the array has a unique fluorescent intensity, beads capturing different analytes can be mixed and run simultaneously in a single tube. This method significantly reduces sample volume and time to results compared to traditional ELISA and Western blot techniques.

[0212] Briefly, target cytokines are captured from lysates, serum, or supernatants by capture antibodies conjugated to beads. Fluorescently labeled detection antibodies bind to the various captured cytokines, and fluorescent signals are recorded during sample acquisition by a flow cytometer. The intensity of the signal depends on the concentration of each cytokine and can be used to calculate the concentration of a specific cytokine using a protein standard curve. By using beads of different sizes and fluorescence to capture different cytokines, the signals recorded for different cytokines can be distinguished by flow cytometric analysis for multiple analytes in a single test sample.

[0213] The fluorescent signal provided by each cytokine captured by the antibody-coated beads is defined as the mean fluorescence intensity (MFI). This value can be converted to the concentration of each cytokine in the test sample using a standard curve generated by measuring the MFI from standards (samples with known concentrations of a given analyte). If cells secrete cytokines after ARDS induction or viral or pseudovirus exposure, increased cytokine concentrations will be detected in the cell culture supernatant, allowing comparison of TPA-treated values ​​with control values ​​to demonstrate that TPA substantially reduces the activation / expression of multiple pro-inflammatory cytokines that would otherwise be activated or elevated (in control subjects / samples) in response to, e.g., ARDS induction and / or natural or artificial viral infection.

[0214] The utility of these screening assays to demonstrate the efficacy and optimal dosing of TPA compounds will enable routine prophylactic and therapeutic treatments to prevent or reduce hyperimmune and hyperinflammatory responses to SARS-CoV-2 and other viral infections in humans. These assays are expected to further demonstrate that TPA may be useful in conjunction with SARS-CoV vaccination programs to reduce the risk of vaccine-induced hyperimmune or inflammatory responses. In particular, TPA may be effective in reducing "antigen-dependent enhancement" (ADE) in SARS-CoV-2 vaccine recipients. ADE has been shown to contribute to an increased risk of hyperimmune and hyperinflammatory responses in vaccine recipients upon subsequent natural exposure to the vaccine's target virus compared with unvaccinated subjects. This has been demonstrated in vaccine recipients for dengue and other viruses and is expected to be a potential complication of coronavirus vaccine programs. It has been noted that more severe COVID-19 cases are associated with a higher incidence of prior endemic hCoV virus infection (including regular 2-3 year cycles in adults with any of the four endemic hCoVs that cause the common cold, which cross-react with SARS-CoV-2 and may contribute to ADE in older, more exposed adults). The same tendency toward hyperimmune and hyperinflammatory responses upon subsequent wild-type virus challenge may occur after COVID-19 vaccination, after vaccine efficacy wanes (following the expected decline in neutralizing anti-SARS-CoV-2 antibodies in vaccine recipients), and may also accompany booster vaccinations. TPA treatment may attenuate these aberrant ADE responses, potentially mitigating the risk or severity of ADE in vaccine recipients who exhibit diminished immunity and are at risk for natural SARS-CoV-2 infection, as well as during booster vaccinations.

[0215] It is well established that increased and upregulated expression of proinflammatory cytokines and chemokines (CSS) mediates excessive organ and tissue damage in ARDS, including that associated with severe COVID-19 disease. CSS is often observed during the acute phase of inflammatory and infectious diseases. Cytokines that are reported to be upregulated in patients with severe COVID-19 disease and likely contribute to organ and tissue damage include, among others, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, CCL2 (MCP-1), CXCL9 (Mig), and CXCL10 (IP-10). Functional roles of proinflammatory cytokines in CSS and tissue / organ damage include, for example: IL-6, IL-8, CCL2 - Activation / recruitment of neutrophils and monocytes IL-17 - Tissue inflammation in autoimmune diseases (MS, IBD) CXCL chemokines (e.g. Mig, IP-10) - Recruitment of NK and T cells to organs / tissues.

[0216] To calibrate and clarify CBA assay results for use in the present invention (e.g., to elucidate and fine-tune TPA efficacy and dosage to reduce the likelihood of CSS), expression levels of a panel of proinflammatory cytokines known to be associated with CSS and ARDS in severe COVID-19 patients are tested and compared between test and control subjects (e.g., cells or individual subjects from animals in which ARDS has been induced or from animals or humans infected with SARS-CoV-2 or pseudoviruses) as described. The panel of proinflammatory cytokines tested would include, for example, human IL-6, IL-8, IL-10, IL-17, IFN-γ, TNF, MCP-1 (CCL2), and Mig (CXCL9). Assays are performed according to well-known, published methods and often include establishing an exemplary profile of baseline proinflammatory cytokines measured in healthy control samples and elevated expression levels for each of the inflammatory cytokines of interest measured in untreated test subjects.

[0217] Clinical studies of TPA compounds for treating and preventing ARDS in humans Clinical studies of the antiviral and anti-ARDS efficacy of TPA compositions and methods in humans will focus directly on SARS-CoV-2 positive subjects who are at elevated risk for or exhibit one or more symptoms of hyperinflammation, CSS, and / or ARDS at the start of the study, as described above. The U.S. Centers for Disease Control and Prevention (CDC) recommends diagnosing SARS-CoV-2 infection using specimens from both the upper respiratory tract (e.g., using nasopharyngeal or oropharyngeal swabs) and the lower respiratory tract (either endotracheal tube or bronchoalveolar lavage). Diagnosis of COVID-19-associated ARDS will primarily be based on RT-PCR analysis of specimens. If RT-PCR is not available, serological tests will be used. Currently, the U.S. Food and Drug Administration (FDA) has approved Roche's SARS-CoV-2 commercial test system (cobas® SARS-CoV-2). This qualitative test requires specimens from nasopharyngeal or oropharyngeal swabs and takes 3.5 hours to obtain results. The RT-PCR-based cobas SARS-CoV-2 test is a dual-target assay that detects both specific SARS-CoV-2 RNA and a highly conserved fragment of the E gene, which is invariant in all members of the Sarbecovirus subgenus. The assay includes full-process negative, positive, and internal controls to ensure specificity and accuracy. On March 21, 2020, the FDA granted another emergency use authorization to Cepheid Inc.'s (USA) Xpert® Xpress SARS-CoV-2, a qualitative test that can provide results within 45 minutes. The test can utilize samples from nasopharyngeal swabs, nasal washes, or aspirates and features automated sample processing, eliminating the need for manual intervention. A result should be considered positive if two or more target genes are present and detected.

[0218] Current SARS-CoV-2 screening methods rely on the abundance of viral genomes in samples, but studies have shown that IgM antibody levels are high in both symptomatic and asymptomatic patients 5 days after the onset of illness. Therefore, combining an IgM ELISA assay with PCR can enhance detection sensitivity and improve both the research and practical clinical methods described herein (e.g., to refine decisions aimed at optimizing TPA administration for individual patients).

[0219] Patient management standards in our human TPA clinical research protocol emphasize the importance of supportive care to prevent complications and nosocomial infections. When patients experience respiratory distress, oxygen or ventilator support is immediately administered. Fluid resuscitation is limited to avoid worsening pulmonary edema and hypoxemia unless there are signs of tissue hypoperfusion. Standard precautions, including respiratory and eye protection, are recommended for all research staff. Lifting of these precautions is permitted only if a particular study subject has two consecutive negative RT-PCR tests at least 24 hours apart (indicating clinical recovery / clearance of the SARS-CoV-2 virus).

[0220] The initial focus of human clinical trials of TPA in SARS-2-mediated ARDS will be on consistent and easily assessable diagnostic indices, including radiological features that correlate with COVID-19 disease severity. Radiologic procedures and interpretation protocols for assessing COVID-19 / ARDS severity, including chest radiography (CXR) and chest computed tomography (CT) scans, are well known and have been widely published, including imaging findings for COVID-19, SARS-CoV, and MERS-CoV-associated pneumonia. The majority of SARS-CoV-2 ARDS patients (up to 75%) exhibit bilateral pneumonia, while the remainder are unilateral. Approximately 14% of patients exhibit multiple patchy and ground-glass opacities. These abnormalities are predominantly peripheral (54%), but may also be present in the lower lobes or be ill-defined. In more severe cases, multiple bilateral consolidations are typically present.

[0221] Chest CT scans more efficiently detect pneumonia in the early stages of COVID-19. The most common patterns of COVID-19 on chest CT scans include multiple glomerular occlusions (56.4%) and bilateral spotty opacities (51.8%). Other patterns consist of focal spotty opacities (28.1%) and interstitial abnormalities (4.4%). Severe cases have more prominent radiological findings on chest CT scans, such as more bilateral spotty opacities (82%), more multiple glomerular occlusions (60%), and more focal spotty opacities (55.1%) compared with non-severe cases. CXR or chest CT abnormalities were not identified in 17.9% of non-severe cases and 2.9% of severe cases. Pure glomerular occlusions can be observed in the early stages. Focal or multifocal GGO lesions may progress to consolidation or GGO lesions with superimposed interlobular / intralobular septal thickening as a crazy-paving pattern during disease progression, and expansion of the consolidation represents disease progression. Pure consolidation lesions are relatively rare. Although rare, pulmonary cavitation lesions, pleural effusion, and lymphadenopathy have also been reported.

[0222] As additional human clinical studies move forward, these pulmonary pathogenicity data will be collected for TPA-treated and control COVID-19 / ARDS subjects, along with parallel data on the manifestations of COVID-19 / ARDS severity and TPA efficiency, as described above.

[0223] While the foregoing invention has been described in detail by way of example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims, which are provided by way of illustration and not limitation. In this regard, the above disclosure has made explanatory and comprehensive references to various publications and other references. Each of these references is incorporated herein in its entirety for all purposes.

[0224] References TIFF2023528804000031.tif218165TIFF2023528804000032.tif224166TIFF2023528804000033.tif221165TIFF2023528804000034.tif224165TIFF2023528804000035.tif119164

Claims

1. A pharmaceutical composition for use in a method for treating or preventing sudden acute respiratory syndrome (SARS) coronavirus infection in a mammalian subject, comprising an antivirally effective amount of a TPA compound, The method comprises administering to a subject at increased risk of SARS-coronavirus infection or a subject exhibiting active SARS-coronavirus infection an antivirally effective amount of a TPA compound to induce a significant antiviral response in the subject that is clinically therapeutic or prophylactic sufficient to prevent or reduce SARS viral infection and / or reduce or eliminate the amount of SARS viral load detectable in the subject.

2. 2. The pharmaceutical composition of claim 1, wherein the SARS coronavirus is a human SARS (hSARS) coronavirus selected from SARS-CoV-2 (COVID-19), SARS-CoV, and Middle East Respiratory Syndrome (MERS) coronavirus.

3. The pharmaceutical composition of claim 2, wherein the hSARS coronavirus is SARS-CoV-2 (COVID-19).

4. The pharmaceutical composition of claim 1, wherein the antiviral TPA compound is effective to induce an antiviral response in a subject sufficient to prevent or reduce SARS-CoV-2 viral infection and / or reduce or eliminate SARS viral load, while preventing or reducing one or more clinical symptoms of COVID-19 disease.

5. The TPA compound is (1) The amount or titer of virus in cells, tissues, or samples from the subject's upper or lower respiratory tract; (2) the amount or titer of virus in the subject's non-respiratory ACE-2 positive cells, tissues or samples, or in the subject's plasma; (3) viral attachment and / or entry into the lungs or other tissues / cells; (4) viral replication in the lungs or other ACE-2 positive cells, tissues or organs of the subject; and / or (5) Viral shedding from upper respiratory tract tissues or samples from infected subjects and administering to a patient in need of treatment an amount and dosage form effective to reduce or eliminate one or more indexes of severity of SARS coronavirus infection selected from the group consisting of: Each of the indices may be determined by observing or measuring the occurrence or value of a subject index in one or more TPA-treated subjects compared to the occurrence or value of the same index in one or more corresponding placebo-treated control subjects; 2. The pharmaceutical composition of claim 1.

6. the SARS coronavirus is a human SARS (hSARS) coronavirus selected from SARS-CoV-2 (COVID-19), SARS-CoV, and Middle East Respiratory Syndrome (MERS) coronavirus; the TPA compound is effective in reducing the amount or titer of hSARS virus in the upper and / or lower respiratory tract of the subject; 2. The pharmaceutical composition of claim 1.

7. the SARS virus is a human SARS (hSARS) coronavirus selected from SARS-CoV-2 (COVID-19), SARS-CoV, and Middle East Respiratory Syndrome (MERS) coronavirus; the TPA compound is effective in reducing the amount or titer of hSARS virus in non-respiratory ACE-2 positive cells, tissues or organs of the subject; 2. The pharmaceutical composition of claim 1.

8. The SARS virus is SARS-CoV-2 (COVID-19), the antiviral TPA compound is effective to induce an antiviral response in a subject that prevents or reduces SARS-CoV-2 viral infection and / or reduces or eliminates SARS viral load as determined by SARS-CoV-2 DNA or other quantitative measure of SARS-CoV-2 levels in nasopharyngeal swab samples taken pre-treatment and post-treatment from an infected subject or by comparison of appropriate test and control samples; 2. The pharmaceutical composition of claim 1.

9. 10. The pharmaceutical composition of claim 8, wherein viral load, as determined by SARS-CoV-2 DNA in nasopharyngeal swab samples or other quantitative measures of SARS-CoV-2 viral load, is reduced by an average of 25-50% or more in treated subjects compared between pre-treatment and post-treatment samples from individual treated patients or groups of treated patients, or between treated subjects and placebo-treated control subjects.

10. 10. The pharmaceutical composition of claim 8, wherein viral load, as determined by SARS-CoV-2 DNA in nasopharyngeal swab samples or other quantitative measures of SARS-CoV-2 viral load, is reduced by an average of 75-95% or more in treated subjects compared between pre-treatment and post-treatment samples from individual treated patients or groups of treated patients, or between treated subjects and placebo-treated control subjects.

11. The pharmaceutical composition of claim 8, wherein viral load, as determined by SARS-CoV-2 DNA or other quantitative measure of SARS-CoV-2 load in nasopharyngeal swab samples from subjects who screened positive for SARS-CoV-2 infection prior to TPA treatment, is reduced by 100% (corresponding to complete clearance of detectable SARS-CoV-2 virus in the upper respiratory tract and indicating a non-contagious status) in at least 50% of TPA-treated subjects within 2 weeks after TPA treatment.

12. The antiviral TPA compound is a compound of formula I: and antivirally active analogs, derivatives, complexes, conjugates, salts, enantiomers and mixtures thereof; During the ceremony, R 1 and R 2 but, hydrogen; The alkyl group contains 1 to 15 carbon atoms. ; and substituted derivatives thereof; R 3 But hydrogen, or a substituted derivative thereof, In the above formula, "lower alkyl" or "lower alkenyl" can contain 1 to 7 carbons, can be straight or branched chain, and can be unsubstituted or substituted with chlorine, fluorine or other halogens, or nitro, amino or other active functional groups.

2. The pharmaceutical composition of claim 1.

13. The antiviral TPA compound has the following formula II: and antiviral active analogs, derivatives, complexes, conjugates, salts, enantiomers and mixtures thereof.

14. The antiviral TPA compounds are phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate; phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol 12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate; phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol 12-myristate; phorbol 13-myristate; phorbol 12,13,20-triacetate; 12-deoxyphorbol 2. The pharmaceutical composition of claim 1, wherein the phorbol ester is selected from phorbol 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol 13-isobutyrate; 12-deoxyphorbol 13-isobutyrate-20-acetate; 12-deoxyphorbol 13-phenylacetate; 12-deoxyphorbol 13-phenylacetate 20-acetate; 12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate 13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; phorbol 13-acetate; and antiviral active analogs, derivatives, complexes, conjugates, salts, enantiomers and mixtures thereof.

15. 2. The pharmaceutical composition of claim 1, wherein the antiviral TPA compound is 12-O-tetradecanoylphorbol-13-acetate.

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