Treatment of coronavirus infection and related cytokine toxicity

Abstract

The present invention relates to novel methods involving the administration of pentosan polysulfate for treating or preventing coronavirus infections and cytokine-associated toxicity, including cytokine toxicity resulting from aberrant activation of the immune system in coronavirus diseases or infections, such as those from SARS-CoV-2.

Description

TECHNICAL FIELD

[0001] Field of the Invention The present invention relates to novel methods for treating or preventing coronavirus infection and cytokine-related toxicity, including cytokine toxicity resulting from abnormal activation of the immune system in coronavirus disease or infection, such as that derived from SARS-CoV-2.

[0002] Related Application This application claims priority from Australian provisional patent applications AU2020900751 and AU2020904824, the entire contents of both of which are incorporated herein by reference. BACKGROUND ART

[0003] Background of the Invention Coronaviruses are a group of viruses that cause disease in mammals and birds. In humans, coronaviruses cause respiratory tract infections that are typically mild, such as some cases of the common cold (among other possible causes, mainly rhinoviruses), although rare forms such as SARS, MERS and SARS-CoV-2 can be fatal. Symptoms differ in other species; in chickens, they cause upper respiratory tract disease, while in cattle and pigs, they cause diarrhea. There are still no vaccines or antiviral drugs for preventing or treating human coronavirus infection.

[0004] Coronaviruses constitute the Orthocoronavirinae subfamily within the Coronaviridae family, Nidovirales order, and Riboviria realm. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases, the largest among known RNA viruses.

[0005] In 2020, the world faced an extreme situation when a highly infectious coronavirus (2019-nCoV; SARS-CoV-2) emerged as a disease called "COVID-19." SARS-CoV-2 infection has spread globally, affecting more than 200 countries, territories, or regions, and has resulted in over 2 million deaths and more than 100 million confirmed cases to date. Symptoms of COVID-19 range from mild to severely life-threatening, with a significant mortality rate.

[0006] Angiotensin-converting enzyme 2 (ACE2) appears to be a receptor target for SARS-CoV-2. ACE2 is an exopeptidase that catalyzes the conversion of angiotensin I to nonapeptide angiotensin or angiotensin II to angiotensin. ACE2 has a broad expression profile and is expressed to varying degrees in almost all human organs. In the respiratory system, ACE2 is mainly expressed on type II alveolar epithelial cells, but is also expressed at lower levels in the oral and nasal mucosa and nasopharynx. ACE2 is also highly expressed on cardiomyocytes, proximal tubular cells of the kidney, and urothelial cells of the bladder, and is abundantly expressed on intestinal cells of the small intestine, particularly in the ileum. As a result, there are multiple potential entry sites for the SARS-CoV-2 virus.

[0007] Because SARS-CoV-2 receptor targets are expressed in multiple different tissue types, COVID-19 disease has been shown to manifest with symptoms affecting the intestinal and vascular systems in addition to the respiratory system. As the disease progresses or worsens, the risk of abnormal inflammation and cytokine storms increases.

[0008] Cytokine-related toxicity, also known as cytokine release syndrome (CRS), is a non-antigen-specific toxicity that results from high levels of immune activation. CRS manifests when a large number of lymphocytes and / or bone marrow cells become activated and release inflammatory cytokines. The result is a potentially fatal immune response consisting of a positive feedback loop between cytokines and lymphocytes. In severe cases, CRS is referred to as a "cytokine storm" and can occur as a result of coronavirus infection.

[0009] Cytokine-related toxicity has been reported to occur in response to a variety of infectious and non-infectious diseases, including graft-versus-host disease (GVHD), acute respiratory distress syndrome (ARDS), sepsis, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS).

[0010] There is a need for new or improved treatments for coronavirus infection and / or conditions related to or caused by coronavirus. Any reference to prior art in this specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art can be reasonably expected to be understood, considered relevant, and / or combined with other parts of the prior art by those skilled in the art. [Overview of the Initiative]

[0011] In the first embodiment, the present invention is: - To provide information to individuals suspected of having or at risk of developing coronavirus infection. - Administer a therapeutically effective amount of sulfated polysaccharide or a pharmaceutically acceptable salt thereof to the subject. Includes, This will enable the treatment or prevention of coronavirus infection in the patient. To provide methods for treating or preventing coronavirus infection.

[0012] In a further embodiment, the present invention provides a method for reducing the severity of a coronavirus infection, the method comprising administering a therapeutically effective amount of a sulfated polysaccharide or a pharmaceutically acceptable salt thereof to a subject in need, thereby reducing the severity of the coronavirus infection in the subject. Preferably, the coronavirus infection is an infection with a betacoronavirus of lineage B, more preferably an infection with SARS-CoV or SARS-CoV-2. More preferably, the infection is an infection with SARS-CoV-2, or a variant or variant thereof, including but not limited to variants D614G, S477N, 20A.EU1, the Danish COVID-19 mink variant, the "UK" variant B1.1.7, the "South Africa" ​​variant B.1.351, the "Brazil" variant P.1 or the "Russia" variant B.1.1.317.

[0013] In another embodiment, the present invention provides a method for treating and / or preventing a disease related to or caused by coronavirus, the method comprising administering a therapeutically effective amount of a sulfated polysaccharide or a pharmaceutically acceptable salt thereof to a subject in need thereof, thereby treating and / or preventing a disease related to or caused by coronavirus.

[0014] Preferably, the disease is associated with or caused by a betacoronavirus of lineage B, more preferably SARS-CoV or SARS-CoV-2. Most preferably, the disease is associated with or caused by SARS-CoV-2, or its variants or variants, including but not limited to variants D614G, S477N, 20A.EU1, or the Danish COVID-19 mink variant. Alternatively, the disease is associated with or caused by a betacoronavirus of lineage C, optionally MERS-CoV.

[0015] Diseases associated with or caused by coronavirus infection may include respiratory diseases, inflammatory diseases (including cytokine-related toxicity), cardiovascular diseases, or gastrointestinal diseases.

[0016] Accordingly, in another embodiment, the present invention provides a method for treating and / or preventing a respiratory illness or condition associated with coronavirus infection, the method comprising administering a therapeutically effective amount of a sulfated polysaccharide or a pharmaceutically acceptable salt thereof to a subject in need thereof, thereby treating and / or preventing a respiratory illness or condition associated with coronavirus infection. Preferably, the respiratory illness or condition is associated with a betacoronavirus of lineage B, more preferably SARS-CoV or SARS-CoV-2. Most preferably, the respiratory illness or condition is associated with or caused by SARS-CoV-2, or its variants or variants, including but not limited to variants D614G, S477N, 20A.EU1, the Danish COVID-19 mink variant, the "UK" variant B1.1.7, the "South Africa" ​​variant B.1.351, the "Brazil" variant P.1 or the "Russia" variant B.1.1.317.

[0017] In this embodiment, the respiratory disease or condition may include airway inflammation, and the method thereby reduces airway inflammation in the subject or improves the subject's ability to cope with the respiratory disease or condition during coronavirus infection.

[0018] Furthermore, this method is a method for treating or preventing cytokine-related toxicity caused by or associated with coronavirus infection, wherein the method is: - To provide subjects who are suspected of having coronavirus infection and are at risk of developing or have developed cytokine-related toxicity. - Administer a therapeutically effective amount of sulfated polysaccharide or a pharmaceutically acceptable salt thereof to the subject. Includes, This provides a method for treating or preventing cytokine-related toxicity in the subject. Preferably, the cytokine-related toxicity is associated with or caused by a betacoronavirus of lineage B, more preferably SARS-CoV or SARS-CoV-2. Most preferably, the disease is associated with or caused by SARS-CoV-2, or its variants or variants, including but not limited to variants D614G, S477N, 20A.EU1, or the Danish COVID-19 mink variant. Alternatively, the disease is associated with or caused by a betacoronavirus of lineage C, optionally MERS-CoV.

[0019] In any embodiment, treatment of coronavirus disease includes treating one or more symptoms associated with infection with coronavirus, or with or caused by coronavirus infection. Therefore, in a further embodiment, the present invention relates to a method for treating one or more of the following: fever, hypotension, tachycardia, muscle pain, headache, vasculitis, gastrointestinal inflammation and dysfunction, or inappropriate cytokine release caused by or related to coronavirus infection, wherein the method is: - To provide subjects who have or are suspected of having coronavirus infection, wherein the individual has or is suspected of having fever, hypotension, tachycardia, muscle pain, headache, vasculitis, gastrointestinal inflammation and dysfunction, or inappropriate cytokine release induced by coronavirus infection. -Administering a therapeutically effective amount of sulfated polysaccharide or a pharmaceutically acceptable salt thereof to the individual. Includes, This treats one or more of the following: fever, hypotension, tachycardia, muscle pain, headache, vasculitis, gastrointestinal inflammation and dysfunction, or inappropriate cytokine release induced by coronavirus infection. The present invention relates to a method. Preferably, the coronavirus infection is an infection with a betacoronavirus, preferably an infection originating from a betacoronavirus from lineage B such as infection with SARS-CoV or infection with SARS-CoV-2. Preferably, the coronavirus infection is SARS-CoV-2 infection, or infection with a variant or mutant thereof including, but not limited to, variant D614G, S477N, 20A.EU1, or the Danish COVID-19 mink variant.

[0020] In a further aspect, the present invention provides: - providing an individual suspected of having cytokine-related toxicity induced by acute respiratory distress syndrome (ARDS), sepsis, systemic inflammatory response syndrome (SIRS), severe viral infection, severe acute respiratory syndrome (SARS) pneumonia, - administering to said individual an effective amount of a sulfated polysaccharide or a pharmaceutically acceptable salt thereof for treating cytokine-related toxicity comprising, wherein the sulfated polysaccharide is selected from the group consisting of pentosan polysulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratan sulfate, heparin, heparan sulfate, dextran polysulfate, fucoidan, lentinan sulfate, mannan sulfate, galactan sulfate, xylomannan sulfate, rhamnan sulfate, curdlan sulfate or sulfated fucan, and pharmaceutically acceptable salts thereof, thereby treating cytokine-related toxicity in said individual, provided is a method for treating cytokine-related toxicity induced by acute respiratory distress syndrome (ARDS), sepsis, systemic inflammatory response syndrome (SIRS), severe viral infection, severe acute respiratory syndrome (SARS) pneumonia.

[0021] The present invention also provides - providing a subject who has been exposed to a coronavirus infection, wherein the subject is at risk of developing fever, hypotension, tachycardia, myalgia, headache or inappropriate cytokine release induced by said exposure, - Administer to the subject an amount effective in preventing one or more of the following: fever, hypotension, tachycardia, muscle pain, headache, or inappropriate cytokine release. Includes, This prevents fever, hypotension, tachycardia, muscle pain, headache, or inappropriate cytokine release induced by exposure to the coronavirus. This also relates to ways to prevent one or more of the fever, low blood pressure, tachycardia, muscle pain, headache, or inappropriate cytokine release induced by coronavirus infection.

[0022] It will be recognized that the methods of the present invention have applicability to the treatment or prevention of various disease severities. In certain embodiments, the symptoms are mild enough for the subject to be treated at home. In other embodiments, the symptoms are more severe and require hospitalization or even treatment in an intensive care unit.

[0023] In a further embodiment of the present invention, a method is provided for preventing or reducing the likelihood of infection with coronavirus (including severe infection). The method comprises administering the sulfated polysaccharides described herein to subjects who are considered to be at risk of coronavirus or who are known to have been exposed to it. Examples of subjects who may be at risk of exposure and infection include subjects with immunodeficiency (including subjects with primary or secondary immunodeficiency), adults over 60 years of age, children under 2 years of age, healthcare workers, adults or children who have had close contact with a person(s) who have a confirmed or suspected coronavirus infection, and people with underlying medical conditions such as lung infections, heart disease, obesity or diabetes.

[0024] The method of the present invention is also useful in reducing the viral load in asymptomatic individuals, thereby reducing the likelihood of such individuals spreading the infection to other individuals. Asymptomatic individuals can be identified via fecal, nasal, and / or pharyngeal swabs that indicate the presence of coronavirus infection.

[0025] In any embodiment described herein, the sulfated polysaccharide is selected from the group consisting of pentosan polysulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratan sulfate, heparin, heparan sulfate, dextrampolysulfate, fucoidan, lentinan sulfate, mannan sulfate, galactan sulfate, xylomannan sulfate, rhamnan sulfate, curdlan sulfate, or sulfated fucane and pharmaceutically acceptable salts thereof.

[0026] In a particularly preferred embodiment of the present invention, the sulfated polysaccharide is pentosan polysulfate or a pharmaceutically acceptable salt thereof.

[0027] In any embodiment of the present invention, sulfated polysaccharides, preferably pentosan polysulfates (PPS), are administered to an individual intravenously, subcutaneously, orally, intramuscularly, via a gastric tube, intranasally, or by inhalation.

[0028] This invention also: - To treat or prevent coronavirus infection; - Reduces the severity of coronavirus infection; - To treat and / or prevent diseases related to or caused by coronavirus infection; - To treat or prevent respiratory diseases, inflammatory diseases (including cytokine-related toxicity), cardiovascular diseases, or gastrointestinal diseases caused by or associated with coronavirus infection; - To treat or prevent cytokine-related toxicity caused by or associated with coronavirus infection; and / or - To treat or prevent one or more of the following symptoms induced by coronavirus infection: fever, hypotension, tachycardia, muscle pain, headache, or inappropriate cytokine release. This invention provides the use of sulfated polysaccharides or pharmaceutically acceptable salts thereof in the manufacture of pharmaceuticals.

[0029] In any use described herein, the sulfated polysaccharide is optionally selected from the group consisting of: pentosan polysulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratan sulfate, heparin, heparan sulfate, dextran polysulfate, fucoidan, lentinan sulfate, mannan sulfate, galactan sulfate, xylomannan sulfate, rhamnan sulfate, curdlan sulfate, or sulfated fucane and pharmaceutically acceptable salts thereof. Preferably, the sulfated polysaccharide is pentosan polysulfate or a pharmaceutically acceptable salt thereof.

[0030] In any use described herein, coronavirus infection is an infection with a betacoronavirus, preferably derived from infectivity of a betacoronavirus of lineage B, such as infection with SARS-CoV or SARS-CoV-2. Preferably, coronavirus infection is SARS-CoV-2 infection, or an infection with a variant or variant thereof, including but not limited to variants D614G, S477N, 20A.EU1, or the Danish COVID-19 mink variant.

[0031] The present invention also, - To treat or prevent coronavirus infection; - To reduce the severity of coronavirus infection; - Treating and / or preventing diseases related to or caused by coronavirus infection; - To treat or prevent respiratory diseases, inflammatory diseases (including cytokine-related toxicity), cardiovascular diseases, or gastrointestinal diseases caused by or related to coronavirus infection; - Treating or preventing cytokine-related toxicity caused by or associated with coronavirus infection; and / or - To treat or prevent one or more of the following symptoms induced by coronavirus infection: fever, hypotension, tachycardia, muscle pain, headache, or inappropriate cytokine release. The present invention provides a pharmaceutical composition for use in the field of [unspecified field], comprising a sulfated polysaccharide, preferably pentosan polysulfate, or a pharmaceutically acceptable salt thereof, or a sulfated polysaccharide, preferably pentosan polysulfate, or a pharmaceutically acceptable salt thereof.

[0032] When used herein, unless the context requires otherwise, the term “comprise,” and its variations such as “comprising,” “comprises,” and “included,” are not intended to exclude further additives, ingredients, integers, or processes.

[0033] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description and accompanying drawings, which are given as examples. [Brief explanation of the drawing]

[0034] Description of the drawing [Figure 1] Inhibition of SARS-CoV-2 replication by polysulfated polysaccharides. The data presented show inhibition by polysaccharides compared to a control. [Figure 2] M100 microscopy images of HCT116 cells administered with FITC-NaPPS (green) and then immunostained for Rab9 (red). Extensive cytoplasmic and perinuclear co-localization of Rab9 with NaPPS was observed in HCT116 cells (arrows). [Figure 3] M100 microscopy images of HCT116 cells given FITC-NaPPS (green) and then immunostained for TGN46 (red), a marker of the trans-Golgi network. TGN36 stained the trans-Golgi in a classical intracisional morphology. NaPPS accumulation was observed just outside the edges of the trans-Golgi stack (arrows). [Modes for carrying out the invention]

[0035] Detailed description of the invention It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0036] Hereinafter, specific embodiments of the present invention will be described in detail. While the present invention will be described in conjunction with its embodiments, it will be understood that the intent is not to limit the present invention to those embodiments. Rather, the present invention is intended to cover all substitutes, modifications, and equivalents that may fall within the scope of the present invention as defined by the claims.

[0037] Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein that could be used in carrying out the present invention. The present invention is by no means limited to the methods and materials described herein. It will be understood that the present invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0038] This invention is based on the remarkable discovery by the inventors that polysulfated polysaccharides are useful in reducing the severity of coronavirus infection and can be used to treat diseases or conditions associated with or caused by coronavirus infection. In particular, the inventors have found that pentosan polysulfate (PPS), a polysulfated polysaccharide, is significantly more effective than other polysulfated polysaccharides in reducing SARS-CoV-2 replication and can therefore be used to reduce or prevent the severity of infection with SARS-CoV-2. The method of the present invention is therefore also useful in reducing the likelihood of subjects developing severe disease and / or cytokine-related toxicity resulting from infection with coronavirus.

[0039] coronavirus As used herein, "coronavirus" refers to members of the Coronavirus Subfamily within the Coronaviridae family and Nidovirales order (International Committee for Taxonomy of Viruses). This subfamily consists of four genera based on their phylogenetic relationships and genomic structure: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Subgroup clusters are labeled as 1a and 1b for alphacoronavirus, and 2a, 2b, 2c, and 2d for betacoronavirus. Alphacoronavirus and betacoronavirus infect only mammals. Gammacoronavirus and deltacoronavirus infect birds, although some of them can also infect mammals. Alphacoronavirus and betacoronavirus typically cause respiratory illness in humans and gastroenteritis in animals. Three highly pathogenic viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, cause severe respiratory syndromes in humans, while four other human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1) induce only mild upper respiratory tract illness in immunocompetent hosts, although some of them can cause severe infections in infants, toddlers, and elderly individuals. Alpha- and beta-coronaviruses can impose a heavy disease burden on livestock; these viruses include porcine infectious gastroenteritis virus, porcine enteric diarrhea virus (PEDV), and the recently emerged porcine acute diarrhea syndrome coronavirus (SADS-CoV). Based on current sequence databases, all human coronaviruses have animal origins: SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, and HCoV-229E are thought to originate from bats; HCoV-OC43 and HKU1 are likely from rodents.

[0040] Coronaviruses include antigen groups I, II, and III. Non-exclusive examples of coronaviruses include SARS coronavirus, MERS coronavirus, infectious gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, mouse hepatitis virus, sialiomyelitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as any other described herein, and including those referenced in Cui, et al. Nature Reviews Microbiology volume 17, pages 181-192 (2019), and Shereen et al. Journal of Advanced Research, Volume 24, July 2020 (published online on March 16, 2020), Pages 91-98.

[0041] Subjects or individuals requiring treatment according to any aspect of the present invention, or requiring administration of any composition described herein, may be individuals exhibiting symptoms of coronavirus infection or diagnosed with coronavirus infection. Furthermore, subjects or individuals may be those clinically or biochemically determined to be infected with coronavirus (e.g., via sequencing of blood samples or sequencing or antibody-based approaches for detecting coronavirus in nasal and / or nasopharyngeal swabs, or from saliva-based assays).

[0042] The subjects may be in the stages of coronavirus infection before the onset of terminal organ failure. Those requiring this may be any person who has coronavirus infection from the onset of clinical progression, before the onset of terminal organ failure. In one embodiment, the subjects have had symptoms of coronavirus infection for 12 days or less and do not have life-threatening organ dysfunction or organ failure. Preferably, the subjects are in the early stages of the disease course, for example, before 14 days from the onset of symptoms, or during the viremia and serologically negative stage.

[0043] The subject may have recovered from the symptoms or signs of an infection, but may still be emitting or harboring viral particles. The subject may have “long COVID” and may be presenting with signs or symptoms of a past coronavirus infection.

[0044] The "subject" or "individual" may also be any animal susceptible to infection by coronavirus and / or susceptible to disease or disorder caused by coronavirus infection. The subject of this invention may be a mammal, and in certain embodiments may be a human, who may be an infant, child, adult or elderly adult.

[0045] "Subjects at risk of coronavirus infection" or "subjects at risk of coronavirus infection" is any subject that may have been or is currently exposed to the coronavirus. "Subject" or "individual" includes any human or non-human animal. In other words, in addition to being useful in the treatment of humans, the compounds of the present invention may also be useful in the veterinary treatment of mammals, including, but not limited to, companion animals and farm animals, such as dogs, cats, horses, cattle, sheep, and pigs, or any animal that can be infected by the coronavirus.

[0046] Those at risk include, but are not limited to, individuals with immunodeficiency, older adults (over 65 years of age), children under 2 years of age, healthcare workers, adults or children who have had close contact with a confirmed or suspected coronavirus infection, and individuals with underlying medical conditions such as lung infection, heart disease or diabetes, or primary or secondary immunodeficiency.

[0047] Treatment of coronavirus infection and related diseases and conditions Among the conditions associated with coronavirus infections, particularly those caused by SARS-CoV-2, is an inappropriate or uncontrolled activation of the immune system, otherwise referred to as a "cytokine storm" or "cytokine-related toxicity."

[0048] Inappropriate or uncontrolled activation of the immune system can lead to a potentially fatal immune response consisting of a positive feedback loop between cytokines and leukocytes, with highly elevated levels of various cytokines. Such uncontrolled immune activation, often called a cytokine cascade, cytokine-related toxicity, or cytokine release syndrome, can be induced by many physical conditions or medical therapies, most notably immunotherapies that specifically utilize the recipient's immune system to fight disease. Cytokine storms have been demonstrated as a result of coronavirus infections.

[0049] Abnormal cytokine release can be a cause of graft-versus-host disease (GVHD), avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis, and systemic inflammatory response syndrome (SIRS). In fact, in many of these conditions, the cause of death is not the disease itself, but rather the effects of the cytokine cascade or storm induced as a result of the disease. For example, cytokine storms are thought to be the cause of the disproportionate deaths of many healthy young adults during the 1918 influenza pandemic; that is, influenza was not the cause of death, but rather the uncontrolled activation of the immune system in response to the infection.

[0050] While different terms may be used to describe cytokine storms, cytokine cascades, or cytokine release syndromes, all of these conditions share a common characteristic: uncontrolled activation of the immune system, which can lead to potentially fatal consequences.

[0051] Current approaches to managing cytokine-related toxicity include the use of immunosuppressants, including monoclonal antibodies that bind to IL-6, corticosteroids, and vasopressors (such as norepinephrine, adrenaline, and dopamine). The problem with this approach, however, is the risk of reducing the effectiveness of immunotherapy in attempting to prevent or control cytokine-related toxicity through immunosuppression.

[0052] Therefore, there is a need for new approaches to treat or prevent cytokine-related toxicity derived from immunotherapy, which promote or achieve the prevention, control, downregulation, and / or termination of cytokine-related toxicity, and reduce or minimize the likelihood of undesirable side effects occurring.

[0053] The inventors have surprisingly discovered that sulfated polysaccharides can be used to control cytokine release as a result of abnormal immune system activation, regardless of whether their activation occurs as a result of coronavirus infection, avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis, and systemic inflammatory response syndrome (SIRS).

[0054] Therefore, in the first embodiment, the method is - To provide individuals suspected of having coronavirus infection, - Administering to the individual an effective amount of sulfated polysaccharide or a pharmaceutically acceptable salt thereof to treat coronavirus infection. Includes, This includes treating coronavirus infection in the individual, Regarding methods for treating coronavirus infection.

[0055] The present invention also intends to use sulfated polysaccharides as a means to prevent the development of coronavirus infection in an individual, particularly when the individual has been exposed to the coronavirus.

[0056] Examples of pro-inflammatory cytokines or pro-inflammatory mediators include interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) (hereinafter collectively referred to as interleukin-1 or IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-17 (IL-17), interleukin-18 (IL-18), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor beta (TGF-β). It will be recognized by those skilled in the art that references to pro-inflammatory cytokines in most embodiments of this disclosure may refer to any one or more pro-inflammatory cytokines known in the art, and may include one or more of the examples of inflammatory cytokines listed above.

[0057] In some embodiments, a reduction in the amount of pro-inflammatory cytokines within an individual helps prevent, control, downregulate, and / or halt the development of cytokine-related toxicity in that individual.

[0058] In some embodiments, the use of sulfated polysaccharides by the method of the present invention affects the state of anti-inflammatory cytokines, anti-inflammatory mediators, or anti-inflammatory factors by, for example, promoting or achieving an increase in the amount of anti-inflammatory cytokines, anti-inflammatory mediators, and / or anti-inflammatory factors in the body. Examples of anti-inflammatory cytokines, anti-inflammatory mediators, and / or anti-inflammatory factors include interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-13 (IL-13), and interferon alpha (IFN-α). Those skilled in the art will understand that references to anti-inflammatory cytokines, anti-inflammatory mediators, and / or anti-inflammatory factors in most embodiments of this disclosure may relate to any one or more anti-inflammatory cytokines, anti-inflammatory mediators, and / or anti-inflammatory factors known in the art, including the examples listed above.

[0059] In some embodiments of this disclosure, the use of sulfated polysaccharides promotes or achieves a reduction in the gene expression of one or more pro-inflammatory cytokines in the body. For example, in some embodiments, administration of sulfated polysaccharides to an individual at risk or suspected of having cytokine-related toxicity promotes or achieves a reduction in the IL-1 gene expression in the body's immune cells (i.e., cells involved in immune responses). Examples of immune cells include lymphocytes, phagocytes, fibroblasts, monocytes, neutrophils, and macrophages.

[0060] In some embodiments of this disclosure, administration of sulfated polysaccharides promotes or achieves a reduction in the secretion or release of one or more pro-inflammatory cytokines by immune cells in the body. For example, in some embodiments, sulfated polysaccharides promote or achieve a reduction in the release or secretion of IL-1 from immune cells.

[0061] In many embodiments, a decrease in the gene expression of pro-inflammatory cytokines (e.g., IL-1 gene expression) leads to a decrease in the secretion of pro-inflammatory cytokines by immune cells (e.g., a decrease in IL-1 secretion), and therefore to lower levels of pro-inflammatory cytokines in the body (e.g., lower levels of IL-1). Lower levels of pro-inflammatory cytokines (e.g., IL-1) in the body promote, achieve, or provide anti-inflammatory effects.

[0062] In some embodiments of this disclosure, administration of sulfated polysaccharides promotes or achieves an increase in the gene expression of one or more anti-inflammatory cytokines in the body. For example, in some embodiments, sulfated polysaccharides promote or achieve increased gene expression of IL-2 by immune cells in the body. In some embodiments, sulfated polysaccharides promote or achieve an increase in the secretion or release of one or more anti-inflammatory cytokines by immune cells in the body. For example, in some embodiments, sulfated polysaccharides promote or achieve increased secretion of IL-2 by immune cells in the body.

[0063] In further embodiments, an increase in the gene expression of one or more anti-inflammatory cytokines (e.g., IL-2) leads to increased secretion of one or more anti-inflammatory cytokines by the body's immune cells, and therefore to a higher amount of anti-inflammatory cytokines in the body. A higher amount of anti-inflammatory cytokines in the body promotes, achieves, or provides an anti-inflammatory effect.

[0064] A reduction in coronavirus infection can be determined using any method known in the art or described herein, which includes measuring the viral load in a sample from a subject after treatment and comparing it to the viral load in a sample from the same subject before treatment. The sample may be any biological sample obtained from the subject and may include blood, saliva, urine, feces, nasal lavage fluid, sputum, and mucous secretions. The sample may be taken from the respiratory tract, preferably the upper respiratory tract, for example, the nose or pharynx (i.e., throat).

[0065] The term “respiratory disease” or “respiratory condition” refers to any one of several diseases that involve inflammation and affect the components of the respiratory system, including the upper respiratory tract (including the nasal cavity, pharynx, and larynx) and the lower respiratory tract (including the trachea, bronchi, and lungs). Inflammation in the upper and lower respiratory tracts may be associated with or caused by viral infections.

[0066] Symptoms of respiratory illness may include cough, excessive mucus production, shortness of breath, or chest tightness with audible wheezing.

[0067] The presence, improvement, treatment, or prevention of respiratory disease may be determined by any clinically or biochemically relevant method of the subject or by biopsy therefrom. For example, parameters to be measured may include the presence or degree of lung function, signs and symptoms of obstruction; exercise tolerance; nocturnal awakenings; number of days absent from school or work; use of bronchodilators; dosage of inhaled corticosteroids (ICS); use of oral glucocorticoids (GC); need for other medications; need for medical treatment; and hospitalization.

[0068] As used herein, the term respiratory infection means an infection of any location in the respiratory tract caused by a coronavirus, preferably SARS-CoV-2.

[0069] Individuals may be identified as having a respiratory infection by viral testing and may exhibit symptoms such as itchy, watery eyes, runny nose, nasal congestion, sneezing, sore throat, cough, headache, fever, malaise, fatigue, and weakness. In one embodiment, a subject with a respiratory infection may not have any other respiratory condition. Detection of the presence or amount of the virus is obtained by PCR / sequencing or serology of RNA isolated from clinical samples (nasal lavage fluid, sputum, BAL).

[0070] The term “treatment” or “to treat” includes the application or administration of polysulfated polysaccharides as described herein for the purpose of delaying, slowing, stabilizing, curing, healing, mitigating, reducing, modifying, treating, mitigating, ameliorating, improving, or influencing a disease or condition, the symptoms of a disease or condition, or the risk (or susceptibility) to a disease or condition.

[0071] The term “to treat” refers to any measure of success in treating or improving an injury, condition or condition, including any objective or subjective parameters such as abatement; remission; lessening; lessening the severity of the disease; stabilization, diminishing or making the injury, condition or state more tolerable to the subject; slowing the rate of degeneration or decay; making the final stage of degeneration less debilitating; or improving the physical or mental well-being of the subject.

[0072] It will be recognized that coronavirus infection may not necessarily lead to severe illness such as a cytokine storm. The present invention therefore intends to use sulfated polysaccharides for the treatment of any symptoms of coronavirus infection, regardless of how mild or severe they may be. That is, the present invention includes a method for treating individuals who have relatively mild symptoms of the infection, such as headache, fever, cough, and fatigue. Such individuals will usually not require hospitalization (and can be treated at home). Other individuals may have sufficiently severe symptoms that may require hospitalization, but it will be recognized that such individuals may not necessarily develop a cytokine storm.

[0073] Furthermore, it should be recognized that not all cases of coronavirus infection, including infection with SARS-CoV-2, are asymptomatic. However, given the inventors' findings, it will be recognized that the method of the present invention finds application in the treatment of infection (including reducing the viral load in the individual) even when the individual is asymptomatic. Such individuals can be easily identified through the use of routine screening of fecal, nasal, and / or nasopharyngeal swabs. Moreover, it will be recognized that this approach reduces the likelihood of an individual spreading their infection to others, and as a result, the method of the present invention can be extended to reduce the likelihood of transmission of coronavirus infection to another individual.

[0074] Given the wide range of symptoms (and their severity) caused by coronaviruses, including those caused by SARS-CoV-2, it will be recognized that a positive response to treatment by the methods described herein may include any amelioration or improvement of the symptoms experienced by the subject.

[0075] For example, a positive response to treatment may be a reduction in the general levels of fatigue, muscle pain, headache, and / or lethargy in the subject. A positive response may also include a reduction in fever and a return to a fever-free state in the subject.

[0076] A positive response to treatment may also be the prevention or reduction of the worsening of respiratory symptoms following a respiratory viral infection. This could be assessed, for example, by comparing the mean change in disease scores from baseline to the end of the study period, based on a questionnaire, or by assessing the lower respiratory tract symptom score (LRSS - chest tightness, wheezing, shortness or breath, and cough) daily following the onset of infection / cold symptoms. Changes from baseline lung function (peak expiratory flow rate, PEF) can also be assessed, and a positive response to treatment may be a significant reduction in PEF. For example, the placebo group might show a significant 15% decrease in morning PEF at the peak of an exacerbation, while the treatment group might show a non-significant decrease in PEF, with a change of less than 15% from baseline.

[0077] A positive response to treatment may also be a reduction in the presence of ground-glass opacities around the lungs or near the pleura (determined, for example, using chest CT imaging techniques).

[0078] Positive responses to treatment may also include an increase in blood oxygenation levels or a return to normal levels.

[0079] Positive responses to treatment may also include improvements in cardiovascular conditions, such as changes in blood pressure and the presence of increased clotting factors.

[0080] The method of the present invention may be further applied once an individual has recovered from an initial coronavirus infection. Such a method may prove useful in treating "long COVID" symptoms, or in individuals with prolonged viral particle shedding, or in individuals with prolonged viral particle shedding. The method of the present invention may also be used to prevent or reduce the likelihood of reinfection with coronaviruses, including reinfection with SARS-CoV-2 (and its variants).

[0081] sulfated polysaccharide As used herein, the term sulfated polysaccharide includes sulfated polysaccharides, sulfated oligosaccharides, and glycosaminoglycans, such as heparin sulfate proteoglycans, which contain glycosaminoglycans linked to a core protein.

[0082] Non-limiting examples of sulfated polysaccharides include pentosan polysulfate (PPS), chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratan sulfate, heparin, heparan sulfate, dextramen polysulfate, fucoidan, lentinan sulfate, mannan sulfate, galactan sulfate, xylomannan sulfate, rhamnan sulfate, curdlan sulfate, and sulfated fucane.

[0083] The present invention also intends to utilize various pharmaceutically acceptable salts of any one of the above-mentioned sulfated polysaccharides, including but not limited to sodium, calcium, potassium, and magnesium salts.

[0084] Therefore, in further terms, this method is - To provide individuals suspected of having cytokine-related toxicity, - Administering to the individual an effective amount of polysulfated polysaccharide or a pharmaceutically acceptable salt thereof to treat the cytokine-related toxicity. Includes, Here, the polysulfated polysaccharide is selected from the group consisting of pentosan polysulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratan sulfate, heparin, heparan sulfate, dextran polysulfate, fucoidan, lentinan sulfate, mannan sulfate, galactan sulfate, xylomannan sulfate, rhamnan sulfate, curdlan sulfate, or sulfated fucane. This is about methods for treating cytokine-related toxicity.

[0085] The present invention also intends to utilize the molecular weight variation of any one of the sulfated polysaccharides described herein, which include molecular weights in the range of 100 to 10,000,000 units by mass, preferably 1,000 to 100,000 units by mass, and more preferably 1,800 to 9,000 units by mass.

[0086] Those skilled in the art will also recognize that the degree of sulfated polysaccharides described herein can vary. This variation in degree of sulfated polysaccharides occurs in both naturally occurring and synthetically produced sulfated polysaccharides. The degree of sulfated polysaccharides can preferably be about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or greater than 2.0. In preferred embodiments, the present invention relates to sulfated polysaccharides for use described herein, wherein the degree of sulfated polysaccharide is high, for example, 1.9.

[0087] Pentosan polysulfate In a particularly preferred embodiment of the present invention, the sulfated polysaccharide is pentosan polysulfate (PPS; also known as hydrogen sulfate [(2R,3R,4S,5R)-2-hydroxy-5-[(2S,3R,4S,5R)-5-hydroxy-3,4-disulfoxyoxan-2-yl]oxy-3-sulfoxyoxan-4-yl]), a semi-synthetically derived heparin-like high molecular weight carbohydrate derivative that is chemically and structurally similar to glycosaminoglycans (GAGs). There are 40 synonyms for pentosan polysulfate listed on PubChem, including BAY-946, HOE-946, pentosan sulfate polyester, polypentose sulfate, polysulfated xylan, PZ-68, SP-54, xylan SP54, and xylan sulfate. PPS is disclosed in U.S. Patent No. 2,689,848, and this compound has been known as a synthetic heparinoid and antithrombotic agent since the early 1960s. PPS was originally developed as a heparin-like drug that inhibits the binding of factor Xa to thrombin through a mechanism independent of AT-III. While it has similar structural properties to heparin, PPS is more sulfated, resulting in a larger overall negative charge. Furthermore, PPS possesses only about 1 / 15th the anticoagulant properties of heparin.

[0088] PPS is most commonly used as an oral formulation for treating interstitial cystitis in humans and as an injectable formulation for treating osteoarthritis in companion animals (Fuller, Ghosh et al., “Plasma and synovial fluid concentrations of calcium pentosan polysulfate achieved in the horse following intramuscular injection,” Equine Veterinary Journal (2002)). Although the compound PPS has 1 / 15th (if) the anticoagulant properties of heparin, it can also be used as an anticoagulant to prevent thrombus formation. PPS has also been used to treat hematomas, hemorrhoids, frostbite, burns, and multi-parameter diseases such as thrombosis and atherosclerosis.

[0089] In the context of interstitial cystitis, PPS is thought to provide a protective coating to the damaged bladder wall because its structure is similar to the natural glycosaminoglycan coating of the bladder's inner lining. In other words, PPS is thought to replace or repair the bladder's inner lining in interstitial cystitis, thereby reducing permeability.

[0090] The mechanism of action of PPS in osteoarthritis is multifactorial, involving both stimulation of cartilage matrix synthesis and prevention of cartilage destruction. It also has systemic effects on blood lipids and fibrinolysis, which can help cleanse the subchondral circulation.

[0091] PPS is obtained by sulfation esterification of beech wood (Fagus sylvatica) hemicellulose and consists of a mixture of polymers having molecular weights ranging from 1,800 to 9,000 units by mass (average 4,700 units). PPS is produced from a chemical solution of polysaccharides (e.g., xylan) extracted from beech bark or other plant sources and then treated with sulfurizing agents and acids such as chlorosulfonic acid or sulfuryl chloride. After sulfation, PPS is usually treated with sodium hydroxide to produce sodium salts.

[0092] Various methods for the preparation of PPS, including WO / 2008 / 107906, WO / 2009 / 047699 and WO / 2012 / 114349, whose entire contents are incorporated herein by reference, are described.

[0093] In some embodiments, the polysulfated polysaccharide for use by the method of the present invention is pentosan polysulfate sodium, manufactured by Bene-PharmaChem GmbH & Co KG, Gerettried, Germany in accordance with specifications submitted to the U.S. FDA and the European Community (EMEA).

[0094] In various embodiments, the molecular weight is in the range of 100 to 10,000,000 units by mass, preferably 1,000 to 100,000 units by mass, and more preferably 1,800 to 9,000 units by mass.

[0095] The basic structure of PPS is a pentose, or (1->4) linked beta-D-xylopyranose unit, which statistically contains a glucuronic acid group every 10 units.

[0096] [ka]

[0097] The following is the structural formula of pentosan polysulfate (PPS) isolated from beech wood hemicellulose. This formula shows that the linear xylan (pentosan) main chain of pentosan polysulfate contains an average of one 4-O-methylglucuronic acid side chain linked at the 2nd position for each 10th xylose (pentose) ring.

[0098] Sodium derivatives are when R=SO3Na. The calcium derivative of PPS (CaPPS) is R=SO3Ca +1 or SO3Ca +2 At that time, the magnesium derivative of PPS (MgPPS) is R=SO3Mg +1or SO3Mg +2 That was the time.

[0099] [ka]

[0100] Pentosan polysulfate (PPS) is available as alkali metal salts or alkaline earth metal salts, for example, calcium or sodium salts, or transition metals such as copper and zinc, and noble metals such as platinum. Therefore, certain complexing ions are alkali metals, for example, Na + The group may be selected from alkaline earth metals, such as Ca2+, Zn2+, Mg2+, Ba2+, and Ag+, Pb2+, Cu2+, Au2+, Pd2+, Pd4+, Pd4+, Pd2+, trivalent metal ions, and quaternary ammonium compound complexes. Examples of the latter compounds are pyridinium chloride, tetraalkylammonium chloride, choline chloride, cetylpyridinium chloride, N-cetyl-N,N,N-trialkylammonium chloride, or derivatives thereof. The preparation of polysulfate polysaccharide-metal complexes is described in detail in U.S. Patent No. 5,668,116, the entire disclosure of which is incorporated herein by reference.

[0101] Accordingly, in further embodiments, the pharmaceutically acceptable salts of pentosan polysulfate used in the method of the present invention are the sodium salt of pentosan polysulfate (NaPPS), the magnesium salt of pentosan polysulfate (MgPPS), and / or the calcium salt of pentosan polysulfate (CaPPS). Preferably, the sodium salt is used in the method of the present invention.

[0102] In further embodiments, the present invention includes the use of pentosan polysulfates having various degrees of sulfatedness. For example, the degree of sulfatedness may be 0.2 to 2 units of sulfuric acid per sugar portion. In preferred embodiments, the degree of sulfatedness may be 1.9 units per sugar portion.

[0103] The present invention also includes biologically active molecular fragments of PPS or analogs or derivatives of PPS.

[0104] A key advantage of the present invention over current approaches to treating cytokine-related toxicity is the well-known safety profile of PPS. Even when administered at high doses for extended periods, PPS has been reported to cause only limited side effects (Nickel et al., 2005). A further advantage is that PPS is bioavailable when administered orally, intravenously, or subcutaneously (in contrast to other gag inhibitors).

[0105] Cytokine-related toxicity As used herein, cytokine-related toxicity refers to a potentially life-threatening and harmful cytokine response to abnormal immune system activation, such as that caused by disease, but also including immunomodulatory therapy.

[0106] Cytokine-related toxicity is also described in the Art using the term cytokine release syndrome (CRS). When sufficiently severe, this syndrome may be referred to as hypercytokinemia or “cytokine storm.” As used herein, CRS defines a systemic inflammatory response in a patient characterized, among other things, by hypotension, fever and / or chills, and potentially leading to death. CRS is thought to be caused by an uncontrolled positive feedback loop between cytokines and immune cells, which results in highly elevated levels of various cytokines. CRS also includes systemic expression of immune system mediators, as well as elevated levels of pro-inflammatory and anti-inflammatory cytokines.

[0107] During a cytokine storm, pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), oxygen free radicals, and coagulation factors, are released by the body's immune cells. Cytokine storms can cause significant damage to the body's tissues and organs. For example, a cytokine storm in the lungs can lead to the accumulation of fluid and immune cells, such as macrophages, in the lungs, ultimately blocking the body's airways and resulting in respiratory distress and even death.

[0108] As described above, the present invention relates to a method for treating or preventing cytokine-related toxicity (or adverse cytokine response). In certain embodiments, the cytokine-related toxicity is severe enough to be classified as a "cytokine storm" or hypercytokinemia. Therefore, in further embodiments, the present invention relates to a method for treating or preventing cytokine-related toxicity (or adverse cytokine response). - To provide individuals who are at risk of or suspected of having hypercytokinemia, - Administer to the individual an effective amount of sulfated polysaccharide or a pharmaceutically acceptable salt thereof to treat hypercytokinemia. Includes, This will treat hypercytokineemia in the individual. This paper discusses methods for treating hypercytokinemia in individuals.

[0109] Hypercytokinemia can be induced by graft-versus-host disease (GVHD), acute respiratory distress syndrome (ARDS), sepsis, systemic inflammatory response syndrome (SIRS), severe viral infection, shock, severe acute respiratory syndrome (SARS), or immunotherapy.

[0110] Therefore, this method also, - To provide individuals at risk or suspected of having graft-versus-host disease (GVHD), acute respiratory distress syndrome (ARDS), sepsis, systemic inflammatory response syndrome (SIRS), severe viral infection, shock, severe acute respiratory syndrome (SARS), or hypercytokinemia induced by immunotherapy. - Administer to the individual an effective amount of sulfated polysaccharide or a pharmaceutically acceptable salt thereof to treat hypercytokinemia. Includes, This will treat hypercytokineemia in the individual. The invention also provides methods for treating hypercytokinemia in individuals with graft-versus-host disease (GVHD), acute respiratory distress syndrome (ARDS), sepsis, systemic inflammatory response syndrome (SIRS), severe viral infections, shock, severe acute respiratory syndrome (SARS), or immunotherapy.

[0111] Administration of sulfated polysaccharides According to various embodiments of the present invention, sulfated polysaccharide formulations are preferably administered to humans at risk of or suspected of having coronavirus infection (including associated cytokine-related toxicity). However, although this specification specifically refers to applications in humans, it will be understood that the present invention is also useful for veterinary purposes. That is, in all embodiments, the present invention is useful for livestock such as cattle, sheep, horses and poultry; companion animals such as cats and dogs; and zoo animals. Accordingly, the general term “subject” or “subject being treated” is understood to include all animals (such as humans, apes, dogs, cats, horses and cattle) that have an enhanced immune response, whether caused by immunotherapy or as a result of another condition.

[0112] The methods described herein may include the use of topical and systemic formulations for oral, intravenous, intramuscular, intra-articular, or subcutaneous administration of sulfated polysaccharides. Various other embodiments may be formulated for administration by transdermal patches, creams, intravenous solutions, eye drops, sprays, liposomes, or any other method of application and ingestion (including through the use of a gastric tube).

[0113] In a further embodiment, sulfated polysaccharides may be adapted for inhalation administration for delivery to the upper or lower respiratory tract, including the nose and nasopharyngeal duct.

[0114] According to some embodiments, liquid (e.g., aqueous) sulfated polysaccharide formulations may be administered by injection. In some embodiments, liquid formulations may be administered orally. In some embodiments, liquid formulations may not be sterilized after use.

[0115] The formulations described herein may be further processed by known methods to produce pharmaceutically acceptable compositions. In certain cases, this may involve using a pharmaceutically acceptable carrier, whether in liquid or solid form, with any of the formulations described herein. For example, the formulations may be further processed to be administered in any suitable liquid or powder form, such as by pills, capsules, liquids, liposomes, lyophilized compositions, or hard or soft chewable tablets. The formulations may be administered, for example, to mammals in one or more dosage forms. The dosage forms of the formulations may be administered in amounts effective to treat one or more diseases.

[0116] The term "administered" means the administration of a therapeutically effective dose of the aforementioned composition, including each of the cells, to an individual. "Therapeutic dose" means the dose that produces the effect of being administered. The exact dose depends on the therapeutic purpose and can be determined by those skilled in the art using known techniques. Adjustments for systemic versus local delivery, age, weight, general health, sex, diet, administration time, drug interactions, and severity of condition may be necessary and can be determined by routine experiments by those skilled in the art, as is known in the art and as described above.

[0117] formulation Methods for preparing various formulations of sulfated polysaccharides will be within the realm of those skilled in the art. In particular, those skilled in the art will be familiar with preparing oral or injectable dosage forms depending on the preferred mode of administration of sulfated polysaccharides.

[0118] In certain preferred embodiments, the method of the present invention involves the use or administration of an oral formulation. The oral formulation may be a capsule, for example, a hard gelatin capsule, and may contain excipients such as microcrystalline cellulose and lubricants such as magnesium stearate. The sulfated polysaccharide may be present in an amount of 10 mg to 1 g, or preferably about 100 mg.

[0119] Various formulations according to the present invention may contain one or more of the aforementioned components in any appropriate concentration or amount. For example, sulfated polysaccharides may be present at a concentration of about 25 mg / mL to about 750 mg / mL, preferably about 25 to about 500 mg / mL, or more preferably about 250 mg / mL. In another example, sulfated polysaccharides may be present in a total amount of about 10 mg to about 5 g. Buffers, such as sodium citrate, citric acid, or other buffers, may be present at concentrations such as about 1 to about 100 mM. For example, in some embodiments, a buffer such as sodium citrate may have a concentration of about 50 mM (14.7 mg / mL) in the formulation, or a buffer such as citric acid may be present at about 55 mM (about 10.5 mg / mL). EDTA may be present at concentrations such as approximately 0.01% to approximately 0.5% w / v, 0.1 mM to approximately 1 mM, approximately 0.25 mg / mL, or more preferably approximately 0.25% w / v.

[0120] Chelating agents may be present at concentrations such as approximately 0.1% to approximately 1 mM. Preservatives may be present at concentrations such as approximately 0.1% to approximately 1%. Antioxidants may be present at concentrations such as approximately 0.1% to approximately 10 mM. Antioxidants may also be present at concentrations such as approximately 0.02% w / v to approximately 5% w / v. Excipients (e.g., pharmaceutical excipients) may be present at any appropriate concentration, e.g., from approximately 1% to approximately 90%.

[0121] In other embodiments of the present invention, various formulations may contain one or more of these components in any appropriate concentration or amount. For example, sulfated polysaccharides, such as PPS, may be present at concentrations ranging from about 25 mg / mL to about 500 mg / mL, or more preferably about 250 mg / mL. Buffering agents may be present at concentrations ranging from about 0.005% to about 5% w / v. Sodium bisulfite may be present at concentrations ranging from about 0.01% to about 1% w / v, about 0.02% to about 1% w / v, 10 mg / mL, or more preferably about 1% w / v. (When added to the formulations of the present invention, sodium pyrosulfite may be converted to sulfur dioxide and sodium bisulfite. In embodiments, pyrosulfite between about 25% and almost all is converted to sulfur dioxide and sodium bisulfite upon addition to the formulations of the present invention.)

[0122] EDTA may be present at concentrations such as approximately 0.01% to approximately 0.5% w / v, 0.1 mM to approximately 1 mM, approximately 0.25 mg / mL, or more preferably approximately 0.25% w / v. Sodium citrate may be present at concentrations such as approximately 0.1% to approximately 4% w / v, or more preferably approximately 1.47% w / v. Citric acid may be present at concentrations such as approximately 0.5% to approximately 2% w / v, or more preferably approximately 1.05% w / v. Antimicrobial agents such as methylparaben may be present at concentrations such as approximately 0.05% to approximately 0.2% w / v, approximately 1 mg / mL, or more preferably approximately 0.1% w / v.

[0123] The formulations of the present invention may be in liquid, solid, or lyophilized form, and may be formulated as aqueous solutions. The formulations may be in a solution having any suitable pH, such as pH 4 to pH 8. In some embodiments, the formulations may have a pH of about 7 to pH 8. For example, the formulations may be in a solution having any suitable pH, such as pH 4 to pH 8. It should be recognized by those skilled in the art that the formulations of the present invention may be lyophilized to produce lyophilized dosage forms using techniques apparent to those skilled in the art in light of this specification. Furthermore, the lyophilized dosage forms may, after reconstitution, be formulated to contain any of the dosage forms of the formulations described herein.

[0124] Exemplary formulations may include one or more of the following: PPS in a concentration of about 25 to about 500 mg / mL; pyrosulfite or bisulfite (e.g., sodium bisulfite) in a concentration of about 0.05% w / v to about 5% w / v; one or more chelating agents in a concentration of about 0.01% w / v to about 0.5% w / v; one or more buffering agents in a concentration of about 0.005% w / v to about 5% w / v; one or more antioxidants in a concentration of about 0.02% w / v to about 1% w / v; one or more antimicrobial agents in a concentration of about 0.05% w / v to about 0.2% w / v; hyaluronic acid; and glucosamine.

[0125] In some embodiments, sodium bisulfite may be present at a concentration of approximately 10 mg / mL. EDTA may be present at a concentration of approximately 0.25 mg / mL. Sodium citrate may be present at a concentration of approximately 14.7 mg / mL. Citric acid may be present at a concentration of approximately 10.5 mg / mL. Methylparaben may be present at a concentration of approximately 1 mg / mL.

[0126] In one example, the dosage form may contain pentosan polysulfate (PPS) at a concentration of approximately 250 mg / mL; sodium bisulfite at a concentration up to approximately 20 mg / mL; and EDTA at a concentration of approximately 0.25 mg / mL. The formulation may be stable in a pH range of approximately 6 to approximately 7.

[0127] An exemplary formulation may contain PPS at a concentration of approximately 250 mg / mL; sodium bisulfite at a concentration up to approximately 10 mg / mL; EDTA at a concentration of approximately 0.25 mg / mL; and methylparaben at a concentration of approximately 1 mg / mL. The formulation may be stable in a pH range of approximately 5.8 to approximately 6.2.

[0128] Another exemplary formulation may contain PPS at a concentration of about 250 mg / mL; sodium bisulfite at a concentration up to about 10 mg / mL; EDTA at a concentration of about 0.25 mg / mL; and methylparaben at a concentration of about 1 mg / mL. The formulation may be stable in a pH range of about 7.8 to about 8.2. In some embodiments, the pH of the formulation may be adjusted with 1% w / v sodium hydroxide.

[0129] In a preferred embodiment, PPS can be formulated in any dosage form, such as a liquid, for oral administration or injectable doses, most preferably for oral administration.

[0130] The oral dosage form of PPS may contain an amount of 25 mg to about 250 mg; preferably about 100 mg to about 200 mg of PPS.

[0131] The oral dosage form may be an immediate-release capsule or a delayed-release capsule.

[0132] Further examples of PPS formulations are described in WO / 2007 / 123800, the full contents of which are incorporated herein by reference.

[0133] dosage Those skilled in the art will be able to readily determine the appropriate dose of sulfated polysaccharides required for the prevention or treatment of cytokine-related toxicity. It will be recognized that the required dose of sulfated polysaccharides depends on the severity of the toxicity (e.g., whether the toxicity is considered to be mild cytokine release syndrome or more severe hypercytokinemia), i.e., that those skilled in the art will determine the appropriate dose on a case-by-case basis. Furthermore, the degree of toxicity will also be influenced by factors such as the severity of the disease causing cytokine-related toxicity, or the severity of the disease being treated with immunotherapy (and the dose of immunotherapy administered). As a further consideration, the dose of sulfated polysaccharides may be adjusted depending on whether the sulfated polysaccharides are intended for prophylactic (i.e., preventive) treatment of cytokine-related toxicity or in accordance with evidence of individuals suffering from cytokine-related toxicity induced by immunotherapy.

[0134] The sulfated polysaccharide preparations used according to the method of the present invention may be formulated for administration to mammals, for example, orally or by injection, in any of the dose ranges described below. Injection may be intravenous, intramuscular, or subcutaneous.

[0135] The dose of the sulfated polysaccharide preparation for use in the method of the present invention may contain sulfated polysaccharide in an amount of about 0.001 to 100 mg / kg / day.

[0136] In preferred embodiments, when the sulfated polysaccharide is PPS, the amount provided in the injectable dose is, for example, 10 mg to about 5 g or about 1 mg / kg to about 5 mg / kg. In some embodiments, a dose of about 3 mg / kg may be administered by injection. In some embodiments, the amount of the dosage form contains enough to inject about 1 mg / kg to about 5 mg / kg of PPS in each injection.

[0137] In some embodiments, the dose of the PPS formulation described herein, for example, the dose for oral administration, may contain an amount of PPS ranging from about 4 mg / kg to about 20 mg / kg. In some embodiments, a dose of about 10 mg / kg may be administered orally. In some embodiments, the amount of the liquid formulation contains an amount sufficient to deliver an oral dose of PPS ranging from about 1 mg / kg to about 50 mg / kg in each administration.

[0138] It should be recognized that lower doses may be appropriate for humans and small mammals, while higher doses may be appropriate for larger animals. Dosage may be based on the mass of the target. For example, the dose may contain approximately 3 mg per kg of body weight of the target, such as a human or horse.

[0139] In certain embodiments, PPS is administered by continuous intravenous infusion over a period of 1 to 3 weeks at doses of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, or 20 mg / kg.

[0140] In further embodiments, PPS is administered by subcutaneous injection at doses of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. When administered subcutaneously, PPS is preferably administered once a day, every other day, three times a week, or once a week.

[0141] Those skilled in the art will recognize that the dosage for a particular formulation depends in part on the salt of PPS contained in the formulation. For example, a formulation containing sodium PPS may have a different dosage than a formulation containing calcium PPS. The calculation of the dosage can be determined by those skilled in the art by taking into account differences in body weight, surface area, and species.

[0142] According to various embodiments of the present invention, the dose may be administered at varying frequencies. The frequency of administration will vary depending on the severity of the coronavirus infection, including the associated cytokine-related toxicity. For example, if sulfated polysaccharides are intended to act as a prophylactic agent to prevent the development of cytokine-related toxicity or other diseases, conditions, or symptoms associated with coronavirus infection, it may be preferable to administer sulfated polysaccharides throughout the duration of the coronavirus infection and until the patient no longer has an active infection. Alternatively, if sulfated polysaccharides are used in response to the development of cytokine-related toxicity, the treatment process may be short-lived and only until the symptoms of cytokine-related toxicity subside.

[0143] The oral doses of the formulations described herein may be administered daily, approximately once every two or three days, approximately twice a week, or weekly. Injectable doses, such as intramuscular, intra-articular, subcutaneous, or intravenous doses, may be administered approximately daily, approximately once every two or three days, approximately twice a week, approximately weekly, approximately every two weeks, approximately monthly, or at other administration frequencies. Such doses may be administered for periods such as approximately four weeks to approximately five weeks, approximately two months, approximately six months, or other treatment periods.

[0144] The doses described herein may also be administered in pulse therapy, for example, by administering the dose regularly (e.g., every three days) for a period of one to three months, then not administering for a period of one to three months, and then regularly (e.g., every day or at some other appropriate intervals) for a period of one to three months. Other administration regimens will be apparent to those skilled in the art in light of this specification.

[0145] Those skilled in the art will recognize that a single dose of the formulations described herein, such as a once-daily dose, may be administered in several divided doses and / or at different times throughout the day. For example, a daily dose may be divided into two halves per day, for example, half in the morning and half in the evening, or three times per day.

[0146] Combination therapy for cytokine-related toxicity The present invention also intends to co-administer sulfated polysaccharides with other agents to prevent or treat cytokine-related toxicity. For example, in one embodiment, corticosteroids such as prednisolone, methylprednisolone, or dexamethasone may be used in combination with sulfated polysaccharides to prevent or treat cytokine-related toxicity.

[0147] In further embodiments, an anti-IL-6 antibody may be used in combination with a sulfated polysaccharide to prevent or treat cytokine-related toxicity. For example, the IL-6 antibody may be tocilizumab.

[0148] Therapies that specifically target TNF-α may also be used in conjunction with sulfated polysaccharides to prevent or treat cytokine-related toxicity. Examples of suitable TNF-α inhibitors are etanercept and infliximab. Angiotensin II receptor blockers (ARBs) and angiotensin-converting enzyme (ACE) inhibitors may also be suitable for downregulating TNFα in cytokine-related toxicity situations, including hypercytokinemia. Examples of suitable ARBs and ACE inhibitors include losartan, telmisartan, irbesartan, olmesartan, valsartan, perindopril, trandolapril, captopril, enalapril, lisinopril, and ramipril.

[0149] In further embodiments, the present invention also aims to prevent or treat cytokine-related toxicity using a combination of sulfated polysaccharides and OX40-Ig (a fusion protein for preventing the rebinding of ligand OX40 to recently activated T cells). [Examples]

[0150] Example 1: Polysulfated polysaccharides inhibit coronavirus replication. material ·Cell culture medium: DMEM F12+10% FCS+penstrep • Infection culture medium: DMEM + TPCK trypsin + penstrep (serum-free); • Calu-3 cells (human lung cancer cell line)

[0151] [Table 1]

[0152] The experiment was conducted as follows: Calu3 cells were treated with 10 different concentrations of polysulfated polysaccharides (or vehicles) either before or after infection. • Cells were infected with either a simulated infection or SARS-CoV-2. • Viral load was analyzed at one time point using the TCID50 assay, and the cytopathic effect induced following continuous passage of the supernatant on target Vero cells was measured.

[0153] The experiment was conducted in 96-well plates consisting of two biological replicas: one uninfected plate and two infected plates (pre+post), each with 60 wells / test compound / cell type (as 10 compound dilutions; 6-series technical replicas) + 6 wells / control (pos+neg; single dilution). Compounds were tested in this format on the same plate as the pre-infection or post-infection treatment.

[0154] Exemplary protocol On day 0, Calu cells were seeded in cell culture medium in a flat-bottomed 96-well plate.

[0155] Day 1: Cells were infected with SARS-CoV-2. Briefly, 0.1 MOI SARS-CoV-2 was diluted in infection culture medium and applied to the cells, then adsorbed at 37°C and 5% CO2 for 30 minutes. Diluted polysulfated polysaccharide or negative control (vehicle) was then applied to the infected cells. Polysulfated polysaccharide was added at a final concentration between approximately 0.03 μM and approximately 55 μM. Cells were incubated in the presence of polysulfated polysaccharide at 37°C and 5% CO2 for 2 days.

[0156] On day 4, Calu culture medium samples were obtained for the purpose of determining the TCID50 (median tissue culture infectious load). Briefly, Calu culture medium was diluted in serum-free DMEM in a round-bottom plate and then plated onto a confluent layer of Cero cells (6 replicas per dilution). The cells were then incubated at 37°C and 5% CO2 for 3 days.

[0157] On day 7, the cytopathic effect (CPE) was calculated to determine TCID50 / ml.

[0158] result The cytopathic effects of each drug treatment concentration were recorded, and the results were analyzed using a Spearman-Karber matrix.

[0159] As shown in Figure 1, viral replication in Calu3 cells was inhibited far more strongly with PPS compared to other polysulfated polysaccharides tested. This was measured by using the supernatant from treated Calu3 cells in Vero cells, which underwent a significant cytopathic effect (CPE), and this was then recorded. Wells were recorded as either positive (CPE) or negative (no virus) four days after receiving the supernatant. Tissue culture infectious load 50% (TCID 50 / mL) was calculated using the Spearman and Kaerber test. Pentosan polysulfate (PPS) demonstrated a nearly four-order-of-magnitude reduction in viral infectivity in the Calu3 SARS-CoV-2 in vitro model.

[0160] These results demonstrate that PPS inhibits SARS-CoV-2 virus replication and reduces viral infectivity, thus suggesting that PPS may be a useful therapeutic agent for treating infections caused by SARS-CoV-2.

[0161] Example 2: PPS inhibits intracellular SARS-CoV-2 assembly in the Golgi and Golgi-related compartments. The SARS-CoV-2 virus is known to enter cells via endosomes and then utilize the host cell's autophagy pathway. There is evidence that once taken up into cells, the virus can disrupt the endogenous function of the autophagy-lysosomal pathway and then accumulate, replicating in autophagosome-like structures.

[0162] Autophagy has both normal and irregular pathways. The latter utilizes trans-Golgi autophagosomes and late endosomes associated with the small GTPase Ras-related protein Rab9.

[0163] In this study, fluorescently labeled NaPPS was added in vitro to cultures of HCT116 colorectal cancer cells. Immunohistochemical staining was then used to compare the intracellular distribution of exogenous NaPPS and endogenous Rab9 endosomes.

[0164] method The fluorescent tag was converted to an ethylamino derivative, covalently bonded to NaPPS, and then coupled to the isothiocyanate ester of fluorescein using a standard method.

[0165] HCT116 cells were cultured on coverslips in DMEM containing 20% ​​fetal bovine serum and antibiotics (pen / strep) at 37°C, 5% CO2, and 5% humidity. Cultures at 20–40% confluence were supplemented with 50 μg / ml FITC-NaPPS for 48 hours. Cells were then fixed with 4% paraformaldehyde in PBS, washed, incubated with mouse antibodies (2 μg / ml) specific to Rab9 or TGN46 (trans-Golgi marker) for 3 hours, washed, and stained with goat anti-mouse IgG antibody conjugated to Alexafluor 594. Samples were washed and counterstained with 0.5 μg / ml DAPI. Coverslips were mounted on slides and observed at m200–m1000 on an Olympus BH2 fluorescence trinocular microscope using an HbO lamp and dichroic bandpass filter cube. The images were captured on a hard drive by a Jenoptik Gryphax camera and rendered using Adobe Photoshop software for contrast enhancement and edge sharpening.

[0166] result Extensive co-localization of Rab9 and FITC-NaPPS was observed in the cytoplasm of interphase and mitotic cells (Figure 2). TGN46 staining revealed that NaPPS accumulates in endosomes near the edges of the Golgi stack (Figure 3).

[0167] conclusion These data provide evidence of a potential mechanism by which NaPPS reduces viral infectivity and replication. NaPPS appears to be an effective treatment for SARS-CoV-2 infection by specifically targeting the autophagosome compartment and other Golgi compartments used by the virus for assembly.

[0168] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

1. Use of pentosan polysulfate or a pharmaceutically acceptable salt thereof in the manufacture of an agent for the prevention or treatment of coronavirus infection, or a disease or condition caused by or associated with coronavirus infection, in an individual requiring such use, wherein coronavirus infection is infection with SARS-CoV-2.

2. Use of claim 1, wherein the disease or condition caused by or related to coronavirus infection is a respiratory disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or a gastrointestinal disease or condition.

3. The use of claim 1, wherein the disease or condition caused by or associated with coronavirus infection is cytokine-related toxicity.

4. The use of claim 3, wherein the cytokine-related toxicity is hypercytokinemia.

5. The use of claim 1, wherein the drug is for the purpose of preventing cytokine-related toxicity in an individual who has been exposed to, has had, or is recovering from a coronavirus infection.

6. The use of any one of claims 3 to 5, wherein cytokine-related toxicity is characterized by fever, hypotension, tachycardia, myalgia, headache, and elevated levels of one or more cytokines selected from the group consisting of IL-1B, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, IFNγ, TNF-α, and granulocyte-macrophage colony-stimulating factor.

7. Use of any one of claims 1 to 6, wherein the disease or condition is characterized as a mild condition that does not require hospitalization.

8. Use of any one of claims 1 to 6, characterized in that the disease or condition is severe and requires hospitalization.

9. The use of claim 1, wherein the individual does not show symptoms of coronavirus infection.

10. Use of any one of claims 1 to 8, wherein the disease or condition is a long-term COVID or prolonged shedding of viral particles.

11. The use of pentosan polysulfate or a pharmaceutically acceptable salt thereof in the manufacture of a drug for the prevention or treatment of fever, hypotension, tachycardia, myalgia, headache or inappropriate cytokine release induced by coronavirus infection or severe acute respiratory syndrome of pneumonia (SARS), wherein coronavirus infection or severe acute respiratory syndrome of pneumonia (SARS) is induced by infection with SARS-CoV-2, respectively.

12. The use of any one of claims 1 to 11, wherein the drug is for administration before or after the onset of coronavirus infection.

13. The use of any one of claims 1 to 12, wherein the drug is administered prophylactically to an individual once they have recovered from an initial infection with coronavirus.

14. The use of pentosan polysulfate or a pharmaceutically acceptable salt thereof in the manufacture of a drug for preventing coronavirus infection or reducing the risk of coronavirus infection in an individual at risk of coronavirus infection, wherein coronavirus infection is infection with SARS-CoV-2.

15. Use of claim 14, which reduces the risk of severe coronavirus infection.

16. Use of claim 14 or 15, wherein individuals at risk are selected from individuals with immunodeficiency (including individuals with primary or secondary immunodeficiency), adults over 60 years of age, children under 2 years of age, healthcare workers, adults or children who have had close contact with a person with confirmed or suspected coronavirus infection, and people with underlying medical conditions.

17. Use of claim 16, wherein the underlying condition is a lung infection, heart disease, obesity, or diabetes.

18. The use of any one of claims 1 to 17, wherein the drug is for intravenous, subcutaneous, oral, intramuscular, via a gastric tube, or intranasal administration or inhalation.

19. The use of any one of claims 1 to 18, wherein the pentosan polysulfate is selected from the group consisting of sodium pentosan polysulfate, calcium pentosan polysulfate, or magnesium pentosan polysulfate.

20. Use of any one of claims 1 to 19, wherein the coronavirus infection is an infection with a variant or variant of SARS-CoV-2, including but not limited to variants D614G, S477N, 20A.EU1, the Danish COVID-19 mink variant, the "UK" variant B1.1.7, the "South Africa" ​​variant B.1.351, the "Brazil" variant P.1, or the "Russia" variant B.1.1.

317.

21. The use of any one of claims 1 to 20, wherein the drug is for administration to an individual in combination with one or more corticosteroids, IL-6 inhibitors, TNF-α inhibitors, or vasopressors.

22. The use of claim 21, wherein the corticosteroid is dexamethasone, prednisolone, or methylprednisolone.

23. The use of claim 21, wherein the IL-6 inhibitor is tocilizumab.

24. The use of claim 21, wherein the vasopressor is one or more of norepinephrine, adrenaline, dopamine, or phenylephrine.

25. The use of claim 21, wherein the TNF-α inhibitor is etanercept or infliximab.

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