WO2022212914A1

WO2022212914A1 - Methods of treating viral diseases

Info

Publication number: WO2022212914A1
Application number: PCT/US2022/023162
Authority: WO
Inventors: David Bar-Or; Gregory Thomas
Original assignee: Ampio Pharmaceuticals, Inc.
Priority date: 2021-04-01
Filing date: 2022-04-01
Publication date: 2022-10-06

Abstract

The present disclosure provides methods of treating a TLR7- and/or TLR-mediated disease and also methods of treating disease in a multi-modal treatment regimen. The methods comprise administering an effective amount of a pharmaceutical composition prepared by removing albumin from a solution of a human serum albumin composition and/or comprising a diketopiperazine with amino acid side chains of aspartic acid and alanine (DA-DKP), such as a low molecular weight fraction of human serum albumin.

Description

METHODS OF TREATING VIRAL DISEASES

FIELD

The present disclosure relates to methods of treating TLR7/8-mediated disease, including viral respiratory disease. The methods comprise administering an effective amount of a pharmaceutical composition prepared by removing albumin from a solution of a human serum albumin composition and/or comprising a diketopiperazine with amino acid side chains of aspartic acid and alanine (DA-DKP), such as a low molecular weight fraction of human serum albumin.

BACKGROUND

Toll-like receptors (TLRs) are key pattern recognition complexes that help the immune system respond to pathogens based on unique microbial molecular structures. However, tissue damage can result in the release of cellular components that mimic these molecular motifs and has been shown to play a role in the pathophysiology of diseases through the activation of TLRs.

Treatments that modulate and/or interrupt TLR signaling may prove beneficial for the treatment of diseases, for example when immune cells are activated by antiviral mechanisms that may help drive hyperinflammation in patients with viral infections, especially respiratory viruses. For example, mounting evidence suggests that clinical outcomes vary greatly for coronavirus disease of 2019 (COVID-19) depending on how the immune system responds to severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) infection. In its mildest forms, SARS-CoV-2 replication is primarily localized to the upper respiratory tract, with limited innate immune response and low viral burden. However, underlying factors such as genetic polymorphisms, autoantibody development, and intrinsic viral mechanisms have been identified that serve to suppress or delay type I interferon production or activity during the course of infection. The resulting maladaptation in antiviral immunity helps drive progression into more severe stages marked by migration of the virus into the lower respiratory tract, elevated viral loads, and dramatic loss of type II pneumocytes.

As the disease enters these phases, ongoing activation of TLRs leads to concomitant activation of innate immune effector mechanisms, including the production of pro- inflammatory cytokines, and up-regulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer (NK) cells. Upregulation of pro- inflammatory cytokines like CXCL10 become predictive of the clinical course, and approximately 20% of these patients go on to develop acute respiratory distress syndrome (ARDS). Moreover, these critical stages are characterized by increased plasma levels of pro- inflammatory cytokines and chemokines, such as IFNy, CXCL10, IL-Ib, and TNFa, indicative of “cytokine storm” development that can eventually lead to multiorgan failure and death. Thus, it appears that failure of the immune system to mount an appropriate antiviral response early in infection drives a pathology involving excessive tissue damage, CD4+ T-cell helper type 1 (Thl) inflammation, and fibrosis.

Diketopiperazines have been reported to exhibit a variety of biological activities. See, e.g, U.S. Patent Nos. 4,289,759 (immunoregulatory agents), 4,331,595 (immunoregulatory agents), 4,940,709 (PAF antagonists), 5,700,804 (inhibitors of plasminogen activator inhibitor), 5,750,530 (inhibitors of plasminogen activator inhibitor) and 5,990,112 (inhibitors of metalloproteases); PCT publication nos. WO 97/36888 (inhibitors of farnesyl-protein transferase) and WO 99/40931 (treatment of central nervous system injury); EP Patent No. 0043219 (immunoregulatory agents); Japanese patent application nos. 63 290868 (PAF antagonists) and 31 76478 (immunosuppressive agents); and Shimazaki et ak, Chem. Pharm. Bull., 35(8), 3527-3530 (1987) (PAF antagonists), Shimazaki et ak, J. Med. Chem., 30, 1709-1711 (1987) (PAF antagonists), Shimazaki et ak, Lipids, 26(12), 1175-1178 (1991) (PAF antagonists), Yoshida et ak, Prog. Biochem. Pharmacol., 22, 68-80 (1988) (PAF antagonists), Alvarez et ak, J. Antibiotics, 47(11), 1195- 1201 (1994) (inhibitors of calpain).

SUMMARY

Disclosed herein are compositions and methods useful to treat diseases mediated by TLR7 and/or TLR8, which recognize single-stranded (ssRNA) RNA viruses. The disclosed compositions and methods are useful to treat symptoms caused by infection with ssRNA viruses and viruses recognized by TLR7 and/or TLR8. For example, the compositions and methods disclosed herein are useful to treat inflammation caused by infection with a virus, for example a respiratory virus and/or a ssRNA respiratory virus. The compositions and methods disclosed herein are also useful to treat other diseases mediated by TLR7 and/or TLR8, such as lupus and/or lupus nephritis. Also disclosed herein are compositions and methods useful to treat or prevent one or more symptoms of a viral infection using a biphasic or multi-modal treatment method, including treating inflammation during a hyperinflammatory stage of a TLR7- and/or TLR8- mediated disease. According to such methods, treatment of a patient occurs by administration of a first drug, during the acute phase of the disease, followed by administration of a pharmaceutical composition comprising DA-DKP before, at or after the onset of a hyperinflammatory stage of the disease. In various aspects, upon administration of the pharmaceutical composition the first drug and the pharmaceutical composition are co-administered to the patient. In various aspects, the first drug is an antiviral, an immune- modifying drug, an anti-depressant, a corticosteroid, or combinations thereof, among other things.

In a first aspect, a method of treating one or more symptoms of a viral infection in a patient is provided. The method comprises administering a first drug to the patient prior to onset of a hyperinflammatory stage of the infection and administering a pharmaceutical composition comprising DA-DKP to the patient before, at, or after the onset of the hyperinflammatory stage.

In a second aspect, a method of treating inflammation during a hyperinflammatory stage of a TLR7- and/or TLR8-mediated disease in a patient is disclosed. The method comprises administering a first drug to the patient prior to onset of a hyperinflammatory stage of the disease and administering a pharmaceutical composition comprising DA-DKP to the patient before, at, or after the onset of the hyperinflammatory stage.

In a third aspect, a method of treating or preventing one or more symptoms of a TLR7- and/or TLR8-mediated disease in a patient, by administering a pharmaceutical composition comprising DA-DKP, is disclosed.

In a fourth aspect, a method of treating or preventing inflammation associated with a TLR7- and/or TLR8-mediated disease in a patient, by administering a pharmaceutical composition comprising DA-DKP, is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and IB show data demonstrating that AMPION^® inhibits CXCL10 release from THP-1 cells. Representative CL075- and CL307-induced CXCL10 chemokine release from PMA-differentiated THP-1 cells cultured in the presence of saline diluent control or AMPION^®. Data are presented as mean pg/ml CXCL10 ± STD of three technical replicates 24 hours (FIG. 1 A) or 72 hours (FIG. IB) post stimulation. * = p-value < 0.05 vs activated saline control by student t-test.

FIGS. 2A and 2B show data demonstrating that AMPION^® inhibits CXCL10 release from THP-1 cells. Representative CL075- and CL307-induced CXCL10 chemokine release from PMA-differentiated THP-1 cells cultured in the presence of saline diluent control or AMPION^®. Data are presented as mean pg/ml CXCL10 ± STD of three technical replicates 24 hours (FIG. 2A) or 72 hours (FIG. 2B) post stimulation. * = p-value < 0.05 vs activated saline control by student t-test.

FIGS. 3A, 3B, and 3C show data demonstrating that AMPION^® inhibits CXCL10 release in THP-1 cells and in Peripheral Blood Mononuclear Cells (PBMC). Box plots for 24-hour 5 pg/ml CL075-induced CXCL10 release and AMPION^® percent inhibitions for PMA-differentiated THP-1 cells and PBMC are shown. Data are presented as CL075- induced CXCL10 pg/ml release for both saline- and AMPION^®-treatment groups from PMA-differentiated THP-1 (FIG. 3 A) or PBMC (FIG. 3B) as well as percent inhibitions in CXCL10 release observed in the AMPION^®-treatment groups calculated versus saline- treated controls (FIG. 3C). Boxes = quartile 1 to 3 boundaries, line = Median, + = Mean. For THP-1, n = 8 independent experiments. For PBMC, n = 9 independent experiments using cells derived from a total of 7 different donors.

FIG. 4 shows data from a relative potency assay of AMPION^® inhibition of CXCL10. The data show dose-dependent reduction in CL075-induced CXCL10 release from PMA-differentiated THP-1 by AMPION^®. Data presented as regression analysis of percent inhibitions ± STD in 24 hour, CL075-induced CXCL10 release versus AMPION^® serially diluted using saline diluent with concentrations listed as nominal in relation to full strength drug product n = 9 for nominal 1 or full-strength product and n = 4 for all other doses.

FIG. 5 shows canonical pathway analysis that demonstrates AMPION^® directional regulation of cytokines and chemokines are predicted to inhibit pathways associated with increased inflammation. Log2 fold-changes and p-values of cytokines and chemokines from the 48-plex cytokine array comparing saline-treated versus AMPION^®-treated PMA- differentiated THP-1 cells activated with 5 pg/ml CL075 for 24h were uploaded into IPA for canonical pathway analysis. Dark grey = pathway predicted to be activated, z-score > 2, black = pathway predicted to be inhibited, z-score < -2, white = directional regulation unable to be predicted, z = 0.

DETAILED DESCRIPTION

Definitions

The term “LMWF” refers to a Low Molecular Weight Fraction of human serum albumin (HSA) that is a composition prepared by separation of high molecular weight components from HSA. For example, LMWF can be prepared by filtration of commercially available HSA solutions wherein molecular weight components of more than 3 kDa, more than 5 kDa, more than 10 kDa, more than 20 kDa, more than 30 kDa, more than 40 kDa, or more than 50 kDa are separated from the HSA solution. In some embodiments, “LMWF” refers to a composition prepared by separation of high molecular weight components from HSA by other techniques, including but not limited to ultrafiltration, column chromatography including size exclusion chromatography, affinity chromatography, anion exchange, cation exchange, sucrose gradient centrifugation, salt precipitation, and/or sonication. In some embodiments, LMWF also refers to a composition that includes components of HSA having a molecular weight less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5000 Da, less than 4000 Da, or less than 3000 Da (corresponding to 50,000 g/mol, 40,000 g/ml, 30,000 g/mol, 20,00 g/mol, 10,000 g/mol, 5,000 g/mol, 4,000 g/mol or 3,000 g/mol, respectively).

As used herein, the terms “patient” and “subject” are interchangeable and generally refer to an animal or a human to which a composition disclosed herein is administered or is to be administered.

As used herein, the term “prodrug” refers to derivatives of a pharmacologically active drug molecule which undergo transformation within the body to produce the pharmacologically active drug, also referred to sometimes as the “parent” drug. Prodrugs are frequently, although not necessarily, pharmacologically inactive until converted to the parent drug.

A “therapeutically effective amount,” “effective amount,” or the like means the amount of a compound or composition that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The “effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated. As used herein, the terms “treat,” “treatment,” “treating,” and derivatives thereof mean to reduce (wholly or partially) the symptoms, duration or severity of a disease. In some embodiments, such terms relate to ameliorating a disease or disorder (/. ., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In other embodiments, such terms refer to ameliorating at least one physical parameter, which may or may not be discernible by the patient. In yet other embodiments, such terms refer to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In still other embodiments, “treating” or “treatment” refers to delaying the onset of the disease or disorder. In accordance with the present state of the art, treat typically does not mean to cure.

Reference will now be made in detail to particular embodiments of compositions and methods. The disclosed embodiments are not intended to be limiting of the claims.

The present disclosure generally relates to methods of treating a viral respiratory disease, for example a TLR7/8-associated disease such as COVID-19, and/or one or more symptoms thereof. In a first aspect, the methods of treatment relate to the administration of the disclosed compositions as mono-therapeutic agents to treat viral respiratory diseases. In a second aspect, the methods of treatment relate to the administration of the disclosed compositions in combination with one or more additional therapeutics for the treatment of a viral respiratory disease.

With respect to the first aspect, the methods comprise administering an effective amount of a pharmaceutical composition comprising aspartyl-alanyl diketopiperazine (DA- DKP) to a subject having a need thereof. In various aspects, the pharmaceutical composition can be prepared by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

With respect to the second aspect, the methods comprise administering an effective amount of a pharmaceutical composition comprising DA-DKP before, during, or after the initiation of a hyperinflammatory stage of viral disease to a subject having a need thereof. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

With further respect to the second aspect, in some embodiments the present disclosure generally relates to methods of treating one or more respiratory viral diseases by administering an effective amount of a pharmaceutical composition comprising DA-DKP before a hyperinflammatory stage of respiratory viral disease to a subject having a need thereof. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

In one embodiment, the present disclosure relates to methods of treating COVID-19 by administering an effective amount of a pharmaceutical composition comprising DA- DKP before a hyperinflammatory stage of COVID-19 to a subject having a need thereof. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

In other embodiments, the present disclosure relates to methods of treating viral diseases by administering an effective amount of a pharmaceutical composition comprising DA-DKP during a hyperinflammatory stage of viral disease to a subject having a need thereof. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

In one embodiment, the present disclosure relates to methods of treating COVID-19 by administering an effective amount of a pharmaceutical composition comprising DA- DKP during a hyperinflammatory stage of COVID-19 to a subject having a need thereof. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

In other embodiments, the present disclosure relates to methods of treating viral diseases by administering an effective amount of a pharmaceutical composition comprising DA-DKP to a subject after a hyperinflammatory stage of viral disease has begun. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

In one embodiment, the present disclosure relates to methods of treating COVID-19 by administering an effective amount of a pharmaceutical composition comprising DA- DKP to a subject after a hyperinflammatory stage of COVID-19 has begun. The pharmaceutical composition can be prepared as noted above, by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

DA-DKP has multiple anti-inflammatory and immune modulating effects including inhibition of multiple pro-inflammatory cytokines, chemokines and signaling molecules at the transcription level, inhibition of the migration and adhesion of T-cells and monocytes, activity at the G-coupled protein receptor level, activity on actin-dependent cytoskeletal events, and reduction in vascular permeability and inhibition of inflammation induced by platelet activating factor, among other effects. DA-DKP can be used to treat lung inflammatory diseases, inflammation related to viral respiratory diseases, and/or to prevent lung inflammation and lung inflammatory diseases related to viral respiratory diseases. DA- DKP can also be used to prevent, reduce in severity and/or duration, and/or otherwise treat cytokine storm or cytokine release syndrome. Cytokine storm or cytokine release syndrome is a systemic inflammatory syndrome characterized by elevated levels of circulating cytokines and immune cell hyperactivation. Innate immune system and Toll-like Receptors

The innate immune response plays a vital role in the detection of infection and linking acute responses to the adaptive branch. Toll-like receptors (TLR) are one of the primary mechanisms by which innate immune cells detect and acutely respond to microbial infection.

Toll-like receptors (TLRs) recognize specific molecular patterns present in molecules that are broadly shared by pathogens, but are structurally distinct from host molecules. The human genome includes 10 known TLRs. The ligands for these receptors are highly conserved microbial molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double stranded RNA (TLR3), CpG motif- containing DNA (recognized by TLR9), and profilin present on uropathogenic bacteria (TLR 11). TLR1, -2, -4, -5, and -6 respond to extracellular stimuli, while TLR3, -7, -8 and -9 respond to intracytoplasmic pathogen-associated molecular patterns (PAMPs, conserved microbial small molecular motifs). The activation of TLRs by their cognate ligands leads to activation of innate immune effector mechanisms, including the production of pro- inflammatory cytokines, and up-regulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer (NK) cells. The consequence of activation of the innate immune system mobilizes and amplifies specific adaptive immune responses involving both T- and B-cell effector functions.

Toll-like receptors -7/-8 are innate immune receptors present in the endosomal compartment that are activated by single-stranded RNA (ssRNA) molecules of viral as well as nonviral origin, inducing the production of inflammatory cytokines necessary for the development of adaptive immunity.

TLR7 is an endosomal sensor that can discriminate non-self, single-stranded ribonucleic acid sequences to trigger an anti-viral response. TLR 7 serves to detect non-self, single stranded ribonucleic acid, and trigger anti-viral immunity. However, dysregulation of TLR7 signaling has been linked to the pathology of inflammatory diseases that present with cytokine storms and acute kidney injury such as sepsis and lupus.

TLR7 in Disease

TLR7 is linked to plasma cytokine storm development, acute kidney injury, and mortality. Cytokine storm is a systemic inflammatory syndrome characterized by high levels of circulating cytokines and hyperactivation of the immune system. This can lead to multiple organ failure and even death. A cytokine storm, or cytokine storm syndrome, can be caused by many infectious and non-infectious etiologies. For example, cytokine storm syndrome can be caused by bacterial and/or viral infection, such as infection by the coronavirus SARS- CoV-2, which causes the disease COVID-19. Cytokine storm symptoms include fever, fatigue, nausea, systemic inflammation, hyperferritinemia, hemodynamic instability, organ failure, and death. Cytokine storm can damage the heart, kidneys, lungs, liver, central nervous system, and other organs, for example by causing cardiac arrest, myocarditis, myocardial injury, cardiomyopathy, arrhythmia, acute kidney injury, proteinuria, and acute respiratory distress syndrome. Furthermore, infection, for example by SARS-CoV-2, can lead to sepsis, which is associated with cytokine storm. Severe COVID-19 patients exhibit cytokine storms associated with acute kidney injury and renal dysfunction. Cytokine storm can be characterized by elevated levels of cytokines including IL-Ib, IL-7, IL-8, IL-9, IL- 10, FGF, G-CSF, GM-CSF, IFN-g, IP-10, MCP-1, MPMA, MIP1-B, PDGF, TNF-a, and VEGF.

TLR7 is also linked to the induction of serum autoantibody products that accelerate glomerulonephritis, exacerbate macrophage activation syndrome, and result in rapid mortality in murine lupus models. Lupus nephritis is linked to TLR7. Lupus nephritis is an autoimmune disease caused in part by the recognition of kidney cells by autoantibodies.

In some embodiments, compositions provided by the present disclosure are useful for the treatment of lupus, including lupus nephritis. Such embodiments are exemplified by methods that can comprise administering an effective amount of a pharmaceutical composition comprising aspartyl-alanyl diketopiperazine (DA-DKP) to a subject having a need thereof. The pharmaceutical composition can be prepared by removing albumin from a solution of a human serum albumin composition. The pharmaceutical composition can also include N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the pharmaceutical composition can comprise AMPION^® (defined herein).

TLR7/8 recognize single strand RNA (ssRNA) viruses, and inflammation caused by any single strand RNA virus can thus be treated with various aspects of the disclosure. Suitable ssRNA viruses include Coronaviridae ( e.g ., SARS-CoV, MERS, SARS-CoV-2, and toroviruses), Hepeviridae (e.g., Hepatitis E), Caliciviridae (e.g., Norovirus, formerly Norwalk virus, and Sapporo virus), Togaviridae ( e.g ., Alphavirus viral diseases (e.g., Chikungunya, Eastern equine encephalomyelitis virus, Getah virus, Mayaro virus, Mucambo virus, O’nyong’nyong virus, Ross river virus, Barmah forest virus, Sagiyama virus, Semliki forest virus, Sindbis virus, Tonate virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus) and Rubivirus (e.g., Rubella virus)), Flaviviridae (e.g., Hepacivirus (e.g., Hepatitis C), Flavivirus (e.g., Dengue virus types 1-4, Hepatitis G virus, Japanese B encephalitis virus, Murray Valley encephalitis virus, Rocio virus, Spondweni virus, St Louis encephalitis, Wesselsbron, West Nile virus (West Nile fever), and Yellow fever virus (yellow fever)), viruses in the Tick- borne virus group (e.g., Absettarov, Hanzalova, Hypr, Kumlinge, Kyasanur forest disease, Louping ill (tick-borne encephalitis), Negishi, Omsk, Powassan (tick-borne encephalitis), Langat (tick-borne encephalitis), Russian spring summer encephalitis), and the Hepatitis G virus group (e.g., Hepatitis G virus)), Picornaviridae (e.g., Enterovirus (e.g., Coxsackievirus, Echovirus, Poliovirus, Enterovirus 68-109, and Rhinovirus A and B), Hepatovirus (e.g., Hepatitis A, alternately human enterovirus type 72), Astroviridae (e.g., Astrovirus species), Mononegavirales (e.g., Henipavirus (e.g., Hendra virus and Nipah virus), Rubulavirus (e.g., Mumps virus and Parainfluenza types 2, 4a and 4b), Morbillivirus, (e.g., Measles virus), Avulavirus(e.g^·., Newcastle disease virus), Metapneumovirus, Pneumovirus (e.g., Respiratory syncytial virus), and Parainfluenza Types 1 to 4), Rhabdoviridae (e.g., Lyssavirus (e.g., Duvenhage and Rabies virus), Retroviridae (e.g., Human T-cell lymphotropic viruses) and Lentivirus (e.g., Human Immunodeficiency virus)), Arenaviridae (e.g., Arenavirus (e.g., lymphocytic choriomeningitis virus)), Bunyaviridae (e.g., Bunyavirus (e.g., Bunyamwera virus), Hantavirus (e.g., Hantaan virus), Nairovirus (e.g., Nairobi sheep disease virus, and Phlebovirus (e.g., sandfly fever Sicilian virus)), and Orthomyxoviridae (e.g., Influenza viruses (e.g., influenza viruses A,B, C, and D) and Thogoto-like viruses (e.g., Thogoto virus)). Diseases mediated by TLR7 and/or TLR8 are diseases caused by infection with ssRNA viruses recognized by TLR7 and/or TLR8, such as inflammation caused by a ssRNA virus, as well as lupus and lupus nephritis.

Inflammation in viral disease

A complication of disease caused by viral infection is overactive immune response, leading to excessive inflammation. This can lead to development of a hyperinflammatory stage of infection that is characterized by cytokine storm, acute lung injury (ALI), and/or acute respiratory distress syndrome (ARDS). Many viruses can cause excessive inflammation, cytokine storm, ALI, and/or ARDS, including respiratory viruses, Herpes simplex viruses, and cytomegaloviruses. Certain respiratory viruses are particularly likely to cause excessive inflammation and even ARDS, such as viruses that cause community- acquired pneumonia and/or pandemics. Respiratory viruses that are especially likely to cause ARDS include influenza viruses, coronaviruses, rhinoviruses, respiratory syncytial viruses, parainfluenzaviruses, human metapneumoviruses, and adenoviruses. These include influenza A viruses (including H1N1 and H3N2 strains), influenza B viruses, Severe Acute Respiratory Distress Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Distress Syndrome coronavirus 2 (SARS-CoV-2), and human adenovirus B21 infections (HAdV-B21). Other emergent viruses, especially those that cause community-acquired pneumonia and/or pandemics, are likely to also cause ARDS. In various aspects, compositions provided by the present disclosure are useful to treat ARDS caused by any virus.

Viral Respiratory Diseases

A viral respiratory disease is an illness caused by a virus that affects the respiratory tract. Such viral respiratory diseases can include Severe Acute Respiratory Distress Syndrome (SARS), Middle East Respiratory Syndrome (MERS), COVID-19, and viral infections associated with asthma, pneumonia, bronchitis and/or tuberculosis. Viruses that can cause one or more viral respiratory diseases include coronaviruses, influenza viruses, respiratory syncytial virus (RSV), parainfluenza viruses, and respiratory adenoviruses. Coronaviruses include SARS-Coronavirus-2 (SARS-CoV-2), SARS-associated coronavirus (SARS-CoV), and Middle East Respiratory Syndrome Coronavirus (MERS- CoV). Coronavirus infections and other viral infections can cause acute respiratory distress syndrome (ARDS), acute lung injury (ALI), interstitial lung disease, pulmonary fibrosis, pneumonia, and reactive airway disease syndrome. Coronavirus infections and other viral infections can cause inflammation in tissues such as lung, brain, heart, kidney, blood vessel, skin, and nerve. Coronavirus infections and other viral infections can cause symptoms such as fatigue, shortness of breath or difficulty breathing, low exercise tolerance, low blood oxygen saturation, cough, sore throat, stuffy or runny nose, joint pain, chest pain, tightness, or discomfort, muscle pain, muscle weakness, fever, heart palpitations, difficulty thinking and/or concentrating, and depression.

COVID-19 infection is a respiratory illness caused by the novel coronavirus SARS- COV-2 and has been classified as a pandemic with no known cure to date. COVID-19 is detected and diagnosed with a laboratory test. The primary symptoms of COVID-19 infection include mild symptoms such as fever, cough, chills, muscle pain, headache, gastrointestinal symptoms, and the loss of taste or smell. Once infected, the virus moves down a patient’s respiratory tract, where the lungs may become inflamed, making breathing difficult and sometimes requiring supplemental oxygen in the more severe cases of the disease.

Respiratory symptoms after a COVID-19 infection include shortness of breath, cough, chest discomfort, low exercise tolerance and low oxygen saturation, all of which point to potential inflammation-related complication sequelae. Infiltrating or resident cells in the immune system (e.g., macrophages, peripheral blood mononuclear cells, etc.) may be responsible for the development of these respiratory long-term consequences. Chronic or prolonged inflammation of the lungs may be responsible for a myriad of respiratory signs and symptoms experienced by patients after a COVID-19 infection. Chest x-rays and CT scans reveal disturbing patterns of perhaps extensive fibrosis and potential loss of elasticity and oxygen diffusion capacity.

The SARS-Cov-2 virus transmits through the respiratory system and can cause a severe dysregulation of the immune response and damage in the lungs. The hyperinflammatory state is thought to lead to prolonged clinical complications, and treatment with immunomodulators at this later point in the disease can be more effective than anti-viral treatment.

Inflammation associated with COVID-19 may trigger even more severe complications including pneumonia, acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS), which is a leading cause of mortality in COVID-19. ARDS is associated with widespread inflammation in the lungs. The underlying mechanism of ARDS involves diffuse injury to cells which form the barrier of the microscopic air sacs (alveoli) of the lung, surfactant dysfunction, and activation of the immune system. The fluid accumulation in the lungs associated with ARDS is partially explained by vascular leakage due to inflammation. An important aspect of ARDS, triggered by COVID-19, is an initial release of chemical signals and other inflammatory mediators secreted by lung epithelial and endothelial cells. Neutrophils and some T-lymphocytes migrate into the inflamed lung tissue and contribute to the amplification/deterioration of ARDS. A decrease in the production of lipid mediators of inflammation (prostaglandins) may impair the resolution of inflammation associated with ARDS (see Fukunaga, et. al., Cyclooxygenase 2 Plays a Pivotal Role in the Resolution of Acute Lung Injury. Journal of Immunology 2005; 174:5033-5039.; Gao et al J Immunol 2017; 199:2043-2054).

The World Health Organization (WHO)’s Clinical Care for Severe Acute Respiratory Infection: COVID-19 Adaptation recommends early intervention with supplemental oxygen for COVID-19 patients with low blood oxygen saturation (Sp02) beginning with the least invasive modality possible ( e.g . hand-held oxygen source) and moving to more invasive modalities (e.g. bilevel positive airway pressure [BiPAP] and/or non-invasive ventilation (NIV)) as severity increases. Treatment during early intervention for COVID-19 patients with respiratory distress requires monitoring of respiratory function with treatment responsive to disease progression. The CDC recommends following the guidelines for treatment of COVID-19 patients with hypoxia in Surviving Sepsis Campaign: Guidelines on the Management of Critically Ill Adults with COVID-19.

Patients who fail to respond to less-invasive treatment of any respiratory viral infection are at a high risk of developing ARDS, a rapidly progressive disease characterized by widespread inflammation in the lungs that results in flooding of the lungs' microscopic air sacs, which are responsible for the exchange of gases such as oxygen and carbon dioxide with capillaries in the lungs. Additional common findings in ARDS include partial collapse of the lungs (atelectasis) and low levels of oxygen in the blood (hypoxemia). The clinical syndrome is associated with pathological findings including pneumonia and diffuse alveolar damage, the latter of which is characterized by diffuse inflammation of lung tissue. The triggering insult to the tissue usually results in an initial release of chemical signals and other inflammatory mediators secreted by local epithelial and endothelial cells.

ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. The underlying mechanism of ARDS involves diffuse injury to cells that form the barrier of the microscopic air sacs of the lungs, surfactant dysfunction, activation of the immune system, and dysfunction of the body's regulation of blood clotting. Diagnosis of ARDS is based on the 2012 Berlin definition:

• lung injury of acute onset, within 1 week of an apparent clinical insult and with progression of respiratory symptoms

• bilateral opacities on chest imaging (chest radiograph or CT) not explained by other lung pathology (e.g. effusion, lobar/lung collapse, or nodules)

• respiratory failure not explained by heart failure or volume overload

• decreased ratio of partial pressure arterial oxygen (PaCk) to fraction of inspired oxygen (FiCk) of less than or equal to 300 mm Hg despite a positive end-expiratory pressure (PEEP) of more than 5 cm EbO.

The severity of ARDS is defined by the Berlin definition as:

• mild ARDS: 201 - 300 mmHg (< 39.9 kPa)

• moderate ARDS: 101 - 200 mmHg (< 26.6 kPa)

• severe ARDS: < 100 mmHg (< 13.3 kPa)

There are no approved treatments for ARDS, and standard of care (SOC) is supportive management.

ARDS caused by a respiratory viral infection, for example a SARS-CoV-2 infection, can lead to damage to many organs, including the lungs, heart, and kidneys. SARS-CoV-2 infects endothelial cells and also leads to systemic inflammation, causing vasculopathy that affects widespread parts of the body. The vasculopathy or other phenomena can cause damage to the heart (myocarditis or arrhythmia), kidneys (acute kidney injury, chronic kidney disease, or renal failure), liver (liver dysfunction), blood vessels (bleeding and blood clots), skin (Kawasaki-like syndrome, rash, hair loss, and urticarial, vesicular, purpuric, and papulosquamous lesions), digestive system (anorexia, nausea, vomiting, diarrhea, and abdominal pain), brain, and nerves (cerebrovascular disease, ataxia, seizure, vision impairment, and nerve pain). Other symptoms include lymphopenia, hypoxia, blood hypercoagulability, multi-organ failure, sepsis, and septic shock.

Conventional pharmaceutical therapies for viral respiratory disease include anti -viral compositions. Anti-viral compositions include protease inhibitors, nucleoside analogs, antibodies (including monoclonal antibodies, neutralizing antibodies, and/or convalescent plasma), and other types of drugs. Administration and Dosing

Compositions provided by the present disclosure can be used for suppressing inflammation in the lungs, thus making them therapeutics for ARDS, ARDS triggered by Covid-19, and ARDS triggered by any viral respiratory disease.

DA-DKP-containing compositions provided by the present disclosure can be administered to a subject by any suitable route of administration to the lungs, including nasal, intratracheal, bronchial, direct instillation into the lung, inhaled and oral. The DA- DKP-containing compositions of the present invention can also be administered enterally ( e.g ., oral, sublingual, buccal, rectal), parenterally (e.g., intravenous, intramuscular, subcutaneous), intranasally, by inhalation, vaginally, or by other routes of administration.

In any of the methods and compositions disclosed herein, forms for administration of the DA-DKP-containing compositions of the present disclosure include nebulized form, aerosolized form, sprays, drops, and powders. The active ingredient may be mixed under sterile conditions with a pharmaceutically-acceptable carrier and with any buffers, excipients or propellants as may be needed to suit a particular composition and/or delivery method. In some embodiments, the form for administration is a sterile liquid that is administered as a nebulized liquid form or intravenously.

Aerosol (inhalation) delivery can be performed using methods standard in the art. Carriers suitable for aerosol delivery are described herein. Devices for delivery of aerosolized formulations include, but are not limited to, pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), and metered solution devices (MSI), and include devices that are nebulizers and inhalers.

The amount of a disclosed therapeutic composition that will be effective in the treatment of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by clinical techniques. For example, in vitro or in vivo assays may be employed to help identify optimal dose ranges. Effective dosage amounts may vary with the severity of the disease or condition, the route(s) of administration, the duration of the treatment, the identity of any other drugs being administered to the subject, the age, size and species of the subject, the discretion of the prescribing health care provider, and like factors.

Compositions provided by the present disclosure comprise DA-DKP having a concentration range with a lower endpoint of about 10 mM, about 20 pM, about 30 pM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 240, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, or about 400 mM; such compositions also comprise DA-DKP having a concentration range with an upper endpoint of about 600 mM, about 580 mM, about 570 mM, about 560 mM, about 550 mM, about 540 mM, about 530 mM, about 520 mM, about 510 mM, about 500 mM, about 490 mM, about 480 mM, about 470 mM, about 460 mM, about 450 mM, about 440 mM, about 430 mM, about 420 mM, about 410 mM, about 400 mM, about 390 mM, about 380 mM, about 370 mM, about 360 mM, about 350, about 340 mM, about 330 mM, about 320 mM, about 310 mM, about 300 mM, about 290 mM, about 280, about 270 mM, about 260 mM, about 250 mM, about 240 mM, about 230 mM, about 220 mM, about 210 mM, or about 200 mM.

An effective amount of DA-DKP in a composition provided by the present disclosure can vary. In some embodiments, the effective amount comprises a range of amounts having a lower endpoint of about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, about 100 pg, about 110 pg, about 120 pg, about 130 pg, about 140 pg, about 150 pg, about 160 pg, about 170 pg, about 180 pg, about 190 pg, about 200 pg, about 210 pg, about 220 pg, about 230 pg, about 240 pg, about 250 pg, about 260 pg, about 270 pg, about 280 pg, about 290 pg, about 300 pg, about 310 pg, about 320 pg, about 330 pg, about 340 pg, about 350 pg, about 360 pg, about 370 pg, about 380 pg, about 390 pg, about 400 pg, about 425 pg, about 450 pg, about 475 pg or about 500 pg. In some embodiments, the effective amount comprises a range of amounts having an upper endpoint of about 500 pg, about 490 pg, about 480 pg, about 470 pg, about 460 pg, about 450 pg, about 440 pg, about 430 pg, about 420 pg, about 410 pg, about 400 pg, about 390 pg, about 380 pg, about 370 pg, about 360 pg, about 350 pg, about 340 pg, about 330 pg, about 320 pg, about 310 pg, about 300 pg, about 290 pg, about 280 pg, about 270 pg, about 260 pg, about 250 pg, about 240 pg, about 230 pg, about 220 pg, about 210 pg, about 200 pg, about 190 pg, about 180 pg, about 170 pg, about 160 pg, about 150 pg, about 140 pg, about 130 pg, about 120 pg, about 110 pg, about 100 pg, about 90 pg, about 80 pg, about 70 pg, about 60 pg, about 50 pg, about 40 pg, about 30 pg, or about 20 pg.

Different doses of the disclosed compositions can be used with different routes of administration. Administration to the lung, for example by nebulizer, can involve doses of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 milliliters. Intravenous administration can involve doses of about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 375, about 380, about 390, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 milliliters (or cubic centimeters). The disclosed compositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, or more times per day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. Disclosed compositions can also be administered for 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, and/or for 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. In some embodiments, the disclosed compositions can be administered once every three months.

In some embodiments, the concentration of DA-DKP in a composition is selected from about 50 mM to about 350 mM and about 110 mM to about 200 mM; the composition also comprises N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof each independently in an amount selected from about 1 mM to about 20 mM and about 1 mM to about 4 mM; and the composition is administered as a multi-dose regimen of between 2 and 6 doses administered every 52 weeks and the amount of each dose is between about 2 mL and about 6 mL, in some embodiments each dose is 4 mL.

While it is possible for a compound of the present disclosure ( e.g ., DA-DKP, N- acetyl -tryptophan (NAT), caprylic acid, caprylate) to be administered alone, in various aspects the disclosed compounds are administered together as a pharmaceutical composition. The pharmaceutical compositions of the present disclosure comprise any combination of one or more compounds of the present disclosure as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, optionally, with one or more other compounds, drugs or other materials. In some embodiments, the compound is DA-DKP. Each carrier is advantageously "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Regardless of the route of administration selected, in embodiments the compounds of the present disclosure are formulated into pharmaceutically-acceptable dosage forms by conventional methods (see, e.g., Remington’s Pharmaceutical Sciences (Easton, Pa: Mack Pub. Co, 1965. Print; 23^rd Ed. (2020) ISBN: 9780128200070)).

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for nebulization, immediately prior to use.

Kits comprising the pharmaceutical products of the present disclosure are also provided. The kits can comprise a DA-DKP composition formulated for administration to the lung including a nebulized form and/or an aerosolized form. The DA-DKP can be prepared as described herein, such as by removing albumin from a solution of a human albumin composition. The kits may contain unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water, immediately prior to use. The kits may also be stored in a condition, wherein the contents are ready for direct use or injection.

The compositions of the present disclosure may further comprise N-acetyl- tryptophan (NAT), caprylic acid, caprylate or combinations thereof. In some embodiments, the compositions comprise NAT. In those embodiments where the disclosed compositions have NAT, caprylic acid, caprylate or combinations thereof, the concentration range of each of NAT, caprylic acid, and/or caprylate have a lower endpoint of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In addition, compositions of the present disclosure having NAT, caprylic acid, caprylate or combinations thereof have a concentration range with an upper endpoint of about 40 mM, about 39 mM, about 38 mM, about 37 mM, about 36 mM, about 35 mM, about 34 mM, about 33 mM, about 32 mM, about 31 mM, about 30 mM, about 29 mM, about 28 mM, about 27 mM, about 26 mM, about 25 mM, about 24 mM, about 23 mM, about 22, or about 21 mM. In some embodiments, the concentration range is about 4 mM to about 20 mM.

In addition, the DA-DKP-containing composition of the present disclosure can also comprise a second drug such as an analgesic (such as lidocaine or paracetamol), an anti inflammatory (such as corticosteroids, such as dexamethasone and betamethasone, non steroid anti-inflammatory drugs (NSAIDs), ibuprofen, naproxen), and/or other suitable drugs.

Synthesis

Methods of making diketopiperazines, such as DA-DKP, are well known in the art, and these methods may be employed to synthesize the diketopiperazines of the present disclosure. See, e.g., U.S. Patents Nos. 4,694,081, 5,817,751, 5,990,112, 5,932,579 and 6,555,543, US Patent Application Publication Number 2004/0024180, PCT applications WO 96/00391 and WO 97/48685, and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369- 2374 (1998).

For instance, diketopiperazines, such as DA-DKP, can be prepared by first synthesizing dipeptides. The dipeptides can be synthesized by methods well known in the art using L-amino acids, D-amino acids or a combination of D- and L-amino acids. In some embodiments, solid-phase peptide synthetic methods are employed. Dipeptides are also available commercially from numerous sources, including DMI Synthesis Ltd., Cardiff, UK (custom synthesis), Sigma-Aldrich, St. Louis, MO (primarily custom synthesis), Phoenix Pharmaceuticals, Inc., Belmont, CA (custom synthesis), Fisher Scientific (custom synthesis) and Advanced ChemTech, Louisville, KY.

Once the dipeptide is synthesized or purchased, it is cyclized to form a diketopiperazine. This can be accomplished by a variety of techniques including, for example, the method provided in U.S. Patent Application Publication Number 2004/0024180, which describes a method of cyclizing dipeptides. Briefly, the dipeptide is heated in an organic solvent while removing water by distillation. In some embodiments, the organic solvent is a low-boiling azeotrope with water, such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol, 2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride, chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal, dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone, 3-pentanol, toluene and xylene. The temperature depends on the reaction speed at which the cyclization takes place and on the type of azeotroping agent used. In some embodiments, the reaction is carried out at 50-200°C, in some embodiments at 80-150°C. The pH range in which cyclization takes place can be 2-9, in some embodiments 3-7.

When one or both of the amino acids of the dipeptide has, or is derivatized to have, a carboxyl group on its side chain ( e.g ., aspartic acid or glutamic acid), the dipeptide is cyclized, for example as described in U.S. Patent No. 6,555,543. Briefly, the dipeptide, with the side-chain carboxyl still protected, is heated under neutral conditions. Typically, the dipeptide will be heated at from about 80°C to about 180°C, in some embodiments at about 120°C. The solvent will be a neutral solvent. For instance, the solvent may be an alcohol (such as butanol, methanol, ethanol, and higher alcohols, but not phenol) and an azeotropic co-solvent (such as toluene, benzene, or xylene). In some embodiments, the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. The heating is continued until the reaction is complete, and such times can be determined empirically. Typically, the dipeptide will be cyclized by refluxing it for about 8-24 hours, in some embodiments about 18 hours. Finally, the protecting group is removed from the diketopiperazine, preferably without the use of strong acids (mineral acids, such as sulfuric or hydrochloric acids), strong bases (alkaline bases, such as potassium hydroxide or sodium hydroxide), and/or strong reducing agents ( e.g ., lithium aluminum hydride), in order to maintain the chirality of the final compound. In that regard, one or more of the compounds described herein contain one or more chiral centers and/or double bonds and, therefore, may exist as stereoisomers such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the compounds described herein encompass all possible enantiomers and stereoisomers including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques.

Dipeptides made on solid phase resins can be cyclized and released from the resin in one step (see, e.g, U.S. Patent No. 5,817,751). For instance, resin having an N-alkylated dipeptide attached can be suspended in toluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) or triethylamine (e.g., 4%). Typically, basic cyclization conditions are utilized for their faster cyclization times.

Other methods of cyclizing dipeptides and of making diketopiperazines can be used in the preparation of diketopiperazines useful in the practice of the present disclosure. In addition, many diketopiperazines suitable for use in the present disclosure can be made as described below from proteins and peptides. Further, diketopiperazines for use in the practice of the present disclosure can be obtained commercially, for example from DMI Synthesis Ltd., Cardiff, UK (custom synthesis).

The DA-DKP compositions and/or products of the present disclosure can be prepared from solutions containing DA-DKP, including from commercially-available pharmaceutical compositions comprising albumin, such as human serum albumin, using methods such as ultrafiltration, chromatography, size-exclusion chromatography (e.g, Centricon filtration), affinity chromatography (e.g, using a column of beads having attached thereto an antibody or antibodies directed to the desired diketopiperazine(s) or an antibody or antibodies directed to the truncated protein or peptide), anion exchange or cation exchange, sucrose gradient centrifugation, salt precipitation, sonication, or other techniques that will remove some or all of the albumin in the solution. The resultant DA-DKP - containing compositions and/or products can be used and incorporated into pharmaceutical compositions as described above. In some embodiments, the disclosed compositions and/or products can be prepared using an ultrafiltration separation method, whereby a human serum albumin composition can be passed over an ultrafiltration membrane having a molecular weight cut-off that retains the albumin while the DA-DKP passes into the resulting filtrate or fraction. This filtrate comprises components having molecular weights selected from less than about 50 kDa, less than about 40 kDa, less than 30 kDa, less than about 20 kDa, less than about 10 kDa, less than about 5 kDa, and less than about 3 kDa. In some embodiments, the filtrate is a LMWF comprising components having molecular weights less than about 5 kDa. This LMWF fraction or filtrate contains DA-DKP which is formed after the dipeptide aspartate-alanine is cleaved from albumin and subsequently cyclized into the diketopiperazine.

AMPION^® (Ampio Pharmaceuticals, Inc., Englewood, CO USA) is a LMWF (<5 kDa fraction) of human serum albumin (HSA). AMPION^® can be produced as described herein, for example by filtering commercially available HSA. Commercially available HSA is produced by fractionation of blood, for example by the Cohn process or its variations. AMPION^® can be produced by filtering commercially available HSA, for example a 5% HSA solution, to remove components above 5 kDa.

Physiologically-acceptable salts of the DA-DKP of the present disclosure may also be used in the practice of the present disclosure. Physiologically-acceptable salts include conventional non-toxic salts, such as salts derived from inorganic acids (such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids (such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic acid, and the like) or bases (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation or organic cations derived from N,N-dibenzylethylenediamine, D-glucosamine, or ethylenediamine). The salts are prepared in a conventional manner, for example by neutralizing the free base form of the compound with an acid.

Combination Therapy

In a second aspect provided by the present disclosure, methods of treating a viral respiratory disease are disclosed, the methods comprising administration of the disclosed compositions in combination with one or more additional therapeutics for the treatment of the viral respiratory disease. In various embodiments, the methods relate to the treatment of a viral respiratory disease by administration of two or more therapeutics, each independently formulated to treat the viral respiratory disease. The two therapeutics can be administered concurrently or at different time points, via the same route or via different routes. In some embodiments, the two therapeutics are selected to administer complimentary, combination therapy to a subject suffering from a viral respiratory disease. Combination therapy is a treatment modality that combines two or more therapeutic agents. The amalgamation of therapeutic agents to treat viral respiratory diseases may enhance efficacy compared to the mono-therapy approach, as the therapeutic agents can be selected to target key pathways in a synergistic and/or additive manner. This approach has many benefits, including potentially reducing drug resistance, while simultaneously providing therapeutic benefits.

In some embodiments, the methods comprise administration of a first drug effective to treat a viral respiratory disease, for example COVID-19, and also administering a pharmaceutical composition provided by the present disclosure, comprising DA-DKP, to treat the same viral respiratory disease. In some embodiments, the methods comprise administration of a first drug before a hyperinflammatory stage of a viral respiratory disease and administering a pharmaceutical composition provided by the present disclosure, comprising DA-DKP, to treat a hyperinflammatory stage of the viral respiratory disease. The pharmaceutical composition comprising DA-DKP can be administered before, at, or after onset of the hyperinflammatory stage of the viral respiratory disease.

The first drug can be one or more antiviral, immune-modifying, anti-depressant, and/or corticosteroid drugs, including combinations thereof. In some embodiments, the first drug is selected from: Protease inhibitors: Paxlovid (PF-07321332) - a protease inhibitor antiviral therapy, nirmatrelvir (an orally active 3C-like protease inhibitor), ritonavir (protease and cytochrome P450-3A4 (CYP3A4) inhibitor); Nucleoside analogs (or prodrugs thereof): Molnupiravir - a prodrug of the synthetic nucleoside derivative N⁴- hydroxycytidine, Remdesivir - a prodrug of nucleoside analog GS-441524, Favipiravir - a prodrug of purine nucleic acid analog favipiravir-ribofuranosyl-5'-triphosphate; Immune- modifying antibodies: Tocilizumab - a monoclonal antibody against the interleukin-6 receptor, Sarilumab - a monoclonal antibody against the interleukin-6 receptor, Lenzilumab - monoclonal antibody against the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF); Neutralizing antibodies: Casirivimab and imdevimab (REGN-COV) - monoclonal antibodies that bind to the spike protein of SARS-CoV-2, Bbamlanivimab and etesevimab - monoclonal antibodies that bind to the spike protein of SARS-CoV-2, Sotrovimab - mAb that binds to the spike protein of SARS-CoV-2, Tixagevimab/cilgavimab (AZD7442) - mAb that binds to the spike protein of SARS-CoV- 2, Convalescent plasma; Other drugs: Baricitinib - a Janus kinase (JAK) inhibitor that reversibly inhibits Janus kinase 1 and Janus kinase 2, Tofacitinib - an inhibitor of the Janus kinase 1 (JAK1) and janus kinase 3 (JAK 3); Anticoagulation drugs: Fluvoxamine - an anti depressant selective serotonin reuptake inhibitor (SSRI), Recombinant ACE-2 - a decoy that is bound by SARS-CoV-2, EXO-CD24 - exosomes carrying CD24 (also known as signal transducer CD24, cluster of differentiation 24, or heat stable antigen CD24); Corticosteroids: Dexamethasone, Prednisone, Methylprednisolone, Hydrocortisone; and combinations of any of the foregoing.

EXAMPLES

AMPION^® reduces CL075- and CL307 -induced CXCL10 release from PMA-differentiated THP-1 cells.

To determine if AMPION^® influences endosomal single stranded ribonucleic acid (ssRNA) sensing pathways, CL075 and CL307 agonists were used to activate TLR7/8 or TLR7, respectively, in PMA-differentiated THP-1 cells. Increases in CXCL10 release indicate progression to hyperinflammation, and decreases indicate inhibition of progression to hyperinflammation.

AMPION^® was manufactured by Ampio Pharmaceuticals, Inc. (Englewood, CO). General cell culture reagents were purchased from ThermoFisher Scientific (Waltham, MA) while X-Vivo 15 serum-free medium was obtained from Lonza (Basel, Switzerland). 0.9% (w/v) sodium chloride was obtained from KD Medical (Columbia, MD). GW9662 and CH223191 antagonists were purchased from MilliporeSigma (St. Louis, MO) with 100 mM stock solutions prepared in DMSO and stored at -80°C prior to use. CL075 and CL307 were purchased from Invivogen (San Diego, CA) and stock solutions prepared using supplied sterile, pyrogen-free water. ELISAs for IPIO/CXCLIO (catalog # KAC2361) were purchased from ThermoScientific. All other reagents were obtained from MilliporeSigma (St. Louis, MO) unless otherwise stated.

Cell Culture and Experimental Treatments

Human THP-1 monocytes (ATCC, Manassas, VA) were passaged in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin (Pen/ Strep) then differentiated using a final concentration of 50 ng/ml phorbol 12-myristate (PMA) in 96-well flat bottom plates seeded at 1 ^c 10⁵ cells per well for 72 hours. Differentiation mediums were then aspirated and replenished as described below.

When CXCL10 release was evaluated 24 hours post-stimulation with CL075, dose- dependent release of chemokine was observed with significant (p-value < 0.05) reductions of 92 ± 3% and 78 ± 6% by AMPION^® treatment at final agonist concentrations of 5 pg/ml and 2 pg/ml, respectively (Fig. 1A). Complete attenuation of CXCL10 release was also observed with AMPION^® treatment at 0.5 pg/ml levels of CL075 but this may have been the result of release measurements at or near the detection limit of the ELISA. As for CL307- induced CXCL10 from PMA-differentiated THP-1 cells, muted chemokine release was observed at this timepoint only at the highest concentration used (1 pg/ml) with a trend towards inhibition (38 ± 29%; p-value = 0.5).

Different dose dynamics were observed when CXCL10 release was tested 72 hours post-stimulation (Fig. IB). Indicative of an asymptotic dose response, high levels of CL075- induced CXCL10 release was observed for all doses, with significant AMPION^® inhibitions ranging from 62 ± 7% to 95 ± 3%. CXCL10 release in response to CL307 exposure at 72 hours was more vigorous and importantly, exhibited dose dependency. Drug-induced inhibitions of 84 ± 1% and 46 ± 23% were observed for 1 pg/ml and 0.1 pg/ml final concentrations of CL307, respectively, with the 1 pg/ml group exhibiting significance (p- value < 0.05).

Together, these data provide evidence that AMPION^® modulates CXCL10 release resulting from the activation of TLR7/8 in macrophage-like THP-1 cells.

AMPION^® reduces CL075- and CL307 -induced CXCL10 release from Peripheral Blood Mononuclear Cells (PBMC).

In a similar fashion, AMPION^® was also found to inhibit CXCL10 release from TLR4-activated PBMC. To establish if AMPION^® can influence ex vivo CL075- and CL307-induced CXCL10, these agonists were used to activate TLR7/8 in PBMC cocultures.

Both frozen and freshly isolated PBMC from consented donors were used in this investigation. When frozen stocks were utilized, cryopreserved vials obtained from Zen-Bio (Research Triangle Park, NC) were thawed using a Thawstar Automated Cell Thawing System (BioLife Solutions, Bothell, WA) and then transferred dropwise to RPMI 1640 medium containing 10% human AB serum, 1% penicillin-streptomycin (Pen/Strep), and 2 U/mL RNase-free DNase (ThermoScientific). Freshly isolated cells were isolated from sodium heparinized whole blood using Polymorphprep (Alere Technologies, Oslo, Norway) and washed with Dulbecco’s phosphate buffer saline. The resulting cell suspensions were then centrifuged at 1000 rpm for 10 minutes and working suspensions prepared at 2 x 10⁶ cells/mL in X-Vivo 15 or RPMI 1640 supplemented with 20% FBS, 2% Pen/Strep, 1% sodium bicarbonate, 7.5% solution, 1% 100 mM sodium pyruvate, 1% 100X MEM non- essential amino acid solution, and 1% 200 mM L-glutamine.

For experimental treatments, 100 mΐ of PBMC suspensions were added to 96-well tissue culture plates or 100 mΐ of the 20% FBS RPMI medium described above was added to THP-1 plate wells. The solutions were then mixed with an equal volume of sterile 0.9% sodium chloride or AMPION^® drug solutions and incubated at 37 °C and 5% CO2 for one hour. Stimulation was then achieved by adding CL075 or CL307 to the final concentrations indicated for an additional 24 to 72 hours before subsequent analysis. For relative potency investigation, 20 mM (10 mM final) concentration of antagonists were added to mediums prior to addition of saline or drug solutions serially diluted in saline. After the indicated stimulation periods described in these Examples, supernatants were collected for in-house CXCL10 ELISA measurements.

As observed in PMA-differentiated THP-1, CXCL10 release induced by CL075 from PBMC is observed 24 hours following exposure (Fig. 2A). However, an inverse relationship of CXCL10 release to CL075 concentration was observed using the donor cells represented in Fig. 2A, suggesting a biphasic response. AMPION^® treatment resulted in significant inhibitions of 93 ± 10%, 55 ± 4%, and 65 ± 2% in CXCL10 release from 5 pg/ml, 2 pg/ml, and 0.5 pg/ml concentrations of agonist, respectively, when corrected for the background chemokine release observed for this donor. In contrast to THP-1, substantial CXCL10 release in response to a final concentration of 1.0 pg/ml CL307 was observed in PBMC cultures after 24 hours with a trend toward AMPION^®-induced inhibition of 82 ± 33% (Fig. 2A). Measuring CXCL10 release from PBMC after 48 hours shows a similar asymptotic response compared to THP-1 cells with AMPION^® inhibitions of 73 ± 2%, 68 ± 4%, and 76 ± 5% in CXCL10 release from 5 pg/ml, 2 pg/ml, and 0.5 pg/ml CL075, respectively (Fig. 2B). Significant inhibition of CXCL10 induced by 1.0 pg/ml CL307 was observed at this timepoint in the drug treatment group (81 ± 6%; p-value < 0.05). These findings demonstrate AMPION^® modulates TLR7/8 signaling in primary cells, such as monocytic and/or other myeloid cells.

Percent inhibition in CXCL10 release normalizes 24-hour CL075-induced PMA- differentiated THP-1 and PBMC data

Due to the magnitude and robustness of AMPION^® inhibition in CXCL10 following 24-hour, 5 pg/ml CL075 activation, this treatment group was selected to determine variability and distributions in CXCL10 release. As shown in Fig. 3A, CL075-induced CXCL10 release from saline-treated PMA-differentiated THP-1 cells exhibits a wide, slightly negative distribution (Median = 4967 pg/ml, IQR = 4432, n = 8) with a distinct reduction seen in the AMPION^® treatment group (Median = 276 pg/ml, IQR = 244, n = 8). CL075-induced CXCL10 release from saline-treated PBMC also exhibits a wide distribution with a strong positive skew (Median = 177 pg/ml, IQR = 756, n = 9 across a total of 7 donors) (Fig. 3B). Donor variability makes direct interpretation of raw release difficult, but a reduction in CXCL10 release was observed (Median = 121 pg/ml, IQR = 427, n = 9).

Fig. 3C demonstrates how normalization within each experiment to percent inhibition in CXCL10 release versus control corrected for observed background chemokine release and transformed the data to a more symmetrical distribution. Once drug response was calculated in this manner, AMPION^® demonstrated an ability to inhibit CL075-induced CXCL10 release from PMA-differentiated THP-1 and PBMC by medians of 92% (IQR = 7, p-value < 0.05 versus hypothetical 0) and 58% (IQR = 34, p-value < 0.05 versus hypothetical 0) respectively.

Together, these data provide evidence that AMPION^® modulates TLR7/8 signaling in both macrophage-like THP-1 cells and ex vivo PBMC cultures. Furthermore, the variability observed in overall CL075-induced CXCL10 release in PMA-differentiated THP-1 and PBMC models suggests that responses to TLR7/8 are highly dependent on cell- type and/or differentiation status. However, AMPION^® inhibition of this pathway is universal, regardless of these dynamics.

PPARy and AhR antagonists reduce AMPION^® drug potency as measured by CL075- induced CXCL10 release.

Inhibition of CXCL10 release from PMA-differentiated THP-1 cells was used to determine the involvement of various pathways. In the first step of this experimental procedure, drug product was serially diluted with saline vehicle and then reductions in 24- hour CL075-induced CXCL10 release were evaluated following percent inhibition normalization. Regression analysis demonstrated that AMPION^® exhibited a log-linear, dose-dependent reduction (R² = 0.986) in chemokine release conducive for relative potency 5 (REP) calculation (Fig. 4). Next, the potency of AMPION^® was established in the presence of antagonists for PPARy (GW9662, MilliporeSigma) or AhR (CH223191, Millipore Sigma) as compared to DMSO vehicle controls. Exposure of cells to 10 mM final concentrations of GW9662 or CH223191 resulted in shifts in the log-linear dose response of AMPION^®- induced CXCL10 inhibition reflective of a loss in drug potency. REP calculated for 3 0 independent experiments demonstrated that antagonism of GW9662 and CH223191 resulted in reduced mean drug potency to 0.29 ± 0.06 and 0.40 ± 0.18, respectively (Table 1; data presented as mean relative potency (REP) ± STD and 95% Cl of three independent experiments versus DMSO controls). AMPION^® thus activates PPARy and AhR transcription factors to inhibit the release of CXCL10 from PMA-differentiated THP-1 cells 5 stimulated with CL075.

Table 1. Disruption of PPARy or AhR signaling decreases the relative potency of AMPION^®.

ELISA array analysis demonstrates that AMPION * inhibits the release of a diverse set of pro-inflammatory cytokines and chemokines from CL075-activated, PMA-differentiated 0 THP-1 cells.

A multi-plex ELISA array was performed to assess the release of 48 cytokines, chemokines, and growth factors, to determine the overall immune response in PMA- differentiated THP-1 cells following CL075 activation and to identify additional drugs effects. The ELISA was Eve Technologies (Calgary, Alberta, Canada) human 5 cytokine/chemokine 48-plex Discovery Assay Array (catalog # HD48). Using this expanded format, a total of 26 inflammatory mediators were found to be significantly (p < 0.05) inhibited by AMPION^®-treatment 24 hours following activation with 5 pg/ml CL075 (Table 2; data are presented as fold change ± STD or percent inhibition (n = 3, p-value = < 0.05 for fold change versus theoretical fold change of 1)). Consistent with the previous findings in this investigation, CXCL10 proved to be the most highly downregulated (97 ± 2% inhibition) target on the array. Other prominent pro-inflammatory cytokines such as IL-6 (92 ± 9%), IL-12 (p40 = 91 ± 1%; p70 = 63 ± 7%), IL-Ib (70 ± 10%), and TNF-a (56 ± 5%) as well as chemokines including MCP (MCP-1 = 90 ± 1%; MCP-3 = 86 ± 5%), MIP (MIP- 1b = 70 ± 10%; MIR-1a = 68 ± 5%), CXCL9 (57 ± 11%) and RANTES (53 ± 1%) were also significantly suppressed. Of note, some reduction in type I and type II interferon was also observed but did not prove significant vs controls.

Table 2. Multiple cytokines and chemokines are significantly suppressed by AMPION^® in CL075-stimulated, PMA-differentiated THP-1 cultures.

These quantified ELISA array results were then utilized to calculate biologically relevant cytokine ratios (Table 3; data presented as mean ratios ± STD, n 3, /i- value = < 0.05), which can be used to assess the immunological state of cells during inflammation. The ratio of IL-12 to IL-10, for example, has been established as a predictive marker of clinical course in multiple sclerosis as well as disease severity in viral infections. As shown in Table 3, treatment of CL075 -activated, PMA-differentiated THP-1 cells with AMP5A significantly reduced the ratio of IL-12p40/IL-10 released from 10.3 ± 3.5 seen in the saline control group to 1.3 ± 0.7 (p-value = 0.01). Similarly, IL-6/IL-10 ratios can be used to predict the prognosis of multiorgan dysfunction syndrome and mortality after trauma as well as the severity of disease in both pneumonia and COVID-19. As with IL-12/IL-10, AMP5A treatment significantly reduced IL-6/IL-10 ratio of these cultures (8.0 ± 4.1 for saline versus 0.6 ± 0.2; p-v alue = 0.04). These findings suggest that AMP5A is acting to biologically modulate the function of these cells in meaningful ways, as measured by phenotypic change, rather than simply inhibiting cytokine release.

Table 3. Significant changes in IL-12p40 and IL-6 to IL-10 ratios induced by AMP5A.

In silico analysis of array cytokines and chemokines reveals that proteins inhibited by AMP ION^® are associated with hyper -inflammatory disease states, immune cell maturation, and pro-inflammatory transcription factor activity.

Gene enrichment analysis was performed by querying Enrichr (Chen etal., Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013; 14: 128.; Kuleshov MV., et al., Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44:W90-7.) or Ingenuity Pathway Analysis (IP A) software using the corresponding gene symbols (Tables 4 and 5; data presented as tables of significantly enriched terms in Wikipathways and Jensens Disease libraries, ranked by enrichment score).

Table 4: Enrichr Wikipathways Analysis.

Table 5: Jensen Disease Enrichment Analysis.

When sorted by enrichment score, the top term returned from the Wikipathways library of Enrichr was the ‘COVID-19 adverse outcome pathway’ (WP4891) followed by others such as ‘cytokine responses and inflammatory response’ (WP530) or ‘toll-like receptor signaling’ (WP75) as well as Tung fibrosis’ (WP3624) and ‘lymphoid development/polarization’ (WP3893 and WP4494). In addition, mining of the Jensen disease-associated databases predicts that these AMPION^®-inhibited cytokines and chemokines are associated with lung disease, influenza, collagen disease, and arthritis.

Canonical pathway analysis performed in Ingenuity Pathway Analysis (IP A) showed that the cytokines and chemokines inhibited by AMPION^® were predicted to significantly overlap with 6 pathways, and of these, ‘dendritic cell maturation’, crosstalk between dendritic cells and natural killer cells’, and ‘NF-kB signaling’ pathways were predicted to be significantly inhibited (black bars, Fig. 5) while IPA predicted activation of ‘liver X receptor/retinoid X receptor activity’ (grey bars, Fig. 5). The data demonstrate that hyperinflammatory disease states, immune cell maturation, and proinflammatory transcription factor activity are suppressed with AMPION^® treatment.

Data and Statistical Analysis

Statistical analysis was performed using the Real Statistics Resource Pack Excel Add-in (http://www.real-statistics.com/) unless otherwise stated. For representative ELISAs, two-tailed, two-sample unequal variance student tests were used to compare groups in Microsoft Excel (Microsoft Corporation, Redmond, WA). For combined inhibition of release analysis, two-tailed, one-sample t-tests (hypothetical value = 0; a = 0.05) were used to establish meaningful percent inhibition measurements from vehicle- controls. For cytokine arrays, two-tailed, one-sample t-tests were used to test for the significance of combined fold changes (hypothetical value = 1; a = 0.05). For potency assays, relative potency was calculated using 4P modeling with ANOVA pure separation and similarity of dose responses was established by f-tests for non-parallelism, non linearity, and significance of response in PLA 3.0 (Stegmann Systems GmbH, Raiffeisenstr, Germany). Box plots were generated, and descriptive statistics performed, using BoxPlotR (shiny.chemgrid.org/boxplotr/). In silico pathway analysis of differentially expressed genes was performed using Enrichr (maayanlab.cloud/Enrichr) or Ingenuity Pathway Analysis (IP A) software (Qiagen Digital Insights, Redwood City, CA). For IP A, calculated differentially abundant cytokines/chemokines were uploaded, and a ‘Core’ expression analysis based on log ratios for analytes with a p-value<0.05 was run using our ‘user dataset’ as reference. IPA calculated overlap p-values and z-scores as confidence metrics when predicting canonical pathway associations. For overlap p-values, we considered the - log(pvalue)>1.3 as significant. IPA calculated z-scores where z > 0 predicts activation, and z < 0 predicts inhibition; an absolute value of 2 was used as a significance cutoff.

Multi-modal COVID-19 treatment

A patient hospitalized due to COVID-19 receives standard of care therapy with the addition of AMPION^® to prevent, reduce the severity or duration of, or treat a hyperinflammatory stage of COVID-19. The patient is administered remdesivir, remdesivir plus dexamethasone, or dexamethasone. The patient may also be administered one or more other antiviral drugs. The patient is administered AMPION^® before, at, or after onset of the hyperinflammatory stage. If the patient experiences rapidly increasing oxygen needs and systemic inflammation, baricitinab, tocilizumab, sarilumab, and/or another immunomodulatory drug may be administered. The antiviral drug or drugs inhibit the viral infection, and AMPION^® treats the hyperinflammatory stage of COVID-19.

Claims

What is claimed is:

1. A method of treating one or more symptoms of a viral infection in a patient, comprising: administering a first drug to the patient prior to onset of a hyperinflammatory stage of the infection; and administering a pharmaceutical composition comprising DA-DKP to the patient before, at, or after the onset of the hyperinflammatory stage.

2. The method of claim 1, wherein the pharmaceutical composition is administered before the onset of the hyperinflammatory stage.

3. The method of claim 1, wherein the pharmaceutical composition is administered at the onset of the hyperinflammatory stage.

4. The method of claim 1, wherein the pharmaceutical composition is administered after the onset of the hyperinflammatory stage.

5. The method of any one of claims 1 - 4, wherein, upon administration of the pharmaceutical composition, the first drug and the pharmaceutical composition are co administered to the patient.

6. The method of any one of claims 1 - 5, wherein the first drug is an antiviral, an immune-modifying drug, an anti-depressant, a corticosteroid, or combinations thereof.

7. The method of any one of claims 1 - 5, wherein the first drug is selected from: a protease inhibitor selected from paxlovid, nirmatrelvir, and ritonavir; a nucleoside analog, or prodrug thereof, selected from molnupiravir, L - hydroxycytidine, remdesivir, favipiravir, and favipiravir-ribofuranosyl-5'-triphosphate; an immune-modifying antibody selected from tocilizumab, sarilumab, and lenzilumab; a neutralizing antibody selected from casirivimab, imdevimab, bamlanivimab, etesevimab, sotrovimab, tixagevimab/cilgavimab, and convalescent plasma; baricitinib or tofacitinib; an anti coagulation drug selected from fluvoxamine, recombinant ACE-2, and EXO-

CD24; a corticosteroid selected from dexamethasone, prednisone, methylprednisolone, and hydrocortisone; and combinations of any of the foregoing.

8. The method of any one of claims 1 - 5, wherein the first drug is a protease inhibitor selected from paxlovid, nirmatrelvir, ritonavir, and combinations thereof.

9. The method of any one of claims 1 - 5, wherein the first drug is a nucleoside analog, or prodrug thereof, selected from molnupiravir, A -hy droxy cy ti dine, remdesivir, favipiravir, favipiravir-ribofuranosyl-5'-triphosphate, and combinations thereof.

10. The method of any one of claims 1 - 5, wherein the first drug is an immune- modifying antibody selected from tocilizumab, sarilumab, lenzilumab, and combinations thereof.

11. The method of any one of claims 1 -5, wherein the first drug is a neutralizing antibody selected from casirivimab, imdevimab, bamlanivimab, etesevimab, sotrovimab, tixagevimab/cilgavimab, convalescent plasma, and combinations thereof.

12. The method of any one of claims 1 - 5, wherein the first drug is selected from baricitinib, tofacitinib, and both baricitinib and tofacitinib.

13. The method of any one of claims 1 - 5, wherein the first drug is an anti coagulation drug selected from fluvoxamine, recombinant ACE-2, EXO-CD24, and combinations thereof.

14. The method of any one of claims 1 - 5, wherein the first drug is a corticosteroid selected from dexamethasone, prednisone, methylprednisolone, hydrocortisone, and combinations thereof.

15. The method of any one of claims 1 - 14, wherein the viral infection is caused by a respiratory virus.

16. The method of any one of claims 1 - 14, wherein the viral infection is caused by a virus selected from the group consisting of an influenza virus, a coronavirus, a rhinovirus, a respiratory syncytial virus, a parainfluenza virus, a human metapneumovirus, an adenovirus, and combinations thereof.

17. The method of any one of claims 1 - 14, wherein the viral infection is caused by a virus selected from the group consisting of an influenza A virus, an influenza B virus, Severe Acute Respiratory Distress Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Distress Syndrome coronavirus 2 (SARS-CoV-2), human adenovirus B21 infections (HAdV-B21), and combinations thereof.

18. The method of any one of claims 1-17, wherein the one or more symptoms are selected from the group consisting of acute respiratory distress syndrome (ARDS), acute lung injury (ALI), interstitial lung disease, pulmonary fibrosis, pneumonia, reactive airway disease syndrome, respiratory distress requiring supplemental oxygen, long COVID, and combinations thereof.

19. The method of any one of claims 5 - 18, wherein the co-administration results in an outcome selected from the group consisting of reduced ventilator time, reduced mortality, improvement in oxygenation parameters, reduced time to resolution of one or more respiratory symptoms, improved pulmonary function, and combinations thereof.

20. The method of any one of claims 5 - 19, wherein the co-administration results in the patient achieving improvement on the World Health Organization COVID- 19 ordinal scale of at least 4, at least 3, at least 2, or at least 1.

21. The method of any one of claims 1-20, wherein the patient has or had respiratory distress requiring supplemental oxygen caused by the viral infection.

22. The method of any one of claims 1-21, wherein the pharmaceutical composition is administered via inhalation.

23. The method of any one of claims 1-21, wherein the pharmaceutical composition is administered intravenously.

24. The method of any one of claims 1-23, wherein the pharmaceutical composition further comprises N-acetyl -tryptophan (NAT), caprylic acid, and/or caprylate.

25. A method of treating inflammation during a hyperinflammatory stage of a TLR7- and/or TLR8-mediated disease in a patient, comprising: administering a first drug to the patient prior to onset of a hyperinflammatory stage of the disease; and administering a pharmaceutical composition comprising DA-DKP to the patient before, at, or after the onset of the hyperinflammatory stage.

26. The method of claim 25, wherein the pharmaceutical composition is administered before the onset of the hyperinflammatory stage.

27. The method of claim 25, wherein the pharmaceutical composition is administered at the onset of the hyperinflammatory stage.

28. The method of claim 25, wherein the pharmaceutical composition is administered after the onset of the hyperinflammatory stage.

29. The method of any one of claims 25 - 28, wherein, upon administration of the pharmaceutical composition, the first drug and the pharmaceutical composition are co administered to the patient.

30. The method of any one of claims 25 - 29, wherein the TLR7- and/or TLR8- mediated disease is caused by a single-stranded RNA virus.

31. The method of any one of claims 25 - 30, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of Coronaviridae, SARS-CoV, MERS, SARS-CoV-2, torovirus, Hepeviridae, Hepatitis E, Caliciviridae, Norovirus, Sapporo virus, Togaviridae, Alphavirus viral diseases, Chikungunya, Eastern equine encephalomyelitis virus, Getah virus, Mayaro virus, Mucambo virus, O’nyong’nyong virus, Ross river virus, Barmah forest virus, Sagiyama virus, Semliki forest virus, Sindbis virus, Tonate virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus and Rubivirus, Rubella virus, Flaviviridae, Hepacivirus, Hepatitis C, Flavivirus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3, Dengue virus type 4, Hepatitis G virus, Japanese B encephalitis virus, Murray Valley encephalitis virus, Rocio virus, Spondweni virus, St Louis encephalitis, Wesselsbron, West Nile virus, Yellow fever virus, Absettarov, Hanzalova, Hypr, Kumlinge, Kyasanur forest disease, Louping ill, Negishi, Omsk, Powassan, Langat, Russian spring summer encephalitis, Hepatitis G virus group, Hepatitis G virus, Picornaviridae, Enterovirus, Coxsackievirus, Echovirus, Poliovirus, Enterovirus 68-109, Rhinovirus A, Rhinovirus B, Hepatovirus, Hepatitis A, Astroviridae, Astrovirus species, Mononegavirales, Henipavirus, Hendra virus and Nipah virus, Rubulavirus, Mumps virus and Parainfluenza types 2, 4a and 4b, Morbillivirus, Measles virus, Avulavirus, Newcastle disease virus, Metapneumovirus, Pneumovirus, Respiratory syncytial virus, and Parainfluenza Type 1, Parainfluenza Type 2, Parainfluenza Type 3, Parainfluenza Type 4, Rhabdoviridae, Lyssavirus, Duvenhage and Rabies virus, Retroviridae, Human T-cell lymphotropic viruses, Lentivirus, Human Immunodeficiency virus, Arenaviridae, Arenavirus, lymphocytic choriomeningitis virus, Bunyaviridae, Bunyavirus, Bunyamwera virus, Hantavirus, Hantaan virus, Nairovirus, Nairobi sheep disease virus, and Phlebovirus, sandfly fever Sicilian virus, and Orthomyxoviridae, Influenza viruses, influenza virus A, influenza virus B, influenza virus C, influenza virus D, Thogoto-like viruses, Thogoto virus, and combinations thereof.

32. The method of any one of claims 25 - 30, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of a coronavirus, an influenza virus, respiratory syncytial virus (RSV), a parainfluenza virus, a human metapneumovirus, and combinations thereof.

33. The method of any one of claims 25 - 30, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of SARS- Coronavirus-2 (SARS-CoV-2), SARS-associated coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and combinations thereof.

34. The method of any one of claims 25 - 30, wherein the TLR7- and/or TLR8- mediated disease is caused by SARS-Coronavirus-2 (SARS-CoV-2).

35. The method of any one of claims 25 - 30, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of an influenza A virus, an influenza B virus, and both an influenza A virus and an influenza B virus.

36. The method of any one of claims 25 - 29, wherein the TLR7- and/or TLR8- mediated disease is selected from the group consisting of acute kidney injury, glomerulonephritis, lupus, lupus nephritis, and combinations thereof.

37. The method of any one of claims 25 - 36, wherein the first drug is selected from: a protease inhibitor selected from paxlovid, nirmatrelvir, and ritonavir; a nucleoside analog, or prodrug thereof, selected from molnupiravir, N⁴- hydroxycytidine, remdesivir, favipiravir, and favipiravir-ribofuranosyl-5'-triphosphate; an immune-modifying antibody selected from tocilizumab, sarilumab, and lenzilumab; a neutralizing antibody selected from casirivimab, imdevimab, bamlanivimab, etesevimab, sotrovimab, tixagevimab/cilgavimab, and convalescent plasma; baricitinib or tofacitinib; an anti coagulation drug selected from fluvoxamine, recombinant ACE-2, and EXO-

38. The method of any one of claims 25 - 36, wherein the first drug is a protease inhibitor selected from paxlovid, nirmatrelvir, ritonavir, and combinations thereof.

39. The method of any one of claims 25 - 36, wherein the first drug is a nucleoside analog, or prodrug thereof, selected from molnupiravir, A -hydroxycytidine, remdesivir, favipiravir, favipiravir-ribofuranosyl-5'-triphosphate, and combinations thereof.

40. The method of any one of claims 25 - 36, wherein the first drug is an immune-modifying antibody selected from tocilizumab, sarilumab, lenzilumab, and combinations thereof.

41. The method of any one of claims 25 - 36, wherein the first drug is a neutralizing antibody selected from casirivimab, imdevimab, bamlanivimab, etesevimab, sotrovimab, tixagevimab/cilgavimab, convalescent plasma, and combinations thereof.

42. The method of any one of claims 25 - 36, wherein the first drug is selected from baricitinib, tofacitinib, and both baricitinib and tofacitinib.

43. The method of any one of claims 25 - 36, wherein the first drug is an anti coagulation drug selected from fluvoxamine, recombinant ACE-2, EXO-CD24, and combinations thereof.

44. The method of any one of claims 25 - 36, wherein the first drug is a corticosteroid selected from dexamethasone, prednisone, methylprednisolone, hydrocortisone, and combinations thereof.

45. The method of any one of claims 30 - 35 or 37 - 44, wherein the patient has or had respiratory distress requiring supplemental oxygen caused by the viral infection.

46. The method of any one of claims 25 - 45, wherein the pharmaceutical composition is administered via inhalation.

47. The method of any one of claims 25 - 46, wherein the pharmaceutical composition is administered intravenously.

48. The method of any one of claims 25 - 47, wherein the pharmaceutical composition further comprises N-acetyl -tryptophan (NAT), caprylic acid, and/or caprylate.

49. A method of treating or preventing one or more symptoms of a TLR7- and/or TLR8-mediated disease in a patient, comprising administering to the patient a pharmaceutical composition comprising DA-DKP.

50. A method of treating or preventing inflammation associated with a TLR7- and/or TLR8-mediated disease in a patient, comprising administering to the patient a pharmaceutical composition comprising DA-DKP.

51. The method of claim 49 or claim 50, wherein the TLR7- and/or TLR8- mediated disease is caused by a single-stranded RNA virus.

52. The method of any one of claims 49 - 51, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of Coronaviridae, SARS-CoV, MERS, SARS-CoV-2, torovirus, Hepeviridae, Hepatitis E, Caliciviridae, Norovirus, Sapporo virus, Togaviridae, Alphavirus viral diseases, Chikungunya, Eastern equine encephalomyelitis virus, Getah virus, Mayaro virus, Mucambo virus, O’nyong’nyong virus, Ross river virus, Barmah forest virus, Sagiyama virus, Semliki forest virus, Sindbis virus, Tonate virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus and Rubivirus, Rubella virus, Flaviviridae, Hepacivirus, Hepatitis C, Flavivirus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3, Dengue virus type 4, Hepatitis G virus, Japanese B encephalitis virus, Murray Valley encephalitis virus, Rocio virus, Spondweni virus, St Louis encephalitis, Wesselsbron, West Nile virus, Yellow fever virus, Absettarov, Hanzalova, Hypr, Kumlinge, Kyasanur forest disease, Louping ill, Negishi, Omsk, Powassan, Langat, Russian spring summer encephalitis, Hepatitis G virus group, Hepatitis G virus, Picornaviridae, Enterovirus, Coxsackievirus, Echovirus, Poliovirus, Enterovirus 68-109, Rhinovirus A, Rhinovirus B, Hepatovirus, Hepatitis A, Astroviridae, Astrovirus species, Mononegavirales, Henipavirus, Hendra virus and Nipah virus, Rubulavirus, Mumps virus and Parainfluenza types 2, 4a and 4b, Morbillivirus, Measles virus, Avulavirus, Newcastle disease virus, Metapneumovirus, Pneumovirus, Respiratory syncytial virus, and Parainfluenza Type 1, Parainfluenza Type 2, Parainfluenza Type 3, Parainfluenza Type 4, Rhabdoviridae, Lyssavirus, Duvenhage and Rabies virus, Retroviridae, Human T-cell lymphotropic viruses, Lentivirus, Human Immunodeficiency virus, Arenaviridae, Arenavirus, lymphocytic choriomeningitis virus, Bunyaviridae, Bunyavirus, Bunyamwera virus, Hantavirus, Hantaan virus, Nairovirus, Nairobi sheep disease virus, and Phlebovirus, sandfly fever Sicilian virus, and Orthomyxoviridae, Influenza viruses, influenza virus A, influenza virus B, influenza virus C, influenza virus D, Thogoto-like viruses, Thogoto virus, and combinations thereof.

53. The method of any one of claims 49 - 51, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from group consisting of a coronavirus, an influenza virus, respiratory syncytial virus (RSV), a parainfluenza virus, a human metapneumovirus, and combinations thereof.

54. The method of any one of claims 49 - 51, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of SARS- Coronavirus-2 (SARS-CoV-2), SARS-associated coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and combinations thereof.

55. The method of any one of claims 49 - 51, wherein the TLR7- and/or TLR8- mediated disease is caused by SARS-Coronavirus-2 (SARS-CoV-2).

56. The method of any one of claims 49 - 51, wherein the TLR7- and/or TLR8- mediated disease is caused by a virus selected from the group consisting of an influenza A virus, an influenza B virus, and both an influenza A virus and an influenza B virus.

57. The method of any one of claims 49 - 56, wherein the patient has or is at risk of developing inflammation of a tissue selected from the group consisting of lung, brain, heart, kidney, blood vessel, skin, and nerve.

58. The method of claim 57, wherein the tissue is selected from the group consisting of lung and kidney.

59. The method of any one of claims 49 - 58, wherein the patient has or is at risk of developing a symptom selected from the group consisting of acute respiratory distress syndrome (ARDS), acute lung injury (ALI), interstitial lung disease, pulmonary fibrosis, pneumonia, reactive airway disease syndrome, and combinations thereof.

60. The method of any one of claims 49 - 59, wherein the patient has or is at risk of developing a symptom selected from the group consisting of fatigue, shortness of breath or difficulty breathing, low exercise tolerance, low blood oxygen saturation, cough, sore throat, stuffy or runny nose, joint pain, chest pain, tightness or discomfort, muscle pain, muscle weakness, fever, heart palpitations, difficulty thinking and/or concentrating, depression, and combinations thereof.

61. The method of claim 60, wherein the patient has experienced the symptom at least four weeks, at least one month, at least two months, or at least three months.

62. The method of any one of claims 49 - 61, wherein the administering results in an outcome selected from the group consisting of reduced ventilator time, reduced mortality, improvement in oxygenation parameters, reduced time to resolution of one or more respiratory symptoms, improved pulmonary function, and combinations thereof.

63. The method of any one of claims 49 - 62, wherein, after the administration, the patient achieves improvement on the World Health Organization COVID-19 ordinal scale of at least 4, at least 3, at least 2, or at least 1.

64. The method of any one of claims 49 - 63, wherein the patient has respiratory distress.

65. The method of claim 64, wherein the patient requires supplemental oxygen.

66. The method of any one of claims 49 - 65, wherein the composition is administered by inhalation.

67. The method of claim 66, wherein the composition is administered by a nebulizer.

68. The method of claim 66 or claim 67, wherein 8 ml of the composition is administered to the patient.

69. The method of any one of claims 66 - 68, wherein the composition is administered quater in die.

70. The method of any one of claims 49 - 65, wherein the composition is administered intravenously.

71. The method of claim 70, wherein 250 cc of the composition is administered to the patient.

72. The method of claim 70 or claim 71, wherein the composition is administered bis in die.

73. The method of any one of claims 49 - 72, wherein the composition further comprises N-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinations thereof.

74. The method of any one of claims 49 - 73, wherein the pharmaceutical composition is prepared by removing albumin from a solution of a human serum albumin composition.

75. The method of claim 74, wherein the albumin is removed by ultrafiltration, sucrose gradient centrifugation, chromatography, salt precipitation, sonication, or combinations thereof.

76. The method of claim 74 or claim 75, wherein the albumin is removed by passing the human serum albumin composition over an ultrafiltration membrane with a molecular weight cut off that retains the albumin, wherein the resulting filtrate comprises DA-DKP.

77. The method of claim 76, wherein the ultrafiltration membrane has a molecular weight cutoff of less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5 kDa or less than 3 kDa.

78. The method of any one of claims 49, 50 or 57 - 77, wherein the TLR7- and/or TLR8-mediated disease is selected from the group consisting of lupus, lupus nephritis, and combinations thereof.