Methods for preventing coronavirus and / or respiratory syncytial virus infections

Abstract

The present invention relates to methods and compositions for preventing or reducing the likelihood of coronavirus (CoV) and / or respiratory syncytial virus (RSV) infection in an individual, preventing or reducing the likelihood or severity of symptoms associated with CoV and / or RSV infection in an individual, reducing the severity and / or persistence of CoV and / or RSV infection in an individual, treating CoV and / or RSV infection in an individual, preventing or reducing viral shedding in individuals infected with CoV and / or RSV infection, or reducing CoV and / or RSV transmission in a population, comprising administering to an individual an effective amount of a macromolecule, the macromolecule comprising a generation 3-5 dendrimer having one or more sulfonic acid or sulfonate-containing moieties attached to one or more surface groups. The present invention also relates to devices for delivering compositions comprising such macromolecules.

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for preventing or reducing the likelihood of coronavirus (CoV) and / or respiratory syncytial virus (RSV) infection in an individual, preventing or reducing the likelihood or severity of symptoms associated with CoV and / or RSV infection in an individual, reducing the severity and / or persistence of CoV and / or RSV infection in an individual, or treating CoV and / or RSV infection in an individual, or preventing or reducing viral shedding in an individual infected with CoV and / or RSV infection, or reducing the transmission of CoV and / or RSV in a population, comprising administering an effective amount of macromolecules to an individual. The present invention also relates to devices for delivering compositions containing macromolecules. [Background technology]

[0002] Viral respiratory infections (VRTIs) are the most common infectious diseases worldwide and represent a major public health concern. Respiratory viruses cause infections at all ages and are major contributors to morbidity and mortality. Disease severity can range from mild, common cold-like illnesses to severe, life-threatening respiratory infections. The burden of VRTIs is often more pronounced in individuals with chronic comorbidities or clinical risk factors.

[0003] In the past, a significant proportion of respiratory illnesses could not be attributed to a specific pathogen. The advent of molecular detection and genotyping technologies has led to a substantial increase in the recognition of non-influenza respiratory viruses involved in several newly identified diseases.

[0004] These potential pathogens include coronaviruses, adenoviruses, rhinovirus species, human respiratory syncytial virus, and human bocavirus. Coronaviruses (CoVs) are ubiquitous worldwide and are associated with a range of conditions from relatively mild respiratory illnesses (e.g., the common cold) to severe acute respiratory syndrome (SARS).

[0005] Coronaviruses are large enveloped viruses with a positive-strand, single-stranded RNA genome. CoV infections pose a serious threat to both humans and animals; they are endemic and are involved in outbreaks of SARS caused by SARS-CoV, Middle East Respiratory Syndrome (MERS) caused by MERS-CoV, and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 in humans. COVID-19 is a newly discovered disease caused by SARS-CoV-2. Some people with SARS-CoV-2 infection remain asymptomatic, while others may develop mild to moderate COVID-19 illness and pneumonia, some patients requiring intensive care support, and in some cases, it can be fatal, especially in the elderly. Symptoms such as fever, cough, and loss of taste, as well as signs such as oxygen saturation and lung auscultation findings, are the first and most readily available diagnostic pieces of information.

[0006] In humans, CoV typically causes acute respiratory infections. Symptoms and severity can range from mild upper respiratory tract infections (e.g., the common cold) to more severe acute respiratory distress syndrome (ARDS), pneumonia, and from single-organ to multiple organ failure. Part of the human CoV toxicity is attributed to the long incubation period and the fact that infected and infectious individuals are asymptomatic or often show only mild symptoms, meaning that many people are unaware they are infected and continue their daily lives, thereby spreading the infection.

[0007] CoV transmission is typically via droplets into the nasal mucosa, after which the virus enters the respiratory tract. Contaminated droplets on the hands can also potentially transmit the virus to the oral and / or nasal mucosa. Currently, hygiene practices are recommended to prevent transmission, and the disease is treated through symptomatic management. Mild symptoms, such as those of the common cold, are usually treated with nonsteroidal anti-inflammatory drugs (NSAIDs). Vaccines have been commercially available and in circulation since the filing of the provisional application of this application. A considerable number of potential drugs have been proposed based on previous research on SARS-CoV, and several early clinical trials are underway; however, currently, no drug has demonstrated very high efficacy in treating infections caused by SARS-CoV-2. The SARS-CoV-2 spike S protein binds to the ACE2 receptor for viral entry, and PIKfyve, TPC2, and cathepsin L are also thought to be important for entry. Recent studies from UCSD have identified 332 highly reliable SARS-CoV-2-human protein-protein interactions, and 66 human proteins or host factors that could lead to new drug development, targeted by 69 existing FDA-approved drugs, drugs in clinical trials, and / or preclinical compounds. In addition, there is a wide range of drugs under development and testing or that have been tested against SARS-CoV-2, including, for example, GM-CSF, IL-6R, CCR5, neutralizing antibodies against the MERS S protein, and drugs such as remdesivir, ribavirin, tyrolone, favipiravir, Kaletra (lopinavir / ritonavir), Prezcobix (darunavir / cobicistat), nelfinavir, mycophenolate, galidesivir, Actemra, OYA1, BPI-002, ifenprodil, APN01, EIDD-2801, baricitinib, camostat mesylate, lycorine, brilacidine, BX-25, and interferons, more specifically IFNβ. While several antiviral compounds have been used to treat COVID-19 and may reduce disease persistence and infection rates, they are not widely used or approved by regulatory bodies due to low efficacy (group clinical trials, WHO), cost, and side effects.

[0008] Respiratory syncytial virus (RSV) is a member of the Pneumoviridae family and is a respiratory virus that most people are infected with by the age of two. In healthy adults, symptoms are mild, but in some individuals, symptoms can be severe (especially in infants and the elderly) and require hospitalization. RSV is involved in more than 60% of acute respiratory infections in children worldwide. The virus can also make individuals susceptible to secondary bacterial infections such as pneumonia or otitis media. In the United States, it is estimated that 11,000 to 17,000 adults die from RSV infection each year, and about 10 times that number are hospitalized annually. RSV infection in adults is usually not a primary infection and is mostly mild to moderate in severity unless the patient has underlying risk factors such as being immunocompromised, having underlying chronic lung or cardiovascular disease, living in a long-term care facility, or being frail. The mortality rate from RSV is high, reaching 30-100%, in solid organ and bone marrow transplant recipients, especially if the infection develops within a few days of transplant surgery. Immunosuppressed or otherwise immunocompromised individuals are at increased risk of severe RSV infection. RSV is the third leading cause of influenza-like illness in the elderly; however, it is the second leading cause of hospitalization.

[0009] After years of research, current therapies to reduce the virus are limited to treating symptoms, and an effective vaccine has yet to be developed. One of the challenges is that many candidate cell receptors have been reported in relation to RSV entry, including annexin II, CX3 chemokine receptor 1, epidermal growth factor receptor (EGF), calcium-dependent lectin, Toll-like receptor 4, intercellular adhesion molecule 1 (ICAM-1), and nucleolins. Some receptors, such as EGF, are thought to be used only by specific strains of RSV. Furthermore, RSV is a rapidly evolving virus, and vaccine development is difficult, especially since RSV is known to evade or suppress B-cell memory in humans.

[0010] Antiviral dendrimers have been developed with activity against HIV, HPV, and HSV in selected animal models; see, for example, WO02 / 079299 and WO2007 / 045009. However, antiviral agents are generally selective in their action against viruses, mainly due to receptor specificity and mode of action. There are no approved broad-spectrum antiviral agents for a wide range of viral pathogens, such as enveloped RNA viruses or negative-strand RNA viruses. Even within families such as herpesviridae, a drug effective against one virus is usually not effective against others; for example, treatments for varicella, EBV, or HSV are not effective against each other. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] WO02 / 079299 [Patent Document 2] WO2007 / 045009 [Overview of the project] [Problems that the invention aims to solve]

[0012] Therefore, there remains a need for drugs that can prevent or reduce the spread of VRTIs, particularly CoV and / or RSV. There also remains a need to reduce the severity and duration of the disease in VRTIs, particularly CoV and / or RSV. [Means for solving the problem]

[0013] The inventors have confirmed that the dendrimer macromolecule SPL7013 has in vitro activity against CoV and RSV. Therefore, SPL7013 and structurally related compounds may be useful in reducing the transmission of CoV and / or RSV, and in preventing or reducing the incidence, severity, and persistence of related conditions. In aspects, the present invention provides a method for preventing or reducing the likelihood of coronavirus (CoV) and / or respiratory syncytial virus (RSV) infection in an individual, the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0014] In an embodiment, the present invention provides a method for preventing or reducing the likelihood or severity of symptoms associated with coronavirus (CoV) and / or respiratory syncytial virus (RSV) infection in an individual, the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0015] In one embodiment, the present invention provides a method for preventing or reducing the possibility of coronavirus (CoV) infection in an individual, the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0016] In one embodiment, the present invention provides a method for preventing or reducing the likelihood or severity of symptoms associated with coronavirus (CoV) infection in an individual, the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0017] In one embodiment, the present invention provides a method for reducing the severity and / or persistence of coronavirus (CoV) infection in an individual, the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0018] In this embodiment, the present invention provides a method for treating coronavirus (CoV) infection in an individual, the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0019] In one embodiment, the present invention provides a method for preventing or reducing viral shedding in individuals infected with coronavirus (CoV), the method being: The method involves administering to an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0020] In one embodiment, the present invention provides a method for reducing the transmission of coronavirus (CoV) in a population, and the method is This involves administering to the respiratory system of a portion of a population an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 1st to 8th generation dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0021] In one embodiment, the present invention provides a method for preventing or reducing the likelihood of respiratory syncytial virus (RSV) infection in an individual, the method being: This involves administering to the respiratory system of an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0022] In an embodiment, the present invention provides a method for preventing or reducing the likelihood or severity of symptoms associated with respiratory syncytial virus (RSV) infection in an individual, the method being: This involves administering to the respiratory system of an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0023] In this embodiment, the present invention provides a method for reducing the severity and / or persistence of respiratory syncytial virus (RSV) infection in an individual, the method being: This involves administering to the respiratory system of an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0024] In this embodiment, the present invention provides a method for treating respiratory syncytial virus (RSV) infection in an individual, the method being: This involves administering to the respiratory system of an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0025] In one embodiment, the present invention provides a method for preventing or reducing viral shedding in an individual infected with respiratory syncytial virus (RSV), the method being: This involves administering to the respiratory system of an individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0026] In one embodiment, the present invention provides a method for reducing the transmission of respiratory syncytial virus (RSV) in a population, the method being: This involves administering to the respiratory system of a portion of a population an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 1st to 8th generation dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0027] In some embodiments, the CoV is selected from alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. In some embodiments, the CoV is betacorinavirus.

[0028] In some embodiments, CoV is SARS-CoV-2 or a subtype of its variant.

[0029] In some embodiments, the RSV is subtype A or subtype B, or a subtype or variant thereof. In some embodiments, the RSV is subtype A.

[0030] In some embodiments, the dendrimer is [ka] And in the formula, at least 50% of R is [ka] Therefore, the pharmaceutically acceptable salt is the sodium salt.

[0031] In aspects, the present invention provides compositions for preventing or reducing the likelihood of coronavirus (CoV) infection in an individual, treating it, reducing the severity and / or persistence of CoV infection in an individual, preventing or reducing viral shedding in an individual infected with CoV, or reducing the transmission of CoV in a population, the compositions are A macromolecule or a pharmaceutically acceptable salt thereof, or a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0032] In an embodiment, the present invention provides a device for delivering nasal, oral, or lung compositions comprising a macromolecule or a pharmaceutically acceptable salt thereof, or a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0033] In some embodiments, the present invention provides a composition comprising an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, together with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimer and Carbopol974 or Carbopol971. The composition contains Carbobol974 or Carbopol971 in a w / w ratio of approximately 1:20 to 1:10 relative to the macromolecule.

[0034] In some embodiments, the present invention provides a composition comprising an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimer and Carbopol974. The composition contains approximately 0.05% w / w to approximately 5% w / w, or approximately 0.05% w / w to approximately 3% w / w, or approximately 0.05% w / w to approximately 2% w / w, or approximately 0.05% w / w to approximately 1% w / w, or approximately 0.05% w / w of Carbopol 974.

[0035] In some embodiments, the present invention provides a composition comprising an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimer and Carbopol971. The composition contains Carbopol 971 in an amount of approximately 0.05% w / w to approximately 1% w / w, or approximately 0.05% w / w to approximately 1.5% w / w, or approximately 0.05% w / w to approximately 1.8% w / w.

[0036] In an embodiment, the present invention provides a nasal moisture barrier dressing comprising a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, The macromolecule contains 3 to 5 generations of dendrimers, along with one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimers.

[0037] Any embodiment described herein shall be made with modifications necessary for any other embodiment unless otherwise specified. For example, those skilled in the art will understand that the examples of macromolecules described above for the methods of the present invention are similarly applicable to the compositions of the present invention.

[0038] The present invention is intended to be illustrative only and should not be limited to the scope of the specific embodiments described herein.

[0039] Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention, as described herein.

[0040] Throughout this specification, unless otherwise specified or required by context, any reference to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be deemed to encompass one or more (i.e., one or more) of those steps, compositions of a substance, groups of steps, or groups of compositions of a substance.

[0041] The present invention is described below by the following non-limiting embodiments and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0042] [Figure 1] The names and structures of the macromolecules SPL-7674, SPL-7615, SPL-7673, BAI-7021, BRI-2999, and BRI-2992 are provided. [Figure 2] The antiviral effect is measured by the reduction of cytotoxicity (CPE) in virus-infected cells and the selectivity of SPL7013 against SARS-CoV-2 (hCoV-19 / Australia / VIC01 / 2020) infection of Vero E6 cells. The notation is as follows: EC50 = 50% effective concentration, EC90 = 90% effective concentration, CC50 = 50% cytotoxic concentration, SI = selectivity index (CC50 / EC50), SD = standard deviation, NC = not calculated, N / A = not applicable. [Figure 3] This paper provides dose-response curves of antiviral activity by SPL7013 against SARS-CoV-2 (hCoV-19 / Australia / VIC01 / 2020) replication in Vero E6 cells, as measured by the reduction in CPE on day 4, and cell viability as a percentage of cell control. A. Cell culture 1 hour before infection - Assay 1 (left panel) and Assay 2 (right panel). B. Cell culture 1 hour after infection - Assay 1 (left panel) and Assay 2 (right panel). [Figure 4] A. The virus and SPL7013 were mixed for 1 hour prior to infection of the cell culture. EC50 and CC50 values ​​and selectivity indices are shown. Points and error bars represent the mean ± SD of the triple measurement readings. B. The amount of virus secreted into the supernatant 8 hours post-infection was determined by TCID50. SPL7013 (0.345 mg / mL, square), remdesivir (5 μM, gray triangle), hydroxychloroquine sulfate (15 μM, circle), and SARS-CoV-2 (hCoV-19 / Australia / VIC01 / 2020) only (black triangle). Each point on the graph represents the viral titer present after one cycle of replication following the addition of the compound at the indicated time after viral infection. The infectious viral titer for SPL7013 was below the limit of detection (LLOD) at all time points. [Figure 5-1] Figure 5. Dose-response and cytotoxic analysis of the SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) antiviral activity of SPL7013 in cells, measured by infectious virus release (Log 10 pfu / mL) on day 4 post-infection in A. Vero E6 cells and B. Calu-3 cells. Points and error bars represent the mean ± SD of triple measurement readings. [Figure 5-2] Continuation of Figure 5. [Figure 6] This provides the antiviral effect of SPL7013 against SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020), as measured by the reduction in mean infectious virus (Log10pfu / mL) 96 hours post-infection in Vero E6 cells. [Figure 7]This study provides the antiviral effect of SPL7013 against SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020), measured by the reduction in mean infectious virus (Log 10 pfu / mL) 16 hours post-infection in Vero E6 cells. SPL7013 (0.0046–30 mg / mL) was incubated with 10⁵ and 10⁴ pfu / mL of SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) for 30 seconds, 1 minute, 5 minutes, and 15 minutes. The treated virus was added to Vero E6 cells, and the amount of infectious virus in the supernatant was determined by plaque assay 16 hours after infection. A. Dose-response of SPL7013 antiviral activity using 10⁴ pfu / mL virus inoculation. Points and error bars represent the mean ± SD of triple measurement readings. B. Log10 reduction in viral load (relative to baseline) with SPL7013 at 10 mg / mL. Columns and error bars represent the mean ± SD of triple measurement readings. [Figure 8] A. Evaluation of SPL7013 against SARS-CoV-2 infection in hACE2 transgenic mice is shown 7 days after nasal administration. B. Inhibition of SARS-CoV, MERS-CoV, and SARS-CoV-2 spike-expressing lentiviral infection in Vero E6 cells by SPL7013. [Figure 9-1] Figure 9. A. Inhibition of human respiratory syncytial virus (HRSV) in Hep-2 cells before and after treatment with SPL7013. B. Cytotoxicity of HRSV in Hep-2 cells before and after treatment with SPL7013. [Figure 9-2] Continuation of Figure 9. [Figure 10]The antiviral effects of SPL7013 and iota-carrageenan against SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) are measured by the decrease in nucleocapsid (ng / mL) on day 4 post-infection in human bronchial epithelial primary cells (HBEpC). Astodrimer sodium (0, 1.1, 3.3, and 10 mg / mL) or iota-carrageenan (0, 6, 60, and 600 μg / mL) were added to cell cultures 1 hour prior to infection. A. Dose-response of SPL7013 antiviral activity is shown. Points and error bars represent the mean ± SD of the triple measurement readings. B. Dose-response of carrageenan antiviral activity is shown. Points represent one multiple measurement. The dotted line indicates the inhibition level achieved in the positive control, SARS-CoV-2 pAb. [Figure 11] A. RT-qPCR results in Vero E6 cells infected with SARS-CoV-2 Slovakia / SK-BMC5 / 2020 virus after treatment with SPL7013. All experiments were independently repeated once (n=2). Results are expressed as the percentage of RNA expression compared to infected untreated control cells. B. Fluorescence focus in Vero E6 cells infected with SARS-CoV-2 Slovakia / SK-BMC5 / 2020 virus after treatment with SPL7013. All experiments were independently repeated once (n=2). Titers were determined using immunofluorescence focus assay. [Figure 12] A. Survival rate of healthy Vero E6 cells after treatment with SPL7013. Cells were pre-incubated with SPL7013 for 1 hour. All experiments were repeated independently once (n=2). Survival rate was assessed using the MTS survival assay. B. Survival rate of SARS-CoV-2 Slovakia / SK-BMC5 / 2020 infected Vero E6 cells after treatment with SPL7013. Cells were pre-incubated with SPL7013 for 1 hour prior to viral infection. The virus was incubated with the cells for 48 hours. All experiments were repeated independently once (n=2). Survival rate was assessed using the MTS survival assay. [Modes for carrying out the invention]

[0043] definition The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "element" means one or more elements.

[0044] Throughout this specification and the subsequent claims, unless the context requires otherwise, the term “comprise,” and variations such as “comprises” and “comprising,” are understood to mean the inclusion of a specified integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0045] As used herein, the term “about” means a quantity, level, value, dimension, size, or volume that varies by up to 30%, 25%, 20%, 15%, 10%, 5%, or 1% relative to the reference quantity, level, value, dimension, size, or volume.

[0046] As used herein, the term “individual” refers to any individual that is susceptible to CoV virus infection and / or RSV virus infection. In certain embodiments, individual is a human, including fetuses, infants, children, early adults, and adults. In some embodiments, individual is a human adult. In one embodiment, individual is an animal. In embodiments, child is one or more of the following ages: under 16 years, under 14 years, under 12 years, under 10 years, under 5 years, under 3 years, under 2 years, under 1 year, under 6 months, under 3 months, and under 1 month. In embodiments, child is 12 years or older. In embodiments, infant is a premature infant. In one embodiment, adult is an elderly adult. In embodiments, adult is one or more of the following ages: over 60 years, over 65 years, over 70 years, over 75 years, over 80 years, over 85 years, and over 90 years. In some embodiments, the individual is human. In some embodiments, the individual is immunocompromised. In some embodiments, the individual has recently undergone surgery. In some embodiments, the individual is 1, 2, 3, 4, 5, 6, 7, 1.5, 2, or 3 weeks post-surgery. In some embodiments, the individual is or will become a transplant recipient. In some embodiments, the individual is or will become a lung transplant recipient, or a bone marrow or stem cell recipient. In some embodiments, the individual has a respiratory condition. In some embodiments, the respiratory condition is selected from one or more of the following: asthma, chronic obstructive pulmonary disease, sleep apnea, emphysema, lung cancer, cystic fibrosis, bronchitis, chronic bronchitis, pneumonia, pleural effusion, pertussis, COVID-19, asbestosis, bronchiectasis, emphysema, silicosis, and tuberculosis.

[0047] As used herein, the terms “prevention” or “prevention” mean reducing the likelihood of contracting or developing an infectious disease or its symptoms. Prevention does not have to be complete and does not mean that the subject will ultimately not contract or develop an infectious disease or its symptoms.

[0048] As used herein, the terms “to treat” or “treatment” mean to obtain at least partially a desired treatment outcome. In embodiments, treatment includes preventing or delaying the onset of one or more symptoms of CoV and / or RSV infection. In embodiments, treatment includes stopping or reducing the onset of one or more symptoms of CoV and / or RSV infection.

[0049] As used herein, the phrase "reduce the severity of an infection" or similar phrases include reducing one or more of the following in an individual: the titer of a virus, the persistence of a viral infection, or the severity or persistence of one or more symptoms of a viral infection in an individual. In embodiments, the viral infection is a CoV and / or RSV virus infection.

[0050] As used herein, the term “persistent CoV and / or RSV infection” refers to the period of time during which an individual has CoV and / or RSV infection, or symptoms caused by CoV and / or RSV infection.

[0051] As used herein, the phrase "macromolecules and their pharmaceutically acceptable salts" is to be used interchangeably with "macromolecules" as the context allows.

[0052] As used herein, “SPL7013” refers to sodium astodrimer (INN,USAN), CAS number 676271-69-5. SPL7013 is also known as 2,6-bis-{(1-naphthalenyl-3,6-disulfonic acid)-oxyacetamide}-2,6-bis-2,6-bis-2,6-bis-(2,6-diamino-hexanoylamino)-2,6-diaminohexanoic acid (diphenylmethyl)-amide, polysodium salt, or It is also known as tetrahexaconta sodium N2,N6-bis{N2,N6-bis[N2,N6-bis(N2,N6-bis{N2,N6-bis[(3,6-disulfonatonaphthalene-1-yloxy)acetyl]-l-lysyl}-l-lysyl)-l-lysyl]-l-lysyl}-N1-(diphenylmethyl)-l-lysineamide.

[0053] As used herein, "astodrimer" refers to CAS number 1379746-42-5, or 2,6-bis-{(1-naphthalenyl-3,6-disulfonic acid)-oxyacetamide}-2,6-bis-2,6-bis-2,6-bis-(2,6-diamino-hexanoylamino)-2,6-diaminohexanoic acid (diphenylmethyl)-amide, or Also known as N2,N6-bis{N2,N6-bis[N2,N6-bis(N2,N6-bis{N2,N6-bis[(3,6-disulfonatonaphthalene-1-yloxy)acetyl]-l-lysyl}-l-lysyl)-l-lysyl]-l-lysyl}-N1-(diphenylmethyl)-l-lysineamide.

[0054] Macromolecules and their pharmaceutically acceptable salts This disclosure includes the use of macromolecules and / or pharmaceutically acceptable salts thereof. Since macromolecules may contain multiple sulfonic acid groups, pharmaceutically acceptable salts may contain multiple cations.

[0055] A pharmaceutically acceptable salt may be any preferred type. Examples of preferred salts include, but are not limited to, metal salts (e.g., aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc salts), organic salts (e.g., organic amines such as N,NI-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethylenediamine, dicyclohexylamine, cyclohexylamine, meglumine, (N-methylglucamine), and procaine), quaternary amines (e.g., choline), sulfonium salts, and phosphonium salts. In certain embodiments, the salt is selected from sodium and potassium, particularly sodium. In embodiments, the salt is a sodium salt (e.g., a polysodium salt).

[0056] Those skilled in the art will understand that many organic compounds can form complexes in the solvents in which they react, or in the solvents in which they precipitate or crystallize. These complexes are known as “solvates.” For example, a complex with water is known as a “hydrate.” When a compound incorporates a solvent, a solvate, such as a hydrate, exists. It will be understood that the macromolecules of the present invention and their salts may exist in the form of solvates. A suitable macromolecule solvate is one in which the associated solvent is pharmaceutically acceptable.

[0057] The macromolecules used in the present invention include 3rd to 5th generation dendrimers having one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface groups of the dendrimer. A dendrimer useful in the present invention may be any suitable 3rd to 5th generation dendrimer capable of presenting one or more sulfonic acid or sulfonate-containing moieties on its surface. In some embodiments, the dendrimer is selected from polylysine, polyglutamate, polyaspartate, polyamidoamine (PAMAM), poly(etherhydroxylamine), polyether, polyester, or poly(propyleneimide) (PPI) dendrimers having 3rd to 5th generations. In some embodiments, the dendrimer has 2nd to 6th generations. In some embodiments, the dendrimer has 3rd to 4th generations. In some embodiments, the dendrimer has 4th generation. In some embodiments, the dendrimer is an amino acid dendrimer selected from the group including polylysine, polyglutamate, and polyaspartate.

[0058] Macromolecules also contain one or more sulfonic acid or sulfonate-containing moieties bonded to one or more surface functional groups of the outermost generation of the dendrimer. For example, if the dendrimer is a polylysine, polyamidoamine, poly(etherhydroxylamine), or poly(propyleneimide)dendrimer, the surface functional group is an amino group; if the dendrimer is a polyglutamate or polyaspartate dendrimer, the surface functional group is a carboxylic acid.

[0059] A dendrimer is a branched macromolecule consisting of multiple branched monomers radiating from a central core. The number of branching points increases as you move from the dendrimer core to its surface and is defined by a continuous layer or "generation" of monomers (or building units). Each generation of building units is numbered to indicate its distance from the core. For example, Generation 1 (G1) is a layer of building units bound to the core, Generation 2 (G2) is a layer of building units bound to Generation 1, Generation 3 (G3) is a layer of building units bound to Generation 2, and so on.

[0060] The outermost generation of building units provides the surface of the dendrimer, presenting a functional group to which at least one sulfonic acid or sulfonate-containing moiety is covalently bonded. The sulfonic acid or sulfonate-containing group may be directly bonded to the surface functional group or bonded to the surface functional group via a linker.

[0061] The dendrimers intended herein can be prepared by methods known in the art. For example, they can be prepared by either convergent means (in which case, effectively, branches are pre-formed and then joined to a core) or divergent means (in which case, layers or generations are continuously constructed outward from the core). Both of these methods will be well understood by those skilled in the art.

[0062] For example, in the case of lysine dendrimers, divergent synthesis can "grow" the dendrimer using amidation chemistry, involving the reaction of the amine group of the lysine residue layer with the carboxyl group of aminoprotected lysine to form the next generation of building units. The protecting group can then be removed to reveal the amino group of the new generation of lysine building units.

[0063] The dendrimer may include any suitable core. As used herein, “core” refers to a portion through which generations of monomers or constructor units are constructed (either through a divergent or convergent process) and may be any portion having at least one reactive or functional site through which layers of monomers or constructor units are successively generated (or to which pre-formed “branchings” are joined).

[0064] The core may be formed from a core precursor having reactive groups suitable for reaction with the constructor unit; for example, the core may be formed from a core precursor having 1, 2, 3, or 4 reactive groups. Some exemplary suitable cores contemplated herein include those formed from a core precursor having 1, 2, 3, or 4 reactive groups independently selected from amino, carboxyl, thiol, alkyl, alkynyl, nitrile, halo, azide, hydroxylamine, carbonyl, maleimide, acrylate, or hydroxyl groups to which layers or generations of constructor units or monomers can be bound.

[0065] In some embodiments, the core is covalently bonded to two construction units via amide bonds, each amide bond being formed between a nitrogen atom present in the core and a carbon atom of an acyl group present in the construction unit. Thus, the core can be formed, for example, from a core precursor containing two amino groups.

[0066] The core components may be the same as or different from the building units.

[0067] Exemplary cores include polyaminohydrocarbons, disulfide-containing polyamines, poly(glycidyl ethers), aminoethanols, ammonia, arylmethyl halides, piperazines, aminoethylpiperazines, poly(ethyleneimines), alkylenes / arylenedithiols, 4,4-dithiobutyric acid, mercaptoalkylamines, thioetheralkylamines, isocyanurates, heterocyclic compounds, macrocyclic compounds, polyglycidyl methacrylates, phosphines, porphines, oxiranes, thioranes, oxetanes, aziridines, azetidines, multiazide functional compounds, siloxanes, oxazolines, carbamates, or caprolactones.

[0068] Some non-limiting examples of the core components intended herein include ammonia, as well as diamino C2-C2 compounds such as ethylenediamine, 1,4-diaminobutane, and 1,6-diaminohexane. 12Alkanes are included. However, it will be understood that the core is not necessarily a linear portion having a single reactive group at each end. Nonlinear, cyclic, or branched core portions are also intended by the present invention. For example, arylmethylamines such as benzhydrylamine (BHA) are suitable cores. In some embodiments, the core is a benzhydrylamine (BHA) group: [ka] It is either or includes it.

[0069] In some preferred embodiments, the core is a benzhydrylamine-lysine core (BHALys). The BHALys core has the following structure: [ka] In dendrimers, it is covalently bonded to the building unit via two nitrogen atoms. BHALys cores are, for example, core precursors: [ka] It can be formed from and has two reactive amino nitrogen atoms.

[0070] In some preferred embodiments, the core is a BHALys core containing an L-lysine residue.

[0071] In some preferred embodiments, the core is a BHALys core containing an L-lysine residue.

[0072] A dendrimer also includes one or more construction units. In some embodiments, the construction units of a dendrimer are Lysine construction unit: [ka] , Amidoamine construction unit: [ka] , ether hydroxyamine construction unit: [ka] , Propyleneimine construction unit: [ka] Glutamate building unit: [ka] Aspartic acid construction unit: [ka] Polyester construction unit: [ka] , and Polyether construction unit: [ka] Selected from.

[0073] In some preferred embodiments, the construction unit is a lysine residue, for example, [ka] That is the case.

[0074] In some preferred embodiments, the construction unit is an L-lysine residue, for example, [ka] That is the case.

[0075] In some embodiments, the dendrimer building unit is lysine or the following formula: [ka] a lysine analog selected from the compounds of formula (I), wherein K is absent or -C 1-6 alkylene-, -C 1-6 alkyleneNHC(O)-, -C 1-6 alkyleneC(O)-, -C 1-3 alkylene-O-C 1-3 alkylene-, -C 1-3 alkylene-O-C 1-3 alkyleneNHC(O)-, and -C 1-3 alkylene-O-C 1-3 alkyleneC(O)-, provided that when L and / or M are absent, J is CH, J is selected from CH or N, L and M are each independently absent or -C 1-6 alkylene- or -C 1-3 alkyleneOC<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ [ka] ,

[0079] A variant 4 having the following structure, where a is an integer 0, 1, 2, 3, 4, or 5, and b and c are independently integers 1, 2, 3, 4, or 5, includes: [ka] In the formula, each # represents a carbonyl residue of a carboxyl group that forms an amide bond with the nitrogen atom of the core or the nitrogen atom of the previous generation construction unit, and any methylene group of the construction unit may be substituted with a methyleneoxy (CH2-O) or ethyleneoxy (CH2-CH2-O) group, provided that this does not result in the formation of a carbonate (-OC(O)-O-) or carbamate (-OC(O)-N-) moiety within the construction unit.

[0080] Other suitable build units / build unit precursors are:

[0081] Analogue 5 has the following structure, where a is an integer from 0 to 2, b and c are the same or different integers from 1 to 4, A1 and A2 are the same or different, selected from NH2, CO2H, OH, SH, X, allyl-X, epoxide, aziridine, N3, or alkyne, and X is F, Cl, Br, or I. [ka] ,

[0082] Analogue 6 having the following structure, where a is an integer from 0 to 2, b and c are the same or different integers from 2 to 6, A1 and A2 are the same or different, selected from NH2, CO2H, OH, SH, X, allyl-X, epoxide, aziridine, N3, or alkyne, and X is F, Cl, Br, or I, [ka] ,

[0083] The analog 7 comprises having the following structure, where a is an integer from 0 to 5, b and c are the same or different integers from 1 to 5, A1 and A2 are the same or different, and are selected from NH2, CO2H, OH, SH, X, allyl-X, epoxide, aziridine, N3, or alkyne, where X is F, Cl, Br, or I. [ka] In the formula, each # represents a carbonyl residue of a carboxyl group that forms an amide bond with the nitrogen atom of the core or the nitrogen atom of the previous generation building unit. Any methylene group in the constructing unit may be substituted with a methyleneoxy (CH2-O) or ethyleneoxy (CH2-CH2-O) group, provided that this does not result in the formation of a carbonate (-OC(O)-O-) or carbamate (-OC(O)-N-) moiety within the constructing unit.

[0084] In some embodiments, the macromolecule is a polylysine dendrimer having a lysine construct unit, particularly a polylysine dendrimer having a benzhydrylamine group, for example, the dendrimer is as follows: [ka] As shown, During the ceremony, [ka] That is the case.

[0085] In some embodiments, the dendrimer contains 3 to 5 generations of building units; for example, in some embodiments, it includes a core and 3 to 5 generations consist of building units. In some embodiments, the dendrimer includes a BHALys core and 3 to 5 generations of lysine building units. In some embodiments, the dendrimer provides 16, 32, or 64 nitrogen atoms on the surface layer of the building units for bonding (either directly or via a linker) with a sulfonic acid or sulfonate-containing moiety. In some embodiments, the dendrimer provides 32 nitrogen atoms on the surface layer of the building units for bonding (either directly or via a linker) with a sulfonic acid or sulfonate-containing moiety.

[0086] The sulfonic acid-containing or sulfonate-containing portion is a portion that can present sulfonic acid or sulfonate groups on the surface of the dendrimer. In some embodiments, the sulfonic acid-containing portion has one sulfonic acid or sulfonate group. In other embodiments, the sulfonic acid-containing portion has two or more sulfonic acid or sulfonate groups, for example, two or three sulfonic acid or sulfonate groups, in particular two sulfonic acid or sulfonate groups. In some embodiments, the sulfonic acid-containing portion contains an aryl group such as a phenyl group or a naphthyl group, in particular a naphthyl group. In some embodiments, the sulfonic acid-containing portion or sulfonate-containing portion contains two sulfonic acid or sulfonate moieties, for example, a naphthyl group substituted by a 3,6-disulfonatonaptyl moiety (also referred to as a naphthyl disulfonate moiety). In some embodiments, a 3,6-disulfonatonaptyl moiety connected to the dendrimer via the 1 position of naphthalene is used.

[0087] If a sulfonate-containing portion is present, the portion will be in ionic form (-SO3 - It may exist in the form of a sodium salt (-SO3Na) or a salt, for example.

[0088] Examples of suitable sulfonic acid or sulfonate-containing portions include, but are not limited to, -NH-(CH2) n SO3 - ,-(CH2) n SO3 - , [ka] It includes, In the formula, n is an integer from 0 to 20, m is an integer from 1 to 2, and p is an integer from 1 to 3. In some embodiments, p = 2.

[0089] In some embodiments, the sulfonic acid-containing portion or the sulfonate-containing portion is [ka] especially [ka] Selected from.

[0090] In some embodiments, two or more sulfonic acid or sulfonate-containing portions are present on the surface of the dendrimer. In some embodiments, at least 5, at least 15, or at least 30 or more sulfonic acid or sulfonate-containing portions are present on the surface of the dendrimer. In some embodiments, 32 or more sulfonic acid or sulfonate-containing portions are present on the surface of the dendrimer.

[0091] In some embodiments, the sulfonic acid or sulfonate-containing moiety is directly bonded to the surface amino groups of the dendrimer. In other embodiments, the sulfonic acid or sulfonate-containing moiety is bonded to the surface amino groups of the dendrimer via linker groups.

[0092] Suitable linker groups include linear or branched alkylene or alkenylene groups in which one or more non-adjacent carbon atoms are optionally substituted with oxygen or sulfur atoms to provide an ether, thioether, polyether, or polythioether, or where X1 and X2 are independently selected from -NH-, -C(O)-, -O-, -S-, and -C(S), and R1 and R2 are independently hydrogen or -C 1-6 A group -X1-(CH2) selected from alkyl groups, where q is an integer from 1 to 10, and the linker contains two or more CH2 groups, and one or more non-adjacent (CH2) groups may be optionally substituted with -O- or -S- to form an ether, thioether, polyether, or polythioether. q -X2 or -X1-(CR1R2) q -X2- is included.

[0093] In some embodiments, the linker is a base-X1-(CH2) q The formula is -C(O)-, where X1 is bonded to a sulfonic acid or sulfonate-containing moiety and selected from the group consisting of O, NH, and S, q is an integer from 1 to 3, and the carbon of the -C(O)- group is bonded to the surface amino group of the dendrimer.

[0094] In some embodiments, the linker is base-X1-(CR1R2) q -C(O)-, where X1 is bonded to a sulfonic acid or sulfonate-containing moiety and selected from the group consisting of O, NH, and S, and R1 and R2 are independently hydrogen or -C 1-6 Selected from alkyl groups, q is an integer between 1 and 3, and the carbon of the -C(O)- group is bonded to the surface amino group of the dendrimer.

[0095] In some embodiments, the linker is #-O-(CR1R2)-C(O)-*, In the formula, R1 is -C 1-6The alkyl group is (e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl), R2 is hydrogen, # indicates bonding with the sulfonic acid-containing moiety, and * indicates bonding with the surface amino group of the dendrimer.

[0096] In some embodiments, the linker is #-O-(CH2) q -C(O)-*, In the formula, q is an integer between 1 and 6, # indicates bonding with the sulfonic acid-containing moiety, and * indicates bonding with the surface amino group of the dendrimer.

[0097] In certain embodiments, the linker is #-O-CH2-C(O)-*, In the formula, # indicates bonding with the sulfonic acid-containing moiety, and * indicates bonding with the surface amino group of the dendrimer.

[0098] In some embodiments, the sulfonic acid- or sulfonate-containing portion is bonded to the surface amino group of the dendrimer via a linker group, and the linker-sulfonic acid / sulfonate portion is [ka] or a pharmaceutically acceptable salt thereof.

[0099] In some embodiments, the sulfonic acid or sulfonate-containing portion is [ka] And the linker is, #-O-(CR1R2)-C(O)-*, In the formula, R1 is -C 1-6 The alkyl group is (e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl), R2 is hydrogen, # indicates bonding with the sulfonic acid-containing moiety, and * indicates bonding with the surface amino group of the dendrimer.

[0100] In some embodiments, the sulfonic acid or sulfonate-containing portion is [ka] And the linker is, #-O-(CH2) q -C(O)-*, In the formula, q is an integer between 1 and 6, # indicates bonding with the sulfonic acid-containing moiety, and * indicates bonding with the surface amino group of the dendrimer.

[0101] Exemplary dendrimers useful in the present invention include formulas I, II, and III: [ka] , [ka] , [ka] , or containing a pharmaceutically acceptable salt thereof, In the formula, each R group is represented by the group of formula IV or by hydrogen. [ka] However, at least one R group is a group of formula IV.

[0102] In certain embodiments, two or more R groups are groups of formula IV, for example, in some embodiments, at least 10 of the R groups are groups of formula IV, at least 15 of the R groups are groups of formula IV, at least 20 of the R groups are groups of formula IV, at least 25 of the R groups are groups of formula IV, or at least 30 of the R groups are groups of formula IV. In some embodiments, all of the R groups are groups of formula IV.

[0103] In some embodiments, the dendrimer is [ka] And in the formula, at least 25% of R is [ka] Therefore, the pharmaceutically acceptable salt is the sodium salt.

[0104] In some embodiments, the dendrimer is [ka] In the formula, R is hydrogen or [ka] And at least 25%, at least 50%, at least 75%, or at least 90% of R is, [ka] Therefore, the pharmaceutically acceptable salt is the sodium salt.

[0105] In some embodiments, macromolecules are given by formula I: [ka] It is a dendrimer of the form R, where R is given by equation IV: [ka] It represents the basis of, In the formula, * represents a bond site with the surface amino group of the dendrimer, and the pharmaceutically acceptable salt is sodium.

[0106] In some embodiments, the dendrimer is [ka] In the formula, R is either hydrogen or the group R'. R' is a bonded sulfonic acid or sulfonate-containing portion, and the sulfonic acid or sulfonate-containing portion is [ka] And the linker is, #-O-(CR1R2)-C(O)-*, In the formula, R1 is -C 1-6 Alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl), R2 is hydrogen, # indicates bonding with a sulfonic acid or sulfonate-containing moiety, and * indicates bonding with a surface amino group of the dendrimer. At least 25%, at least 50%, at least 75%, or at least 90%, or all of R is R', and the pharmaceutically acceptable salt is the sodium salt.

[0107] In some embodiments, the dendrimer is [ka] In the formula, R is either hydrogen or the group R'. R' is a bonded sulfonic acid or sulfonate-containing portion, and the sulfonic acid or sulfonate-containing portion is [ka] And the linker is, #-O-(CH2) q -C(O)-*, In the formula, q is an integer from 1 to 6, # indicates bonding with a sulfonic acid or sulfonate-containing moiety, * indicates bonding with a surface amino group of the dendrimer, and at least 25%, at least 50%, at least 75%, or at least 90%, or all, of R is R', and the pharmaceutically acceptable salt is the sodium salt.

[0108] Certain dendrimers of formula I have all R groups as groups of formula IV (SPL7013). SPL7013, also known as sodium astodrimer, has the following structure: [ka] It holds.

[0109] In some embodiments, the macromolecule is an astrodrimer. In some embodiments, the macromolecule is a pharmaceutically acceptable salt of the astrodrimer. In some embodiments, the pharmaceutically acceptable salt is SPL7013 (astodrimer sodium).

[0110] Certain dendrimers of formula II have all R groups as groups of formula IV (SPL7320). Certain dendrimers of formula III have all R groups as groups of formula IV (SPL7304).

[0111] The synthesis of the dendrimers of formulas I, II, and III is described in WO02 / 079299.

[0112] In some embodiments, the macromolecules are not SPL-7674, SPL-7615, SPL-7673, BAI-7021, BRI-2999, and BRI-2992. The structures of these molecules are shown in Figure 1.

[0113] coronavirus As used herein, the family Coronaviridae, commonly known as "coronavirus" or "CoV," consists of enveloped, positive-sense, single-stranded RNA viruses. The Coronaviridae family has two subfamilies: the Retovirinae and the Orthocoronaviridae. The phylogenetic development of coronaviruses is outlined in the Coronaviridae Study Group (2020).

[0114] In one embodiment, the CoV is selected from the genus alpha-coronavirus (alpha-CoV), beta-coronavirus (beta-CoV), gamma-coronavirus (gamma-CoV), and delta-coronavirus (delta-CoV).

[0115] In one embodiment, alpha-CoV is selected from coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), infectious gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and feline infectious peritonitis virus (FIPV).

[0116] In one embodiment, beta-CoV is selected from human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome-associated coronavirus (SARS-CoV), severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), mouse hepatitis virus (MHV), and / or bovine coronavirus (BCoV).

[0117] In one embodiment, CoV can infect humans.

[0118] In one embodiment, the CoV capable of infecting humans is selected from SARS-CoV-2, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, SARS-CoV, and MERS-CoV, or subtypes of their variants.

[0119] In one embodiment, CoV has a mortality rate of approximately 0.001 to 10% in humans. In one embodiment, CoV has a mortality rate of approximately 0.01 to 9% in humans. In one embodiment, CoV has a mortality rate of approximately 0.01 to 9% in humans. In one embodiment, CoV has a mortality rate of approximately 0.01 to 7% in humans. In one embodiment, CoV has a mortality rate of approximately 0.01 to 6% in humans.

[0120] In one embodiment, CoV has a median daily time-varying basic reproduction number (Rt) of about 1.3 to about 5 in humans when minimal social restrictions are in place. In one embodiment, CoV has an Rt of about 1.4 to about 4 in humans when minimal social restrictions are in place. In one embodiment, CoV has an Rt of about 1.4 to about 3 in humans when minimal social restrictions are in place. In one embodiment, CoV has an Rt of about 1.4 to about 2.6 in humans when minimal social restrictions are in place. In embodiments, Rt is calculated as described in Kucharski et al 2020.

[0121] In one embodiment, CoV is SARS-CoV-2 or a subtype or variant thereof. In one embodiment, SARS-CoV-2 is SARS-CoV-2 subtype L as described in Tang et al., 2020. In one embodiment, SARS-CoV-2 is SARS-CoV-2 subtype S as described in Tang et al., 2020. In one embodiment, SARS-CoV-2 is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020 or a variant thereof. In one embodiment, SARS-CoV-2 includes the sequence or variant thereof described in the NCBI reference sequence: NC_045512.2. In one embodiment, SARS-CoV-2 includes the sequence or variant thereof described in GenBank: MN908947.3. In an embodiment, SARS-CoV-2 is B.1.1.7 (also known as 20I / 501Y.V1 or VOC202012 / 01) or a variant thereof. In an embodiment, SARS-CoV-2 is B.1.351 (also known as 20H / 501Y.V2) or a variant thereof. In an embodiment, SARS-CoV-2 is P1 (also known as 20J / 501Y.V3) or a variant thereof. In an embodiment, SARS-CoV-2 is B.1.526 or a variant thereof. In an embodiment, SARS-CoV-2 is B.1.427 or a variant thereof. In an embodiment, SARS-CoV-2 is B.1.429 or a variant thereof. The B.1.1.7, B.1.351, P.1, B.1.427, and B.1.429 variants are classified as variants of concern by the CDC.

[0122] Examples of SARS-CoV-2 variants are described, for example, in Shen et al., 2020 and Tang et al., 2020. Foster et al (2020), based on genomic analysis, have identified three variants, A, B, and C. In some embodiments, SARS-CoV-2 is SARS-CoV-2 variant A. In some embodiments, SARS-CoV-2 is SARS-CoV-2 variant B. In some embodiments, SARS-CoV-2 is SARS-CoV-2 variant C.

[0123] In one embodiment, the variant is at least 90% identical to the parent sequence. In one embodiment, the variant is at least 92% identical to the parent sequence. In one embodiment, the variant is at least 93% identical to the parent sequence. In one embodiment, the variant is at least 94% identical to the parent sequence. In one embodiment, the variant is at least 95% identical to the parent sequence. In one embodiment, the variant is at least 96% identical to the parent sequence. In one embodiment, the variant is at least 97% identical to the parent sequence. In one embodiment, the variant is at least 98% identical to the parent sequence. In one embodiment, the variant is at least 99% identical to the parent sequence. In some embodiments, the parent strain is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020. In some embodiments, the parent strain is betaCoV / Wuhan / WIV04 / 2019. In some embodiments, the parent strain is SARS-CoV-2 Slovakia / SK-BMC5 / 2020. In some embodiments, the parent strain is SARS-CoV-2 2019-nCoV / USA-WA1 / 2020. In embodiments, the parent strain is B.1.1.7. In embodiments, the parent strain is B.1.351. In embodiments, the parent strain is P1.

[0124] CoV infection can cause respiratory, intestinal, hepatic, and neurological disorders in different animal species, including camels, cattle, cattle, and bats.

[0125] CoV can be transmitted from one individual to another through contact of virus droplets with mucosa. Typically, the virus droplets are airborne and inhaled through the respiratory tract including the nasal airway. Typically, the individual is a human individual. In some embodiments, the individual is a domestic animal or a pet. Typically, during infection, CoV can be found in the upper respiratory tract, such as the nasal cavity. In some examples, CoV can be found in the lower respiratory tract, such as the bronchi and / or alveoli.

[0126] In embodiments, a CoV infection can cause one or more symptoms selected from one or more of fever, cough, sore throat, shortness of breath, viral excretion, respiratory failure, runny nose, nasal congestion, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS). In embodiments, ARDS is selected from mild ARDS (defined as 200 mmHg < PaO2 / FiO2 ≤ 300 mmHg), moderate ARDS (defined as 100 mmHg < PaO2 / FiO2 ≤ 200 mmHg), and severe ARDS (defined as PaO2 / FiO2 ≤ 100 mmHg).

[0127] In embodiments, a SARS-CoV-2 infection can cause one or more symptoms selected from one or more of fever, cough, sore throat, shortness of breath, viral excretion, respiratory failure, runny nose, nasal congestion, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS).

[0128] In one embodiment, the macromolecule reduces an individual's NEWS (National Early Warning Score) or NEWS2 score. In another embodiment, the macromolecule or a pharmaceutically acceptable salt thereof reduces an individual's viral load. Those skilled in the art will understand that the viral load can be measured by any method known to those skilled in the art, including, for example, measurement by quantitative reverse transcription PCR (RT-qPCR) against the relevant viral nucleotide sequence. In one embodiment, the viral load is reduced to above 20 CT (cycle threshold), above 30 CT, above 35 CT, or above 40 CT.

[0129] In one embodiment, macromolecules reduce the individual's CoV antibody titer. In one embodiment, IgA, IgG, and / or IgM antibody titers are reduced to below a detectable level, as measured by ELISA. In some embodiments, the antibodies are against protein S or N. In some embodiments, the sample to be tested is collected from an oral swab, nasal swab, blood sample, throat swab, or lung fluid.

[0130] In some embodiments, macromolecules are retained within the lungs and do not leach into the systemic circulation. In some embodiments, the percentage of macromolecules reaching the systemic circulation is less than 10%, less than 25%, less than 50%, and less than 70%. Systemic delivery refers to the delivery of a pharmacokinetically active drug from the lungs into the blood, either directly via absorption into the pulmonary capillaries or after absorption into the pulmonary lymphatic capillaries.

[0131] In one embodiment, CoV is not SARS-CoV. In one embodiment, CoV is not alpha-CoV. In one embodiment, CoV is not canine coronavirus.

[0132] Respiratory syncytial virus As used herein, "orthopneumovirus," commonly known as "respiratory syncytial virus" or "RSV," is a negative-strand, single-stranded RNA virus. RSV is a member of the Pneumoviridae family. RSV primarily infects respiratory epithelial cells. As outlined in Borchers et al (2013), there is a single RSV serotype with two major antigenic subgroups, A and B. The subtype can be determined based on the reactivity of F and G surface proteins to monoclonal antibodies. RSV infection can cause symptoms in primates, humans, rats, mice, cattle, guinea pigs, ferrets, and hamsters.

[0133] In one embodiment, RSV is human RSV (HRSV). In another embodiment, HRSV is HRSV long.

[0134] In one embodiment, the RSV is selected from RSV subtype A (RSVA) or RSV subtype B (RSVB).

[0135] In embodiments, RSVA is selected from the GA1, GA2, GA3, GA4, GA5, GA6, and GA7 clades, as described by Melero et al (2013). In embodiments, RSVA is selected from GA2 and GA5. In embodiments, the GA2 clade includes the NA1, NA2, CB-A, and ON1 genotypes. In embodiments, RSVB is one or more of GB1, GB2, GB3 / SAB3, GB4, and BA. In embodiments, RSVB is the BA clade.

[0136] In an embodiment, RSVA is a member of one of the 23 genotypes identified by Ramaekers et al 2020. In an embodiment, RSVA is selected from genotypes A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, and A23. In an embodiment, RSVB is a member of one of the 6 RSVB genotypes identified by Ramaekers et al 2020. In an embodiment, RSVB is selected from genotypes B1, B2, B3, B4, B5, and B6.

[0137] In embodiments, RSV infection causes one or more of the following symptoms: nasal congestion or runny nose, loss of appetite, cough, mucus when coughing (yellow, green, or gray mucus), sneezing, sore throat, mild headache, fever, wheezing, rapid or dyspnea, bluish discoloration of the skin (cyanosis), severe asthmatic symptoms in individuals with asthma, acute bronchitis, severe bronchitis, airway inflammation, airway obstruction, chronic obstructive pulmonary disease, cardiomegaly, bacteremia, pneumonia, acute otitis media, and recurrent otitis media.

[0138] RSV infection can lead to secondary infections such as bacteremia, pneumonia, acute otitis media, and recurrent otitis media.

[0139] RSV can be transmitted from one individual to another through contact between viral droplets and mucous membranes. Typically, viral droplets are airborne and inhaled through the respiratory tract, including the nasal tract. Typically, the individual is a human individual. In some embodiments, the individual is livestock or a pet. In some embodiments, the livestock is a cattle. Typically, during infection, RSV can be found in the upper respiratory tract, e.g., the nasal cavity. In some examples, RSV can be found in the lower respiratory tract, e.g., the bronchi and / or alveoli.

[0140] In one embodiment, a macromolecule or a pharmaceutically acceptable salt thereof reduces the viral load of an individual. Those skilled in the art will understand that the viral load can be measured by any method known to those skilled in the art, including, for example, measurement by quantitative reverse transcription PCR (RT-qPCR) against the relevant viral nucleotide sequence. In one embodiment, the viral load is reduced to above 20 CT (cycle threshold), above 30 CT, above 35 CT, or above 40 CT.

[0141] In one embodiment, macromolecules reduce the RSV antibody titer of an individual. In one embodiment, IgA, IgG, IgM, and / or IgE antibody titers are reduced to below a detectable level, as measured by ELISA. In some embodiments, the sample to be tested is collected from an oral swab, nasal swab, blood sample, throat swab, or lung fluid.

[0142] In some embodiments, the macromolecules are retained within the lungs and do not leach into the systemic circulation.

[0143] In some embodiments, the percentage of macromolecules reaching systemic circulation is less than 10%, less than 25%, less than 50%, and less than 70%. Systemic delivery refers to the delivery of a pharmacokinetically active drug from the lungs into the blood, either directly via absorption into the pulmonary capillaries or after absorption into the pulmonary lymphatic capillaries.

[0144] Treatment for RSV may include one or more of the following: hospitalization, intensive care, ICU admission, intubation, and oxygen support.

[0145] Method and Use The present invention relates to methods and compositions for preventing or reducing the likelihood of CoV and / or RSV infection in an individual, preventing or reducing the likelihood of symptoms associated with CoV and / or RSV infection in an individual, reducing the severity and / or duration of CoV and / or RSV infection in an individual, treating CoV and / or RSV infection in an individual, preventing or reducing viral shedding in an individual infected with CoV and / or RSV infection, or reducing the transmission of CoV and / or RSV in a population, comprising administering an effective amount of macromolecules to an individual.

[0146] In one embodiment, the macromolecule described herein is intended for administration to the respiratory system. As used herein, the term “respiratory system” refers to the passage formed by the mouth, nose, throat, and lungs through which air passes during respiration. References to the respiratory system include both the upper and / or lower respiratory system. In one embodiment, the macromolecule described herein is intended for administration to the upper respiratory system. In one embodiment, the macromolecule described herein is intended for administration to the lower respiratory system. Those skilled in the art will understand that the upper respiratory system includes one or more of the nasal cavity, oral cavity, sinuses, throat, pharynx, and larynx. Those skilled in the art will understand that the nasal cavity includes one or more of the vestibular region, olfactory region, superior turbinate, middle turbinate, inferior turbinate, and nasopharynx. Those skilled in the art will understand that the lower respiratory system includes one or more of the trachea, primary bronchi, and lungs. In some embodiments, the macromolecule is delivered by the nose. In embodiments, administration of a macromolecule includes administration to the mucous membrane of one or more areas of the respiratory tract. In embodiments, the macromolecule is administered to the nasal cavity. In embodiments, the macromolecule described herein is administered to the nasal mucosa. In embodiments, the macromolecule is administered to one or more of the nasal turbinates, nasopharynx, and / or oropharynx. In embodiments, the macromolecule described herein is administered to the oral mucosa. In embodiments, the macromolecule described herein is administered to the mucous membrane of the primitive bronchi. In embodiments, the macromolecule described herein is administered to the mucous membrane of the lungs.

[0147] The lungs are known to be a particularly harsh environment for the stability of active agents. Small molecules quickly pass through the lung epithelium and are taken up into the vascular system. Particle size is important for reaching the relevant diseased structures within the lungs. Another difficulty encountered when delivering large particles to the lungs is that the action of the cilia in the lungs tends to quickly remove the drug delivered to the lungs and excrete it via feces. Thus, a particular advantage of some embodiments of the present invention is that the dendrimer is not degraded or quickly excreted by the cilia after administration to the lung environment. In some embodiments, the macromolecule is retained in the lungs for a long period of time. In some embodiments, the macromolecule is retained in the lungs for up to 1 month, 1 week, or 1 day.

[0148] In some embodiments, the macromolecule is administered locally. In embodiments, the macromolecule is administered locally to the epidermis or the eye. In some embodiments, local administration does not extend to administration to the respiratory tract.

[0149] In some embodiments, the macromolecule is administered to the skin. For example, the macromolecule can be administered topically to one or more of the hand, wrist, forearm, face, and neck.

[0150] In some embodiments, the macromolecule is delivered via a parenteral route (e.g., intravenous, subcutaneous, or intramuscular) for systemic delivery. In some embodiments, the macromolecule is delivered by bolus or infusion. In some embodiments, the macromolecule is delivered via injection. In some embodiments, the macromolecule is delivered intravenously.

[0151] In some embodiments, macromolecules are applied to surfaces. In some embodiments, macromolecules are applied to surfaces including textiles, polymers, wood, ceramics, glass, concrete, skin, human tissue, mucous membranes, and bone, as well as metals, polymers such as paint, plastics, and rubber. In some embodiments, macromolecules are applied to personal protective equipment (PPE), including gloves, masks, gowns, and scrubs. In some embodiments, macromolecules are applied to wipes and tissues. In some embodiments, macromolecules are applied to surgical / medical fields, including patients, tables, and equipment. Surgical / medical fields may be for human or veterinary use.

[0152] composition In some embodiments, compositions comprising macromolecules and pharmaceutically acceptable carriers are used. The compositions described herein are suitable, for example, for nasal, pulmonary, ocular, cutaneous, and / or parenteral administration.

[0153] The pharmaceutical composition may also contain polymeric excipients / additives or carriers, such as polyvinylpyrrolidone, microcrystalline cellulose / carboxymethylcellulose, Ficol (a polymeric sugar), hydroxyethyl starch (HES), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin), dextran, PVP, inulin, polyethylene glycol, and pectin. The pharmaceutical composition may also contain amino acids or sugar carriers, such as glycine, leucine, alanine, mannitol, and trehalose. The composition may further contain diluents, buffers, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), flavoring agents, taste blockers, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamine, fatty acids and fatty esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc, and other suitable cations). Other pharmaceutically acceptable excipients and / or additives suitable for use in compositions according to the present invention are listed in "Remington: The Science & Practice of Pharmacy", 19th sup.th ed., Williams & Williams, (1995), "Physician's Desk Reference", 52nd sup.nd ed., Medical Economics, Montvale, NJ (1998), and "Handbook of Pharmaceutical Excipients", Third Ed., Ed. AH Kibbe, Pharmaceutical Press, 2000.

[0154] The carrier, excipient, or diluent may comprise one or more of any and all conventional solvents, dispersions, fillers, solid carriers, aqueous solutions, coatings, viscosity modifiers, isotonic agents, and absorption enhancers or retarders, activity enhancers or retarders, etc. The use of such media and agents in pharmaceutically active substances is well known to those skilled in the art, and is described, for example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Unless any conventional carrier and / or diluent is incompatible with the active ingredient, their use in the compositions of the present invention is intended.

[0155] In some embodiments, the macromolecule composition includes a rheological modifier, particularly polyacrylic acid (carbomer), such as Carbopol® polymers like Lubrizol's Carbopol® 971P, 974P, or 71G, or Noveon Polycarbophil, or equivalents thereof. In some embodiments, the rheological modifier is Carbopol® 974P. They may be homopolymers of acrylic acid or crosslinked with pentaerythritol allyl ether, sucrose allyl ether, or propylene allyl ether. In embodiments, it is a carbomer. In embodiments, it is carboxypolymethylene. In embodiments, it is an acrylic acid polymer. Those skilled in the art will understand that the chains may have different lengths, different degrees of crosslinking, molecular weight, etc., and may be of different grades for specific uses (e.g., pharmaceuticals designated by P). In some embodiments, the Carbopol polymer is the NF (National Formulatory) version. Those skilled in the art will recognize when it is appropriate to use pharmaceutical and non-pharmaceutical grades. Rheological modifiers may be present in amounts of 1–10% w / w, particularly about 2–5% w / w, or 0.01–0.1% w / w. In some embodiments, the rheological modifier is Carbopol. Carbopol rheological modifiers are present in amounts of 0.01%–1% w / w, or about 0.01–0.1%, particularly 0.05%–0.1%, particularly 0.05% w / w, etc. In some embodiments, Carbopol is Carbopol 974. In some embodiments, Carbopol974 is present in amounts such as 0.05% w / w to about 5% w / w, or about 0.05% w / w to about 3% w / w, or about 0.05% w / w to about 2% w / w, or about 0.05% w / w to about 1% w / w, or about 1%, or about 0.05% w / w. In some embodiments, Carbopol is Carbopol971.In some embodiments, Carbopol971 is present in amounts such as 0.05% w / w to about 1% w / w, or about 0.05% w / w to about 1.5% w / w, or about 0.05% w / w to about 1.8%. In some embodiments, the rheological modifier is cellulose, for example, hydroxypropyl methylcellulose or microcrystalline cellulose / carboxymethylcellulose. In some embodiments, the rheological modifier is hydroxypropyl methylcellulose. In some embodiments, hydroxypropyl methylcellulose is present in amounts such as 0.01% to 1% w / w, or about 0.05% to 0.5% w / w, particularly about 0.1%. In some embodiments, the rheological modifier is microcrystalline cellulose / carboxymethylcellulose. In some embodiments, microcrystalline cellulose / carboxymethylcellulose is present in amounts such as 0.5% to 5% w / w, or about 1% to 3% w / w, particularly about 2% w / w. Rheological modifiers help the composition possess bioadhesive / mucosal adhesive properties.

[0156] Macromolecular compositions may also contain chelating agents such as polyaminocarboxylic acids. Particularly useful chelating agents are ethylenediaminetetraacetic acid (EDTA) and its salts. Preferred amounts of chelating agents are in the range of 0.001% to 2% w / w, particularly 0.005% to 1% w / w. In some embodiments, the chelating agent is present in low amounts such as 0.001% to 0.1% w / w, particularly about 0.005%. Other components that may be included in the gel composition include preservatives such as parabens, e.g., methylparaben and propylparaben, or mixtures thereof, in amounts up to 1% w / w. Preferred amounts of parabens are in the range of 0.01% to 0.5% w / w, particularly 0.01% to 0.2% w / w. In some embodiments, methylparaben is present in amounts such as 0.05% to 0.2% w / w, particularly about 0.18%. In some embodiments, methylparaben is present in amounts such as 0.14% to 0.23% w / w. In some embodiments, propylparaben is present in amounts such as 0.01% to 0.05% w / w, particularly about 0.02%. In some embodiments, propylparaben is present in amounts such as 0.015% to 0.0025% w / w. In some embodiments, benzalkonium chloride is present in amounts ranging from 0.01% to 0.1 w / w%, particularly about 0.05%.

[0157] Other components that may be included in the composition include, for example, solvents such as water, pH adjusters such as hydroxides and / or hydrochloric acid, and softeners and wetting agents such as glycerin and propylene glycol, in amounts up to 5%. In some embodiments, glycerin (glycerol) is present. In some embodiments, glycerin is present in amounts such as 0.1% to 5% w / w, 0.5% to 2% w / w, and especially about 1% w / w. In some embodiments, propylene glycol is present. In some embodiments, propylene glycol is present in amounts such as 0.1% to 5% w / w, 0.5% to 2% w / w, and especially about 1% w / w.

[0158] In embodiments, the composition, when delivered by a nasal spray device as described herein, creates a moisturizing and protective barrier within the nasal cavity. In embodiments, the composition, when delivered by a nasal spray device as described herein, creates a moisturizing and protective barrier on the nasal mucosa.

[0159] Respiratory compositions (for the nose and mouth) In some embodiments, administering macromolecules to the respiratory system may involve delivering the macromolecules to diseased lungs via oral or nasal routes, to the upper respiratory system via nasal routes, or to the nasal cavity and / or nasal mucosa via nasal routes. For example, in some embodiments, macromolecules may be delivered by inhalation, such as through inhalation via the mouth and / or nose. In some embodiments, macromolecules may be delivered by intratracheal infusion or inhalation. Thus, macromolecules can deliver pharmaceutically active drugs to the respiratory system without requiring separate targeting agents that target diseased tissues or cells.

[0160] For example, in some embodiments, the pharmaceutical composition may be an aerosol composition, a spray composition, a dry powder composition, an aqueous composition, or a blown composition. In some embodiments, the pharmaceutical composition may be contained in a pressurized metered-dose inhaler, a dry powder inhaler, a nebulizer, a spray, etc. In embodiments, the composition is suitable for administration by nasal spray, oral spray, inhaler, or nebulizer. For further consideration, see Zarogoulidis et al (2012).

[0161] In some embodiments, the macromolecule is formulated for nasal delivery. In some embodiments, the macromolecule is formulated for delivery to the nasal cavity. In some embodiments, the macromolecule is formulated for delivery to the nasal mucosa. In some embodiments, the composition is formulated for delivery to one or more of the nasal turbinates, nasopharynx, and / or oropharynx.

[0162] In some embodiments, the pharmaceutical composition may be suitable for intranasal delivery, such as an aqueous nasal spray composition or a dry powder nasal spray. The nasal spray composition may contain a purified aqueous solution of the activator together with a preservative and an isotonic agent. Such a composition may be adjusted to a pH and isotonic state suitable for the nasal mucosa. In some embodiments, macromolecules are delivered as powder, gel, liquid, aerosol, or emulsion. In some embodiments, the pH of the composition is about 4.5 to about 7.42. In some embodiments, the pH of the composition is about 5 to about 7. In some embodiments, the pH of the composition is about 5 to about 6.5. In some embodiments, the pH is about 5.5 to about 6.5. In other embodiments, the pH is about 7.4.

[0163] In some embodiments, the osmotic pressure of the composition is about 200 to about 700 Osmol / kg. In some embodiments, the osmotic pressure of the composition is about 300 to about 600 Osmol / kg. In some embodiments, the osmotic pressure of the composition is about 300 to about 700 Osmol / kg. In some embodiments, the osmotic pressure of the composition is about 200 to about 400 Osmol / kg, more preferably about 280 Osmol / kg. Osmotic pressure adjusting agents include NaCl, lysine, CaCl2, and sodium citrate, and pH adjusting agents include H2SO4, NaOH, tromethamine, and HCl. In some embodiments, the osmotic pressure of the composition is about 200 to about 400 mOsmol.

[0164] In some embodiments, the composition contains methylparaben in an amount of about 0.14% to about 0.23%.

[0165] In some embodiments, the composition contains propylparaben at a concentration of about 0.015% to about 0.025%.

[0166] In embodiments, the nasal spray composition has antiviral activity against CoV. In embodiments, the nasal spray composition inactivates more than 90%, more than 92%, more than 95%, more than 99%, or more than 99.9% of CoV. In embodiments, the nasal spray composition inactivates more than 90%, more than 92%, more than 95%, more than 99%, or more than 99.9% of SARS-CoV-2. In embodiments, the nasal spray composition inactivates more than 90%, more than 92%, more than 95%, more than 99%, or more than 99.9% of the CoV that causes COVID-19. In embodiments, the nasal spray composition has antiviral activity against RSV virus. In embodiments, the nasal spray composition inactivates more than 90%, more than 92%, more than 95%, more than 99%, or more than 99.9% of RSV. In embodiments, inactivation occurs at least 1 minute after exposure to the composition described herein. In embodiments, the nasal spray composition helps to provide a moisture layer to keep the nasal tissue hydrated. Keeping the nasal tissue hydrated protects it from drying out and damage, making it more difficult for viruses to penetrate.

[0167] In some embodiments, macromolecules are formulated for delivery to the lungs. Neutral pH and tonicity are important factors for lower respiratory delivery, as the lungs have poor buffering capacity and therefore avoid bronchoconstriction in patients with respiratory disorders.

[0168] In some embodiments, the pharmaceutical composition may be a dry powder having a particle size greater than 0.5 μm and less than 50 μm. In some embodiments, the particle size is less than 5 μm and greater than 1 μm.

[0169] In some embodiments, the macromolecule can have a particle size of less than about 100 nm. In other embodiments, the macromolecule can have a particle size of about 1 to about 10 nm, about 2 to about 8 nm, and about 3 to about 6 nm by DLS. In some embodiments, the macromolecule can have an average size of about 5 nm by DLS (1 mg / ml in 10 - 2 M NaCl). In some embodiments, the macromolecule can have a molecular weight of less than 30 kDa, about 10 to about 30 kDa, and about 10 to about 20 kDa.

[0170] Examples of components suitable for nasal or oral delivery are provided in Table 1 below. [Table 1]

[0171] The rapid mucociliary clearance in the nasal cavity, as well as the presence of nasal lysozyme and macrophages, can pose challenges to mucosal delivery. A mucoadhesive excipient may be required. Depending on the intended mode of administration, the composition can include a bioadhesive. In embodiments, the bioadhesive is a mucoadhesive polymer. The bioadhesive can change the viscosity, rheology, and / or ciliary beat frequency (CBF). Examples of mucoadhesive polymers include poly(acrylate), chitosan, cellulose and derivatives such as carboxymethyl cellulose and hydroxypropyl cellulose, hyaluronic acid derivatives, pectin, tragacanth, starch, poly(ethylene glycol), sulfated polysaccharides, carrageenan, sodium alginate, polyvinyl alcohol, polyvinyl pyrrolidone, acacia gum, alginic acid, and gelatin. In embodiments, the composition can include a nasal mucosa adhesive component.

[0172] However, viscosity should not obstruct airflow. In some embodiments, the viscosity of the composition is 1 to 10,000 cP, or 1 to 1,000 cP, or 100 to 1,000 cP, or 100 to 500 cP, or 100 to 400 cP, or 150 to 300 cP, or 150 to 250 cP, or 1 to 200 cP, or 1 to 100 cP, or 1 to 50 cP, or 1 to 25 cP, or 1 to 10 cP. In preferred embodiments, the viscosity of the composition is about 1 to about 10 cP (in contrast, SPL7013 gel for vaginal use has a viscosity of 20,000 to 60,000 cP). In some embodiments, the kinematic viscosity of the solution is less than 1,000, or 500 mmHg. 2 s -1 It is less than.

[0173] For pulmonary delivery, viscosity should be low. In some embodiments, viscosity is less than 200 cP. In some embodiments, viscosity is less than 100 cP.

[0174] For nasal delivery, viscosity should be low. In some embodiments, viscosity is less than 100 cP. In some embodiments, viscosity is less than 50 cP. In some embodiments, viscosity is less than 20 cP. In some embodiments, viscosity is less than 15 cP. In some embodiments, viscosity is less than 10 cP.

[0175] In one embodiment, the nasal composition comprises the formulations shown in Table 2. [Table 2]

[0176] In some embodiments, the pharmaceutical composition may also include any other therapeutic components, surfactants, propellants, stabilizers, etc. The carrier must be pharmaceutically acceptable in the sense that it is compatible with the other components of the composition and is not excessively harmful to its recipient.

[0177] In some embodiments, the pharmaceutical composition may produce particle sizes greater than 0.5 μm and less than 50 μm. In some embodiments, the particle size is less than 5 μm, less than 1 μm, or less than 10 μm.

[0178] In one embodiment, the average particle size is approximately 0.21 to approximately -200 μm. In one embodiment, the average particle size is approximately 1 to approximately 200 μm. In one embodiment, the average particle size is approximately 1 to approximately 50 μm. In one embodiment, the average particle size is approximately 1 to approximately 20 μm. In one embodiment, the average particle size is approximately 1 to approximately 5 μm.

[0179] In some embodiments, particle sizes of 1 to about 5 μm are well-suited for delivery to the lower respiratory tract, while particles of 5 to 10 μm are mainly deposited in the trachea and bronchi, and particles with a diameter of >10 μm are mainly deposited in the nose. Generally, particles with a median aerodynamic diameter of less than 10 μm can reach the lower respiratory tract during nasal breathing. The composition may be a liquid, gel, or powder.

[0180] In some embodiments, a Dv90 suitable for lower airway delivery is about 5–20 μm. In some embodiments, a Dv50 suitable for lower airway delivery is about 5–10 μm. In some embodiments, a Dv10 suitable for lower airway delivery is about 1–5 μm. In some embodiments, a Dv10 suitable for nasal delivery is about 10, 15, or greater than 20 μm.

[0181] In some embodiments suitable for nasal delivery, Dv50 is greater than about 20, 40, or 60 μm. In some embodiments suitable for nasal delivery, Dv90 is greater than about 60, 80, or 1000 μm.

[0182] In some embodiments suitable for nasal delivery, about 10% to about 0.5% of the particles are about 10 μm or smaller. In some embodiments suitable for nasal delivery, about 10% to about 0.5% of the particles are about 10 μm or smaller. In some embodiments suitable for nasal delivery, about 7% to about 0.5% of the particles are about 10 μm or smaller. In some embodiments suitable for nasal delivery, about 5% to about 0.5% of the particles are about 10 μm or smaller. In some embodiments suitable for nasal delivery, about 10% to about 0.5% of the particles are about 5 μm or smaller. In some embodiments suitable for nasal delivery, about 7% to about 0.5% of the particles are about 5 μm or smaller. In some embodiments suitable for nasal delivery, about 6% to about 0.5% of the particles are about 5 μm or smaller. In some embodiments suitable for nasal delivery, about 5% to about 0.5% of the particles are about 5 μm or smaller. In some embodiments suitable for nasal delivery, less than 10% of the particles are about 6 μm or smaller. In some embodiments suitable for nasal delivery, less than 10% of the particles are about 5 μm or smaller. In some embodiments suitable for transnasal delivery, less than 5% of the particles are about 5 μm or smaller. In some embodiments suitable for transnasal delivery, less than 5% of the particles are about 5 μm or smaller.

[0183] Ophthalmic composition The macromolecules of the present invention can be delivered in any composition suitable for application to the eye, such as a solution, ointment, gel, lotion, sustained-release polymer, or by coating, binding, or impregnating contact lenses. In embodiments, the composition can be delivered to the eye by eye drops. In embodiments, the composition can be delivered to the eye by spray.

[0184] "Suitable for ocular application" means that no component of the composition causes long-lasting adverse effects on the eye or the subject being treated. Mild irritation or transient effects such as "tingling" at administration may occur without long-lasting adverse effects. Macromolecules can be formulated as simple aqueous solutions. Alternatively, macromolecules can be formulated in solutions, gels, lotions, or ointments to have one or more physiologically suitable osmotic pressures and pH values ​​by including, for example, salts and buffers, as well as other components such as preservatives, gelling agents, viscosity modifiers, ophthalmic lubricants, mucosal adhesion polymers, surfactants, and antioxidants.

[0185] The macromolecules of the present invention are retained on or within the epithelium for a certain period of time, allowing them to diffuse from the epithelium. Such diffusion provides sustained release of the drug into the ocular environment, allowing the antiviral activity of the macromolecules to be delivered over a period of time and not rapidly washed away by ophthalmic fluid and physical washing. The macromolecules can be released from the epithelium for a period of time exceeding 10 minutes, more specifically for a period exceeding 1 hour, and more specifically for a period exceeding 6 hours.

[0186] In some embodiments, the present invention provides compositions comprising a macromolecule as described herein and at least one pharmaceutically acceptable carrier that provides an eye-friendly pH and osmotic pressure.

[0187] Suitable ophthalmologically acceptable salts that can be used as osmotic agents include salts having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfate ions. Examples of suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfate, and ammonium sulfate.

[0188] Suitable ophthalmologically acceptable pH adjusters and / or buffers include acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trihydroxymethylaminomethane; and buffers such as dextrose citrate, sodium bicarbonate, and ammonium chloride.

[0189] Suitable preservatives include stabilized ammonium compounds such as benzalkonium chloride, cethihelimethylammonium chloride, and cetylpyridinium chloride; mercury compounds such as phenylmercury acetate; imidazolidinyl urea; parabens such as methylparaben, ethylparaben, propylparaben, or butylparaben; phenoxyethanol, chlorophenoxyethanol, phenoxypropanol, chlorobutanol, chlorocresol, phenylethyl alcohol, ethylenediaminetetraacetic acid, sorbic acid, and salts thereof.

[0190] Suitable gelling agents or viscosity regulators include gelling agents that increase viscosity when they come into contact with tears, for example, lacrimation caused by blinking or tearing. Such gelling agents are used to reduce macromolecule loss due to tear drainage, which can allow macromolecules to increase their residence time and, consequently, absorption in the epithelial layer of the eye or eyelid. Suitable gelling agents include gellan gum, particularly low-acetylated gellan gum, alginate gum, or chitosan. Viscosity modifiers may also include alkylcelluloses such as methylcellulose or ethylcellulose, hydroxyalkylcelluloses such as hydroxyethylcellulose or hydroxypropylmethylcellulose, hyaluronic acid or its salts, chondroitin sulfate or its salts, polydextrose, cyclodexyllin, polydextrin, maltodextrin, dextrin, gelatin, collagen, polygalacturonic acid derivatives such as pectin, natural gums such as xanthan gum, carob, acacia, trangacanth and carrageenan, agar, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, acrylamide, acrylic acid, and polycyanoacrylate polymers, as well as film-forming polymers such as methyl methacrylate and 2-hydroxy-ethyl methacrylate polymers. Viscosity modifiers or gelling agents may be present in amounts of 0.1% to about 6.5% w / w of the composition, particularly about 0.5% to 4.5% w / w of the composition.

[0191] Suitable lubricants include polyvinyl alcohol, methylcellulose, hydroxypropyl methylcellulose, and polyvinylpytridone.

[0192] Suitable mucosal adhesive polymers include hydroxypropyl methylcellulose, carboxymethylcellulose, poly(methyl methacrylate), polyacrylamide, polycarbophil, polyethylene oxide, sodium alginate, and dextrin.

[0193] Suitable ophthalmologically acceptable surfactants include non-10mc surfactants such as polyoxyethylene fatty acid glycerides and plant oils containing polyoxyethylene (60) hydrogenated castor oil, as well as polyoxyethylene alkyl ethers and alkylphenyl ethers such as octoxynol 10 and octoxynol 40.

[0194] Suitable antioxidants include ascorbic acid and sodium metabisulfate.

[0195] The ophthalmic ointment may also contain one or more thickeners such as liquid paraffin, yellow petrolatum, solid paraffin, and / or lanolin.

[0196] In embodiments, the ophthalmic compositions described herein are suitable for treating and / or preventing CoV infection. In embodiments, the ophthalmic compositions described herein are suitable for preventing, reducing, or isolating CoV virus shedding in individuals having CoV infection.

[0197] In embodiments, the ophthalmic compositions described herein are suitable for treating and / or preventing RSV infection. In embodiments, the ophthalmic compositions described herein are suitable for preventing, reducing, or isolating RSV virus shedding in individuals having RSV infection.

[0198] The compositions of the present invention can be formulated with carriers, diluents, and excipients commonly used in the art, as described above in topical ophthalmic compositions; however, it is well known that many commonly used preservatives have drawbacks when used in topical ophthalmic compositions. For example, some preservatives cause eye irritation, and when used in long-term therapy, they can cause eye damage. Furthermore, some preservatives are ineffective against certain strains of bacteria that cause spoilage of the composition. Parabens are generally considered unsuitable for ophthalmic compositions due to their irritating properties. In some cases, eye drop compositions are formulated without preservatives to reduce irritation. However, such compositions must be packaged for single use or refrigerated after opening.

[0199] In some embodiments, the ophthalmic compositions described herein consist of an aqueous solution of a macromolecule together with at least one pharmaceutically acceptable excipient, the at least one excipient providing a pH of 7.0 to 7.6 and an osmotic pressure of 240 to 310 mOsm / kg, in particular an osmotic pressure isotonic with tears. In other embodiments, the composition comprises an aqueous solution of a macromolecule together with at least one pharmaceutically acceptable excipient, the at least one excipient providing a pH of 7.0 to 7.5 and an osmotic pressure of 240 to 310 mOsm / kg, but without preservatives other than the macromolecule.

[0200] Other compositions In embodiments, the compositions described herein are suitable for dermal administration and can be formulated as aqueous, gel, or cream compositions.

[0201] In embodiments, the compositions described herein are suitable for use as surface sprays, washes, or wipes, including manual washing and surgical field preparation.

[0202] In embodiments, the compositions described herein are embedded in, coated on, or bonded to personal protective equipment (PPE), such as masks, gloves, or surgical gowns, or mask filters.

[0203] In some embodiments, the macromolecules described herein are formulated into compositions suitable for parenteral delivery. For example, for intravenous delivery, the composition may be an aqueous composition, such as Ringer's solution, physiological saline, water, or dextrose solution, or may be diluted with 0.9% physiological saline or 5% dextrose for use.

[0204] In some embodiments, the composition is formulated as a lozenge or a throat gargle. Lozenge compositions are described, for example, in Umashankar et al (2016) and Vera et al (2014).

[0205] The compositions described herein may, for convenience, be provided in unit dosage forms and may be prepared by any method well known in the field of pharmacy. As used herein, a dosage unit form refers to a physically distinct unit suitable as a single dose for the individual being treated, each unit containing a predetermined amount of the active ingredient, calculated to produce the desired prophylactic or therapeutic effect, together with the necessary pharmaceutical carrier and / or diluent.

[0206] All methods involve the step of combining a macromolecule with a carrier constituting one or more accessory components. Generally, compositions can be prepared by combining a macromolecule with a liquid carrier to form a solution or suspension. Such dosage forms are intended to be administered over a period of time (e.g., from about a few seconds in inhalation doses to about 2-6 hours in parenteral doses, and from a few seconds in bolus doses to 24 hours in infusion doses).

[0207] Effective amount The methods of this disclosure require the administration of an effective amount of a macromolecule or a composition containing a macromolecule. “Effective amount” means the amount necessary to at least partially achieve the desired response, or to delay the onset of an infection, inhibit its progression, or completely halt it. An effective amount for a human patient may, for example, be in the range of about 0.5 mg to about 5 mg. An effective amount for a human patient may, for example, be in the range of about 0.5 mg to about 5 mg per nostril action.

[0208] In some embodiments, the effective dose is in the range of approximately 0.04 mg to approximately 1 g, approximately 10 mg to approximately 500 mg, approximately 10 mg to approximately 100 mg, or approximately 100 mg to approximately 500 mg. In some embodiments, the effective dose is in the range of approximately 0.5 to 5 mg. In some embodiments, the effective dose is in the range of approximately 0.5 to 1.5 mg. In some embodiments, the effective dose is approximately 1 mg. In some embodiments, the effective dose is approximately 0.5 mg.

[0209] In some embodiments, the effective dose is approximately 0.1 mg to approximately 1 g / m². 2 , about 1mg to about 100mg / m 2 , about 10mg~about 100mg / m 2 , or approximately 10 mg to approximately 500 g / m² 2 It is within the range.

[0210] In some embodiments, the macromolecule is delivered at a dose of 0.1 to 10 mg / kg per day. In another embodiment, the macromolecule is delivered at a dose of 10 mg / kg per day. In yet another embodiment, the macromolecule is delivered at a dose of 0.1 to 1 mg / kg per day.

[0211] In some embodiments, the macromolecule is delivered via injection of 0.01 to 5 g / day. In other embodiments, the macromolecule is delivered via injection of 0.1 to 2 g / day. In some embodiments, the macromolecule is delivered via injection of 1 to 2 g / day. In some embodiments, the macromolecule is delivered via injection of 0.5 to 1 g / day.

[0212] In some embodiments, the macromolecule-containing composition is formulated to contain an amount of macromolecule effective in establishing an in vivo concentration of the macromolecule in the range of about 0.050 to about 25 μM. In vivo concentration refers to a tissue concentration such as plasma concentration, lung fluid concentration, or lung tissue concentration. In some embodiments, the macromolecule-containing composition is formulated to contain an amount of macromolecule effective in establishing an in vivo concentration of the macromolecule of about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, or about 15 μM. In some embodiments, the macromolecule-containing composition is formulated to contain an amount of macromolecule effective in establishing an in vivo concentration of the macromolecule of at least 0.5 μM, at least 0.75 μM, at least 1 μM, or at least 2 μM. Combinations of these values ​​can form ranges with upper limits of about 20 μM, about 17 μM, or about 15 μM. In some embodiments, compositions containing macromolecules are formulated to contain an amount of macromolecule effective in establishing in vivo concentrations of the macromolecule in the range of about 0.1 to about 100 μM, about 0.5 to about 50 μM, or about 1 to about 25 μM.

[0213] In some embodiments, a single dose of 10-50 mg / kg will achieve an effective concentration. In another embodiment, a single dose of 20-40 mg / kg will achieve an effective concentration. In yet another embodiment, a single dose of 30 mg / kg will achieve an effective concentration.

[0214] Typically, when injected, macromolecules undergo EC 50 Exceeding EC, preferably EC 90 The macromolecule is injected at a rate that exceeds the limit and establishes and / or maintains an in vivo concentration that avoids undue side effects.

[0215] In some embodiments, the macromolecule is injected at a rate that establishes and / or maintains an in vivo concentration of at least 0.08 μM, at least 0.9, at least 0.75 μM, at least 1 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 10 μM, or at least 20 μM. In some embodiments, the macromolecule is injected at a rate that establishes and / or maintains an in vivo concentration of at least 0.001 mg / ml, at least 0.005 mg / ml, at least 0.01 mg / ml, at least 0.02 mg / ml, at least 0.03 mg / ml, at least 0.04 mg / ml, at least 0.05 mg / ml, at least 0.1 mg / ml, at least 0.2 mg / ml, or at least 0.3 mg / ml.

[0216] In some embodiments, the macromolecule is injected at a rate that establishes and / or maintains an in vivo concentration of the macromolecule in the range of about 0.01 to about 100 μM, about 0.5 to about 50 μM, about 1 to about 50 μM, about 2 to about 50 μM, about 5 to about 50 μM, or about 10 to about 50 μM.

[0217] In some embodiments, the macromolecule is injected at a rate that establishes and / or maintains an in vivo concentration of the macromolecule in the range of about 0.001 mg / ml to about 2 mg / ml, about 0.01 mg / ml to about 1 mg / ml, or about 0.05 to about 0.5 mg / ml.

[0218] In some embodiments, macromolecules are injected at a desired rate to establish and / or maintain an in vivo concentration of the macromolecule at a desired concentration. For example, when targeting a concentration of about 400 mg / L, the injection rate may, in some embodiments, range from about 500 to about 3000 mg / hour, about 1000 to about 2000 mg / hour, or about 1500 to about 1600 mg / hour (e.g., 1584 mg / hour) (e.g., 400 mg / L × 3.96 L / hour). For example, when targeting a concentration of about 200 mg / L, the injection rate may, in some embodiments, range from about 250 to about 1500 mg / hour, about 500 to about 1000 mg / hour, or about 750 to about 800 mg / hour (e.g., 792 mg / hour) (e.g., 200 mg / L × 3.96 L / hour).

[0219] In some embodiments, the effective amount is formulated as a macromolecule of about 0.1% to about 10% w / w, or about 0.5% to about 10% w / w, or about 0.5% to about 5% w / w, or about 0.5% to about 3% w / w, or about 1% to 3% w / w. In some embodiments, the effective amount is formulated as a macromolecule of about 0.5% w / w, about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, or about 5% w / w. In some embodiments, the composition contains a macromolecule of about 0.5 mg / ml, or about 1 mg / ml, or about 2 mg / ml, or about 2.5 mg / mL, or about 5 mg / mL, or about 10 mg / ml, about 20 mg / mL, or about 30 mg / mL.

[0220] In some embodiments, the composition is administered in volumes of about 0.1 to about 50 ml, about 0.2 ml to about 1 ml, about 1 to about 25 ml, about 0.025 ml to about 0.2 ml, or about 5 ml. In some embodiments, the composition is administered in volumes of about 0.025 ml, 0.05 ml, 0.1 ml, or about 0.2 ml.

[0221] When used in a delivery system, the amount of the antiviral composition contained in the delivery system according to this disclosure may be, for example, about 0.10 g to about 2 g, or about 0.1 g to about 0.5 g, or about 0.1 g to about 0.25 g.

[0222] In some embodiments, when the composition is for nasal delivery, the dose may be administered in two actions (sprays), i.e., one to each nostril. In some embodiments, when the composition is for nasal delivery, the dose may be administered in volumes of about 5 μL to about 200 μL, about 5 μL to about 150 μL, about 5 μL to about 100 μL, 5 μL to about 80 μL, 5 μL to about 70 μL, 5 μL to about 50 μL, 5 μL to about 40 μL, 5 μL to about 30 μL, or about 5 μL to about 10 μL per nostril. In a preferred embodiment, the dose is administered in a volume of about 100 μL per nostril. Macromolecules may be administered in a dosing regimen that provides the desired effect. For example, a drug, macromolecule, or composition may be administered 1 to 8 times per day, 1 to 6 times per day, 1 to 5 times per day, 1 to 4 times per day, 1 to 3 times per day, or once per day. In some embodiments, a drug, macromolecule, or composition may be administered 1 to 4 times per day. In some embodiments, the macromolecule or composition is administered into each nostril (for example, 4 times per day includes 4 doses into each nostril). In some embodiments, a drug, composition, or macromolecule is administered for about 1 to 2 weeks, about 1 month, about 3 months, or about 6 months. In some embodiments, a drug, composition, or macromolecule is administered once per day, 4 times per day, 6 times per day, or 8 times per day. In some embodiments, a drug, macromolecule, or composition may be administered up to 4 times per day. In some embodiments, a drug, macromolecule, or composition may be administered up to 8 times per day. In some embodiments, the drug, composition, or macromolecule is administered for a maximum of 10 consecutive days. In some embodiments, the drug, composition, or macromolecule is administered for a maximum of 20 consecutive days. In some embodiments, the drug, composition, or macromolecule is administered for a maximum of 30 consecutive days.

[0223] Delivery device In embodiments, the present invention provides a device for delivering nasal, oral, or lung compositions containing macromolecules as described herein. The device described herein can deliver macromolecules to the upper and / or lower respiratory tract. In embodiments, the device can deliver macromolecules to the nasal cavity. In embodiments, the device can deliver one or more doses. In embodiments, the device is reusable.

[0224] In some embodiments, the devices described herein include the compositions described herein.

[0225] In embodiments, the device is a nasal delivery device. In embodiments, the device is an oral delivery device. In embodiments, the nasal delivery device is selected from a spray, an inhaler, a nebulizer, or a nasal irrigation device.

[0226] In one embodiment, the device is a nasal spray. In an embodiment, the nasal spray is a pump spray. Such a pump may include an actuation means. In an embodiment, the nasal spray described herein is a positive displacement pump. In an embodiment, the pump is actuated by pushing the actuation means toward the bottle, causing a piston to move downward within the metering chamber. A valve mechanism at the bottom of the metering chamber prevents backflow into the dip tube. Thus, the downward movement of the piston pushes air (before priming) or liquid outward through the actuator, creating pressure within the metering chamber that produces the spray. When the operating pressure is released, a spring causes the piston and actuator to return to their original positions. The metering chamber ensures accurate dispensing, and an opening swivel chamber at the tip of the actuator aerosolizes the metered dose. Because these pumps do not have measures in place to prevent microbial contamination during use, compositions often contain preservatives, most often benzalkonium chloride (BAC) or parabens. In some embodiments, the device uses silver as a preservative. In some embodiments, the device uses a silver wire, a silver-coated spring, and a ball at the tip of the actuator. Such a system can prevent microorganisms from contaminating the composition during long dosing intervals. Another approach is to use tip sealing technology to prevent backflow into the device. In some embodiments, the total volume discharged by each operation of the device is about 25 to about 200 μL per operation. In some embodiments, the volume discharged by each operation is about 50 to about 150 μL per operation. In one embodiment, the volume discharged by each operation is about 150 μL per operation. In one embodiment, the volume discharged by each operation is about 100 μL per operation. In one embodiment, the volume discharged by each operation is about 50 μL per operation.

[0227] In some embodiments, each operation produces an average particle size of approximately 10 to approximately 200 μm. In some embodiments, each operation produces an average particle size of approximately 20 to approximately 180 μm. In some embodiments, each operation produces an average particle size of approximately 40 to approximately 160 μm. In some embodiments, each operation produces an average particle size of approximately 60 to approximately 110 μm.

[0228] In some embodiments, particle size is measured at operating speeds of approximately 60 mm / sec to approximately 110 mm / sec. In some embodiments, particle size is measured at operating speeds of approximately 60 mm / sec to approximately 90 mm / sec. In some embodiments, particle size is measured at operating speeds of approximately 60 mm / sec to approximately 80 mm / sec. In some embodiments, particle size is measured at operating speeds of approximately 60 mm / sec. In some embodiments, particle size is measured at operating speeds of approximately 80 mm / sec. In some embodiments, particle size is measured at a distance of approximately 30 mm to approximately 80 mm from the dispersion point to the perpendicular laser path. In some embodiments, particle size is measured at a distance of approximately 40 mm to approximately 80 mm. In some embodiments, particle size is measured at a distance of approximately 50 mm to approximately 70 mm. In some embodiments, particle size is measured at a distance of approximately 55 mm to approximately 65 mm. In some embodiments, particle size is measured using an operation of 60 mm / s and a distance of 40 to 70 mm.

[0229] In some embodiments, each operation generates a droplet size distribution Dv10 of at least 10 μm (i.e., 10% of particles have a diameter of less than 10 μm) or at least 15 μm (i.e., 10% of particles have a diameter of less than 15 μm) at a distance of 40 to 70 nm and an operation speed of 60 mm / second. In some embodiments, each operation generates a droplet size distribution Dv50 (median) of at least 50 μm or at least 70 μm at a distance of 40 to 70 nm and an operation speed of 60 mm / second. In some embodiments, each operation generates less than 5% or less than 10% of particles less than 10 μm.

[0230] In some embodiments, the distance is measured from the actuator. In some embodiments, the distance is measured from the distribution opening within the actuator.

[0231] In embodiments, the device is an oral delivery device. Those skilled in the art will understand that the oral delivery device may be a lung-oral delivery device, for example, as described in Ibrahim et al (2015) or Chandel et al (2019). In embodiments, the oral delivery device is selected from a spray, an inhaler, a nebulizer, or an oral rinse. In embodiments, the device can deliver one or more doses. In embodiments, the device is reusable. In embodiments, the spray is a multi-dose spray.

[0232] In one embodiment, the oral device is an oral spray. In another embodiment, the oral spray is a pump spray.

[0233] In an embodiment, the device is an inhaler. In an embodiment, the inhaler is a metered-dose inhaler. In an embodiment, the inhaler is a multi-dose inhaler. In an embodiment, the inhaler is a dry powder inhaler. Examples of inhalers can be found in Chandel et al (2019).

[0234] In some embodiments, the total volume discharged by each operation of the inhaler is approximately 5 to 150 μL per operation. In some embodiments, the total volume discharged by each operation of the inhaler is approximately 10 to 110 μL per operation. In one embodiment, the total volume discharged by each operation of the inhaler is approximately 20 μL to 100 μL per operation. In one embodiment, the total volume discharged by each operation of the inhaler is approximately 100 μL per operation. In one embodiment, the total volume discharged by each operation of the inhaler is approximately 40 μL to 80 μL per operation.

[0235] In an embodiment, each inhaler operation produces an average particle size of approximately 0.01 to approximately 7 μm. In an embodiment, each nebulizer operation produces an average particle size of approximately 0.01 to approximately 5 μm. In an embodiment, each nebulizer operation produces an average particle size of approximately 0.5 to approximately 5 μm. In an embodiment, each nebulizer operation produces an average particle size of approximately 1 to approximately 5 μm. In an embodiment, each nebulizer operation produces an average particle size of approximately 2 to approximately 4 μm.

[0236] In an embodiment, the nebulizer is a jet nebulizer. In an embodiment, the nebulizer is an ultrasonic nebulizer. In an embodiment, the nebulizer is a vibrating mesh nebulizer. In an embodiment, the nebulizer is a respiratory-activated nebulizer. In an embodiment, the nebulizer is a respiratory-enhancing nebulizer. In an embodiment, the nebulizer is selected from Spiriva Respimat®, AERx® Pulmonary Drug Delivery System, AeroEclipse® II BAN (Monaghan Medical Corporation), CompAIR® NE-C801 (OMRON Healthcare Europe BV), I-neb AAD System (Koninklijke Philips NV), Micro Air® NE-U22 (OMRON Healthcare Europe BV), PARI LC® Plus (PARI international), PARI eFlow® rapid (PARI international), and AKITA® Inhalation System (Activaero).

[0237] In some embodiments, the total volume delivered by the nebulizer is about 5 to about 150 μL per operation. In some embodiments, the total volume delivered is about 10 to about 110 μL per operation. In one embodiment, the total volume delivered is about 20 μL to about 100 μL per operation. In one embodiment, the total volume delivered is about 100 μL per operation. In one embodiment, the total volume delivered is about 40 μL to about 80 μL per operation.

[0238] In one embodiment, the nebulizer produces an average particle size of approximately 0.01 to approximately 7 μm. In another embodiment, the nebulizer produces an average particle size of approximately 0.01 to approximately 5 μm. In yet another embodiment, the nebulizer produces an average particle size of approximately 0.5 to approximately 5 μm. In yet another embodiment, the nebulizer produces an average particle size of approximately 1 to approximately 5 μm. In yet another embodiment, the nebulizer produces an average particle size of approximately 2 to approximately 4 μm.

[0239] Nasal spray In some embodiments, the macromolecules or compositions described herein are delivered to the nasal cavity and / or nasal mucosa via a nasal spray device. The nasal spray device of the present invention comprises the composition of the present invention. The operation of the nasal spray device described herein, comprising the composition described herein, delivers a moisturizing protective barrier to the nasal mucosa, which helps to keep the nasal mucosa moist and acts as a physical barrier against respiratory viruses.

[0240] In embodiments, the compositions described herein are packaged in a container closure system that includes an integrated spray pump unit that delivers a precisely measured amount of the composition as a spray when in operation. In embodiments, dispersion as a spray is achieved by passing the composition through a nasal actuator and its opening.

[0241] In this embodiment, the container holds about 1 mL to about 50 mL of composition. In this embodiment, the container holds about 4 mL to about 40 mL of composition. In this embodiment, the container holds about 8 mL to about 25 mL of composition. In this embodiment, the container holds about 10 mL to about 20 mL of composition. In this embodiment, the container holds about 10 mL to about 15 mL of composition. In this embodiment, the container holds about 10 mL of composition. In this embodiment, the device is a multi-dose nasal spray device.

[0242] In an embodiment, the nasal spray device contains a composition for about 20 to about 120 sprays. In an embodiment, the nasal spray device contains a composition for about 40 to about 100 sprays. In an embodiment, the nasal spray device contains a composition for about 60 to about 80 sprays. In an embodiment, the nasal spray device contains a composition for 80 sprays. In a preferred embodiment, the measured amount of composition is about 100 μL.

[0243] The non-sterile, pre-filled nasal spray device consists of SPL7013 formulated into a mucosal adhesive formulation containing a small amount of preservative, which adheres to at least the nasal turbinates, nasopharynx, and / or oropharynx. The mucosal adhesive composition adheres to the nasal cavity, where respiratory viruses causing respiratory illnesses such as the common cold, influenza, and more severe COVID-19 first attach and begin to multiply. As shown in the experiments described herein, SPL7013 has antiviral activity against CoV and RSV and can therefore function as a physical barrier against respiratory viruses such as CoV and RSV, helping to reduce exposure to respiratory viral pathogens and reduce viral load. Reducing the infectious viral load can help prevent the acquisition or transmission of infection. Due to its physical size and negative charge, SPL7013 is not absorbed into the bloodstream after topical nasal application. The inactivated virus is naturally cleared through nasal mucus.

[0244] In an embodiment, the nasal spray device comprises a composition comprising the formulation described herein. In an embodiment, the formulation is Variation 1 as described herein. In an embodiment, the formulation is Variation 2 as described herein. In an embodiment, the formulation is Variation 3 as described herein. In an embodiment, the formulation is Variation 4 as described herein. In an embodiment, the formulation is Variation 5 as described herein.

[0245] In embodiments, the nasal spray device described herein, comprising the composition described herein, delivers a nasal moisture barrier when activated. As used herein, “nasal moisture barrier” refers to a substance applied to the nasal passages (nostrils) to provide a protective moisture barrier to the external environment, to moisturize the nasal mucosa, and to soothe it. In embodiments, the nasal moisture barrier has the Global Medical Device Nomenclature code 47679.

[0246] In embodiments, the nasal moisture barrier described herein includes one or more of the following: i) properties that moisturize the nasal mucosa, ii) properties that inactivate CoV, and iii) properties that reduce the viral load of CoV.

[0247] In embodiments, the nasal moisture barrier described herein includes one or more of the following: i) properties that moisturize the nasal mucosa, ii) properties that inactivate SARS-CoV-2, and iii) properties that reduce the viral load of SARS-CoV-2.

[0248] In embodiments, the nasal moisture barrier described herein includes one or more of the following: i) properties that moisturize the nasal mucosa, ii) properties that inactivate RSV, and iii) properties that reduce the viral load of RSV.

[0249] Simultaneous administration In some embodiments of this disclosure, macromolecules or salts thereof may be the sole active ingredient used, while in other embodiments, the macromolecules used may be used in combination with one or more further active ingredients, for example, further activators for preventing, treating, or reducing the likelihood of viral infection. In one embodiment, the virus can infect an individual via the respiratory tract. In one embodiment, the virus can infect an individual via the respiratory tract and is selected from coronaviruses, rhinoviruses, respiratory syncytial viruses, influenza viruses, syncytial viruses, parainfluenza viruses, adenoviruses, metapneumoviruses, and enteroviruses. In one embodiment, the virus is CoV. In one embodiment, the virus is RSV.

[0250] In embodiments, the activator is selected from one or more of the following: antiviral activators, vaccines, immunomodulators, bronchodilators, antibacterial agents, neuraminidase inhibitors, cap-dependent endonuclease inhibitors, adamantane, anticoagulants, drugs that promote platelet formation, angiotensin-converting enzyme inhibitors, vitamins, convalescent plasma therapy, and / or anti-inflammatory agents.

[0251] As used herein, the term “antiviral agent” refers to a compound that is directly or indirectly effective in specifically interfering with at least one of the following viral actions: viral penetration into eukaryotic cells, viral replication within eukaryotic cells, viral assembly, or viral release from infected eukaryotic cells; or that is effective in nonspecifically inhibiting an increase in viral titer in a eukaryotic or mammalian host system, or in nonspecifically reducing viral titer levels. It also refers to a drug that prevents or reduces the likelihood of contracting a viral infection.

[0252] In the embodiment, the antiviral agent is selected from the antiviral agents described in Gordon et al., 2020 or Ghareeb et al (2021). In embodiments, the antiviral agent is selected from one or more drugs including carrageenan, GM-CSF, IL-6R, CCR5, MERS S protein, and ribavirin, tyrolone, favipiravir, Kaletra (lopinavir / ritonavir), Prezcobix (darunavir / cobicistat), nelfinavir, mycophenolate, galidesivir, Actemra, OYA1, BPI-002, ifenprodil, APN01, EIDD-2801, baricitinib, camostat mesylate, lycorine, brilacidine, BX-25, amostat, umifenovir, lopinavir, ritonavir, pulconalil, and favipiravir, interferon (e.g., IFNβ), combined antimalarial chloroquine, and the antibiotic azithromycin.

[0253] In the embodiment, the anti-inflammatory agent is selected from one or more of indomethacin, tocilizumab, a JAK inhibitor, and ruxolitinib.

[0254] In embodiments, the activator is selected from one or more of acetaminophen, motabizumab, albuterol, epinephrine, ribavirin, and palivizumab.

[0255] Examples of carrageenan are described, for example, in CA2696009. In one embodiment, the carrageenan is selected from iota-carrageenan, kappa-carrageenan, and lambda-carrageenan. In one embodiment, the carrageenan is iota-carrageenan.

[0256] In embodiments, the activator reduces the symptoms of one or more RSV. In embodiments, when the virus is RSV, the activator is selected from one or more of acetaminophen, motavizumab, albuterol, epinephrine, ribavirin, and palivizumab.

[0257] In one embodiment, the antibacterial agent is an antibiotic. In another embodiment, the antibiotic is a broad-spectrum antibiotic.

[0258] In one embodiment, the immunomodulator is an immunosuppressant, a cytokine inhibitor, an antibody, or an immunostimulant. The immunomodulator can suppress inflammation of the airways.

[0259] Macromolecules or their salts can also be used in combination with nonsteroidal anti-inflammatory drugs (NSAIDs). For example, NSAIDs may be used to treat the symptoms of CoV and / or RSV infections, while macromolecules or their salts may be used to prevent the transmission of the virus to another individual.

[0260] The present invention is described more fully here with reference to the accompanying embodiments. However, it should be understood that the following description is illustrative and should not be taken in any way as a limitation on the generality of the present invention. [Examples]

[0261] Example 1: SPL7013 CPE-based antiviral assay method: The virus strain: SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020 was donated by the Peter Doherty Institute for Infection and Immunity (Melbourne, Australia). Documentation received with the parent stock indicated that prior to receipt, the virus had been passaged twice in Vero cells as follows. The working stock was prepared by two further passages in Vero cells in viral growth medium containing L-glutamine-free minimal essential medium supplemented with 1% (w / v) L-glutamine, 1.0 μg / mL TPCK-trypsin (Worthington), 0.2% BSA, and 1% insulin transferrin selenium (ITS). The SARS-CoV-2 2019-nCoV / USA-WA1 / 2020 strain was supplied by BEI Resources (NR-52281). The virus originated from African green monkey kidney vero E6 cells or lung homogenates from human angiotensin-converting enzyme 2 (hACE2) transgenic mice.

[0262] Cells: African green monkey kidney (Vero) cells (ATCC-CCL81) were passaged to create cell bank stocks in cell growth medium containing L-glutamine-free minimal essential medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum and 1% (w / v) L-glutamine. The cell stocks were frozen overnight at -80°C and then transferred to liquid nitrogen for longer-term storage. Vero cells were passaged for up to 13 passages, after which new working cell bank stocks were recovered from liquid nitrogen for further use.

[0263] Preparation of test and control compounds: SPL7013 was dissolved in water at a concentration of 40 mg / mL, vortexed, and visually inspected to confirm complete dissolution. The positive control compound, remdesivir, was prepared as a 10 mM stock in DMSO and stored at -20°C.

[0264] Cell preparation for assay: Vero cells (ATCC-CCL81) were placed in a 96-well plate with 2 × 10⁶ cells in 100 μL of seeding medium (minimum essential medium supplemented with 1% (w / v) L-glutamine, 1% ITS, and 0.2% BSA). 4 Seeds were sown in individual wells for 24 hours. The plates were incubated overnight at 37°C with 5% CO2.

[0265] Addition of test and control samples to assay plates: 1400 μL of viral growth medium (minimum essential medium supplemented with 1% (w / v) L-glutamine, 1% ITS, 0.2% BSA, 1 μg / mL TPCK-trypsin, and 1 × Pen / Strep) was added to rows A and columns 3-11 of a v-bottom-border PCR plate. The compound (40 mg / mL) was added to column 2 (1300 μL). A 1:3 serial dilution was prepared by transferring 700 μL of the compound from column 2 to column 3, from column 3 to column 4, and so on up to column 10, with the last dilution discarded. 50 μL from each compound dilution series was added to rows B-G of the assay plate. SPL7013 was added to the assay plate 1 hour before or 1 hour after infection.

[0266] Virus addition: 50 μL of SARS-CoV-2, diluted in viral growth medium to produce a moi of 0.05, was added to the plates. It had been previously determined that this moi would provide 100% CPE after 4 days. Antiviral activity was evaluated by adding the virus to rows B, C, and D, and cytotoxicity was evaluated by adding viral growth medium without virus to rows E, F, and G. The plates were incubated at 5% CO2, 37°C for 4 days, and then CPE was evaluated.

[0267] Determination of cell necrosis effect (CPE): After 4 days of incubation, viable cells were determined by staining with MTT. 100 μL of 3 mg / mL MTT solution was added to a plate and incubated in a 5% CO2 incubator at 37°C for 4 hours. The wells were aspirated until dry using a multi-channel manifold attached to a vacuum chamber, and the formazan crystals were dissolved by adding 200 μL of 100% 2-propanol at room temperature for 30 minutes. Absorbance was measured at 540–650 nm using a plate reader.

[0268] Effective 50% concentration (EC 50 Determination of: The percentage of cytoprotection achieved by the positive control and test samples in virus-infected cells was calculated using the following formula. Cell protection percentage = ([ODt]virus - [ODc]virus / [ODc]mock - [ODc]virus) × 100 During the ceremony: [ODt] Virus = Optical density measured in a well used to test the effect of a predetermined concentration of test material or positive control on virus-infected cells. [ODc] Virus = Optical density measured in the well used to test the effect of a negative control on virus-infected cells. [ODc] Mock = Optical density measured in a well used to test the effect of a negative control on simulated infected cells.

number

[0269] 50% cytotoxic concentration (CC 50 CC is defined as the concentration of the test compound that reduces the absorbance of simulated infected cells by 50% compared to the control value. 50 The value was calculated as the ratio of (ODt) mock / (ODc) mock. The above calculation was performed using the IDBS XLFit4 Excel Add-in (ID Business Solutions Inc., Alameda, CA).

[0270] Pre-infection prophylaxis assay: Nine concentrations of SPL7013 (astodrimer sodium) and remdesivir control were prepared in assay medium (AM) by 3-fold serial dilution and added to Vero cells in triple count. After 1 hour, 50 μl of AM containing the minimum MOI of SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020 (experimentally determined to provide 100% CPE at 4 days post-infection) was added to the control wells containing only the compound and virus [Multiple MOI of infection (MOI) = 0.05]. Equal volumes of AM were added to the wells containing only cytotoxic cells and cells. Remdesivir was used as a positive control.

[0271] Post-infection treatment assay: AM containing the minimum MOI of SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020, experimentally determined to provide 100% CPE 4 days after infection, was added to the virus-only control well. [Multiple Infection MoI (MOI) = 0.05]. Equivalent volumes of AM alone were added to the cytotoxic and cell-only wells. After 1 hour, nine concentrations of SPL7013 (astodrimer sodium) and remdesivir control were prepared by 3-fold serial dilution in assay medium (AM) and added to Vero cells by triple count.

[0272] result: The experimental results are provided in Table 3. The data is from SPL7013 EC 50 However, it was demonstrated that it is in the micromolar range [25 μM and 24 μM], indicating that it is an effective antiviral agent for the prevention and treatment of viral infection. In addition, an SI of approximately 3.5 in the pre-infection and post-infection assays indicates the selectivity of SPL7013 against SARS-CoV-2. The cytotoxicity of the control in this assay was greater than expected, and an SI of 5 or higher may be expected in the replicates.

[0273] In comparison, chloroquine has an IC of 8 μM. 50 and 261 CC 50It has been reported to possess and produce 30 SIs against SARS (Keyaerts et al 2004). More recent studies have shown that chloroquine activity against beta-CoV / Wuhan / WIV O4 / 2019 is EC 50 = 1.13 μM, CC 50 >100 μM, SI > 88.50, and remdesivir is (EC 50 =0.77μM, CC 50 >100μM, SI>129.87) (Cell Research volume 30, pages 269-271 (2020 and EMA Compassionate Use Application Procedure No. EMEA / H / K / 5622 / CU, EC by Gilead) 50 A value of 0.137 μM was reported. These assays are not directly comparable because they involved fewer replications and shorter incubation periods. [Table 3]

[0274] Example 2: SPL7013 Viral Strategy 25 mg / mL of SPL7013 (astodrimer sodium) is added to an equal volume of SARS-CoV-2. 5 TCID 50 The virus-compound mixture was incubated with the virus / mL unit. The virus-compound mixture was incubated at 37°C for 60 minutes and then incubated with TCID. 50 For assay-based quantification of infectious virus titers, pre-seeded Vero cells in 96-well plates were immediately titrated. The plates were incubated at 37°C for 3 days in a humidified 5% CO2 atmosphere. Virus-induced CPE was visually scored. TCID of the virus suspension was also used. 50 The efficacy was determined using the method of Reed and Muench (1938). The viricidal effect was quantified as a percentage and log reduction in viral titer compared to the titer of SARS-CoV-2 assay medium alone. Controls included AM, AM+ virus, and 60 mM sodium citrate as a positive control.

[0275] SPL7013 showed virucidal activity at 25 mg / mL in this assay. The assay indicated that the compound halted all viral replication.

[0276] Example 3: Astodrimer sodium (SPL7013) inhibits SARS-CoV-2 replication in vitro. This study evaluated the antiviral activity of astrodrimer sodium against SARS-CoV-2 in vitro. When astrodrimer sodium was added to cells 1 hour before or 1 hour after infection, it inhibited SARS-CoV-2 replication in Vero E6 cells and reduced the virus-induced cell necrosis effect at a 50% effective concentration (EC) in the range of 0.090–0.742 μM (0.002–0.012 mg / mL). 50 It was found to possess ) the following properties. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also effective in virucidal evaluation when mixed with the virus for 1 hour before cell infection (EC 50 (1.83 μM [0.030 mg / mL]). The results of the addition time test showed that infectious viruses were below the lower limit of detection at all time points tested, which is consistent with compounds that inhibit the early stages of viral invasion. These data were similar across all studies and were consistent with the potent antiviral activity of astodrimer sodium, which is attributed to the inhibition of virus-host cell interactions.

[0277] method: Virus, cell culture, astodrimer sodium, and control: SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020 was donated by the Peter Doherty Institute for Infection and Immunity (Melbourne, Australia). Virus stocks were prepared at 360 Biolabs (Melbourne, Australia) by two passages in Vero cells in viral growth medium containing 1% (w / v) L-glutamine, 1.0 μg / mL L-(tosylamido-2-phenyl)ethylchloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemical, NJUSA), 0.2% bovine serum albumin (BSA), and 1% insulin-transferrin-selenium (ITS) in L-glutamine-free minimal essential medium (MEM).

[0278] The SARS-CoV-2 2019-nCoV / USA-WA1 / 2020 strain was isolated from an oropharyngeal swab of a respiratory patient who developed clinical disease (COVID-19) in Washington, USA in January 2020, and supplied by BEI Resources (NR-52281). The virus was derived from lung homogenates from African green monkey kidney vero E6 cells or from hACE2 human angiotensin-converting enzyme 2 (hACE2) transgenic mice.

[0279] Vero E6 and human Calu-3 cell lines were cultured in L-glutamine-free minimal essential medium (MEM) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (w / v) L-glutamine. Vero E6 and Calu-3 cells were passaged for up to 10 passages for antiviral and antiviral testing. Hanks equilibrium salt solution (HBBS) supplemented with 2% fetal bovine serum (FBS) was used for infection. Antiviral assays for the 2019-nCoV / USA-WA1 / 2020 strain were performed using an infection multiplicity (moi) of 0.1. Virus inoculation for the antiviral assay was performed on 2.5 × 10⁶ cells. 4 1.5 mL added to each well 4 , 10 5 , and 106 pfu / mL.

[0280] Viral inoculation for the antiviral assay is 10 4 , 10 5 , and 10 6 The concentration was pfu / mL. After the prescribed incubation period, the solution was passed through a 20% sucrose cushion (Beckman SW40 Ti rotor) to pelletize, resuspended in 1.5 mL of MEM, and then the cells were 2.5 × 10⁶. 4 It was added to each well.

[0281] Astodrimer sodium was prepared in water at a concentration of 86.29 mg / mL or 100 mg / mL and stored at 4°C. Astodrimer sodium has a molecular weight of 16581.57 g / mol. The purity of the compounds used in these tests was assessed as 98.79% by ultra-high performance liquid chromatography (UPLC). Remdesivir (MedChemExpress, NJ, USA) was used as a positive control for inhibition of virus-induced cell necrosis (CPE) and for the time of the added plaque assay.

[0282] Virus-induced cell necrosis inhibition assay: African green monkey kidney (Vero E6, ATCC-CRL1586) cell stocks were prepared in cell growth medium containing L-glutamine-free MEM supplemented with 10% (v / v) heat-inactivated FBS and 1% (w / v) L-glutamine. Vero E6 cell monolayers were placed in 96-well plates with 2 × 10⁶ cells in 100 μL of growth medium (MEM supplemented with 1% (w / v) L-glutamine and 2% FBS). 4 Cells were seeded at 1 cell / well and incubated overnight at 37°C in 5% CO2. SARS-CoV-2 infection was established by infecting a cell monolayer using a MOI of 0.05. Astodrimer sodium or remdesivir were sequentially diluted 1:3 nine times, and each compound concentration was evaluated by triple assay for both antiviral efficacy and cytotoxicity. Astodrimer sodium was added to Vero E6 cells 1 hour before or 1 hour after SARS-CoV-2 infection. Cell cultures were incubated at 37°C in 5% CO2 for 4 days before evaluation of CPE. The viral growth medium was MEM supplemented with 1% (w / v) L-glutamine, 2% FBS, and 4 μg / mL TPCK-treated trypsin. On day 4, the virus-induced CPE and cytotoxicity of the compound were determined by measuring viable cells using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (MP Biomedicals, NSW, Australia). Absorbance was measured at 540–650 nm using a plate reader.

[0283] Antiviral plaque assay evaluation and nucleocapsid ELISA: In the antiviral evaluation, astodrimer sodium was added to cells 1 hour before exposure to the virus, at the time of exposure, and 1 hour after exposure. In both antiviral and antiviral assays, cells were washed 6 hours after infection to remove any remaining virus in the astodrimer sodium and / or supernatant. In this manner, cell cultures were incubated after initial infection, and the supernatant was collected after 16 hours or 4 days. The amount of virus in the supernatant was determined by plaque assay (plaque-forming units [pfu]) and nucleocapsid enzyme-coupled immunosorbent assay (ELISA). The plaque assay used utilized a 2% carboxymethylcellulose overlay, cell fixation with 4% paraformaldehyde, and staining with 0.1% crystal violet, as described in van den Worm et al (2012). The nucleocapsid ELISA assay was performed as described in Bioss Antibodies, USA (BSKV0001). Cytotoxicity of astodrimer sodium was assessed on day 4 by measuring intracytoplasmic lactate dehydrogenase (LDH) activity using an LDH detection kit (Cayman Chemical), along with 0.5% saponin used as a cytotoxicity-positive control.

[0284] Viral toxicization assay: Astodrimer sodium was sequentially diluted 1:3 nine times and tested in triple measurement wells. SARS-CoV-2 was mixed with diluted astodrimer sodium at an MOI of 0.05 and incubated in 5% CO2 at 37°C for 1 hour. The virus-compound mixture was added to a monolayer of Vero E6 cells in a 96-well plate and incubated in 5% CO2 at 37°C for 4 days. On day 4, virus-induced CPE was measured by the MTT assay as described above. For viral toxicization evaluation, astodrimer sodium concentrations (0.0046 to 30 mg / mL) were incubated with SARS-CoV-2 2019-nCoV / USA-WA1 / 2020 for a period ranging from 5 seconds to 2 hours. To neutralize the effect of astodrimer sodium, unbound compounds were separated from the astodrimer:virus mixture by pelletizing the pre-incubation mixture through a 20% sucrose cushion (Beckman SW40 Ti rotor). The supernatant containing astodrimer sodium was removed (i.e., neutralizing the effect of SPL7013), and the pelleted virus was then gently resuspended and added to Vero E6 or Calu-3 cell cultures. Viral infection, cell culture, and cytotoxicity assessment were performed as described in the plaque assay section above.

[0285] Effective concentration (EC 50 and EC 90 ) and cytotoxicity (CC 50 ) determination: 50% or 90% reduction in virus-induced CPE (EC, respectively) 50 or EC 90 The concentration of the compound that resulted in ) was calculated using the formula described in Example 1. A 50% decrease in cell viability (CC) occurred after 4 days of culture. 50 The concentration of the compound that produced the result was also calculated using the formula described in Example 1.

[0286] Time-of-Adsorption Assay (TOA): Vero E6 cell monolayers were grown in MEM supplemented with 1% (w / v) L-glutamine and 2% FBS. To ensure robust infection, cell cultures were infected with SARS-CoV-2 at a MOI of 1. The virus was adsorbed at 4°C for 1 hour, and then parallel cultures were incubated at 37°C for 0 min, 15 min, 30 min, 60 min, 2 min, 4 min, and 6 min before adding 0.345 mg / mL astodrimer sodium, 15 μM hydroxychloroquine, 5 μM remdesivir, or a negative control (assay medium only). At 0 min, the test or control was added immediately after pre-adsorption of the virus. Eight hours after viral infection (one replication cycle), the virus was collected from the cells at each time point. The supernatant containing the virus was retained, and the viral titer was determined at each time point via a viral yield assay.

[0287] Viral yield reduction assay: Viral titer from TOA test is compared to the median tissue culture infectious dose (TCID). 50 ) was quantified as TCID. 50 TCID is a measure of viral titer, representing the titer of the virus that causes infection in 50% of tissue culture samples. Vero E6 cell monolayers were grown in MEM supplemented with 1% (w / v) L-glutamine and 2% FBS. Viruses collected from each time point were added to three wells and successively diluted 3-fold across the plate for a total of nine different viral concentrations. Six of the wells contained only assay medium (i.e., no virus) and served as controls. The plates were incubated for 3 days, and then the cell monolayers were observed under a microscope using visual scoring of virus-induced CPE, which was used as the endpoint. TCID of the viral suspension 50 This was determined using the method of Reed and Muench (1938). Virus yield was expressed at each time point as a percentage of the virus growth compared to the growth without compound addition.

[0288] result: Viral-induced cell necrosis inhibition assay: In two independent viral-induced CPE inhibition assays, astodrimer sodium dose-dependently inhibited SARS-CoV-2 (hCoV-19 / Australia / VIC01 / 2020) replication in Vero E6 cells (Figures 2 and 3). Astodrimer sodium inhibited viral replication when added either 1 hour before or 1 hour after SARS-CoV-2 infection. Astodrimer sodium was initially tested in the range of 0.0013–8.63 mg / mL (0.078–520.4 μM). In a repeat set of the assay, astodrimer sodium was tested in the range of 0.0001–0.86 mg / mL (0.008–52.0 μM) to further characterize the lower end of the dose-response curve. Effective and cytotoxic concentrations, as well as selectivity indices, from the assays are shown individually as averages in Figure 2 for CPE determination. The selectivity index (SI) for astodrimer sodium against SARS-CoV-2 in the CPE test ranged from 793 to 2197 in the initial assay, where the compound was added 1 hour before and 1 hour after infection, respectively, and >70 to >80 in the repeated assay, where cytotoxicity was not observed up to the highest concentration tested (0.86 mg / mL). The positive control, remdesivir, was also active in the CPE inhibition assay and had an SI of >33.

[0289] Antiviral efficacy: To determine the ability of astodrimer sodium to inhibit a globally diverse range of SARS-CoV-2 strains, the compound was evaluated against 2019-nCoV / USA-WA1 / 2020 virus in Vero E6 cells and human Calu-3 cells. Antiviral readout information was based on virological endpoints of infectious virus or viral nucleocapsid released into the supernatant after infection. As shown in Table 4 and Figure 5, astodrimers inhibited 2019-nCoV / USA-WA1 / 2020 strains at EC levels of 0.019–0.032 mg / mL and 0.0320–0.037 mg / mL, respectively, when determined by plaque assays in Vero E6 cells or Calu-3 cells. 50Inhibition was observed. These data are consistent with astodrimer-mediated inhibition of replication of Australian SARS-CoV-2 isolates in vitro. Dose-response data by ELISA of nucleocapsid released into the supernatant were similar to infectious virus release data in each cell line (data not shown). Remdesivir, a positive control, was also active in the plaque assay. [Table 4]

[0290] Viral efficacy: The study was conducted to determine whether astodrimer sodium could reduce viral infectivity by irreversibly inactivating SARS-CoV-2 before infection of Vero E6 cells. Astodrimer sodium treatment showed a similar level of antiviral efficacy to the CPE study (Figure 2, Figure 4A) at 1.83 μM (0.030 mg / mL) EC 50 The study included SI values ​​of 130 and n=1. The antiviral activity in the early, mid, and late stages of viral replication was evaluated by adding the compound at different time points after infection (0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours). The amount of virus secreted into the supernatant 8 hours after infection was measured using TCID. 50The determination was made by the following: Viral toxic assays investigated whether astodrimer sodium could reduce viral infectivity by irreversibly inactivating SARS-CoV-2 before infection of Vero E6 cells and human respiratory Calu-3 cells. After incubation of the virus with the astodrimer for up to 2 hours and neutralization of the astodrimer, the astodrimer-exposed virus was added to cell cultures. After either 16 hours or 96 hours (day 4), the cell culture supernatant was collected to assess progeny viral infectivity, which was determined by the amount of infectious virus and nucleocapsid secreted. The SARS-CoV-2 replication life cycle is completed in approximately 8 hours (Ogando et al., 2020), and in these tests, sampling was performed at 16 hours (2 life cycles) or day 4 (12 life cycles) post-infection. By allowing for a possible 12 rounds of infection, the sampling point at day 4 (96 hours) identified that exposure to 106 pfu / mL SARS-CoV-2 with astodrimer sodium for 1–2 hours resulted in a dose-dependent reduction in viral infectivity. 10–30 mg / mL of astodrimer sodium achieved a maximum >99.999% (>5log10) reduction in infectivity in Vero E6 cells and a >99.9% (>3log10) reduction in infectivity in Calu-3 cells compared to untreated virus (data not shown). SARS-CoV-2 infectivity was also reduced by up to >99.999% in Vero E6 cells when the incubation time with 106 pfu / mL virus in astodrimer (10–30 mg / mL) was reduced to 15–30 minutes (data not shown).

[0291] Astodrimer sodium (1-30 mg / mL) and 10 4 , 10 5 , and 10 6 Incubation for a minimum of 5 seconds with pfu / mL viral inoculation yielded evidence of reduced infectivity, and 10-15 minutes of exposure was sufficient to achieve a >99.9% reduction in viral infectivity, with greater reductions achieved at lower viral inoculations (>99.999%). 4(Virus inoculation at pfu / mL, 10-30 mg / mL astodrimer sodium, and incubation time of 10-15 minutes) (Table 5, Figure 6). When evaluated 16 hours after infection of cells with astodrimer-exposed virus, >10 mg / mL of astodrimer sodium was found to inactivate >99.9% of SARS-CoV-2 (10⁴ pfu / mL) within just 1 minute of exposure (Table 6, Figure 7). 30-second exposure to the virus with astodrimer sodium did not show any detectable virucidal effect. [Table 5] [Table 6]

[0292] Timing of Addition Assay (TOA): To further investigate the mechanism of action of astodrimer sodium, a TOA assay was performed. The compound was added to virus-infected cells at the early, mid, and late stages of the SARS-CoV-2 replication lifecycle, which is completed in approximately 8 hours. Addition of 0.345 mg / mL of astodrimer sodium in the time range of 0 minutes to 6 hours after infection resulted in virus levels below the lower limit of detection at all time points (Figure 4B). This result was in contrast to the infectious virus levels detectable at all equivalent time points in positive controls (remdesivir and hydroxychloroquine sulfate) and virus-only cultures. Hydroxychloroquine sulfate, at 15 μM, had no recognizable effect on viral replication at any time point. Remdesivir (5 μM, EC) 50 (Approximately 5 to 10 times) If added within 15 or 30 minutes after infection, <1 log 10 TCID 50 This inhibited virus replication.

[0293] Consideration: Astodrimer sodium demonstrated potent antiviral activity against a variety of SARS-CoV-2 cells in vitro. Antiviral activity was demonstrated by reduction of CPE, release of infectious virus, and release of viral nucleocuspid proteins. Antiviral activity was demonstrated when astodrimer sodium was added to cells before infection and when the compound was added to cells already exposed to SARS-CoV-2. Irreversible virucidal activity was demonstrated when astodrimer sodium was mixed with the virus for only 1 minute.

[0294] Of particular note is that the SI of astodrimer sodium is significantly higher in antiviral assays compared to other antiviral compounds being investigated for SARS-CoV-2 activity (Pizzorno et al., 2020).

[0295] Remdesivir was used as an antiviral positive control in CPE inhibition and antiviral assays, and experimental EC 50 This was consistent with publicly available data generated from different clinical isolates of SARS-CoV-2 (Wang et al., 2020).

[0296] The antiviral data are consistent with astodrimer sodium being a potent inhibitor of early events in the viral life cycle. The antiviral assay data suggest that the antiviral activity of astodrimer sodium is consistent with its binding to the virus, thereby irreversibly inactivating the virus and blocking infection.

[0297] The complete elimination of viral infection at all time points in the TOA assay is consistent with the fact that astodrimer sodium is a potent antiviral agent that inhibits the early stages of viral infection and replication.

[0298] The virucidal activity of astodrimer sodium demonstrated that it irreversibly inhibits the initial stages of viral infection and replication. These test results suggest potent inhibition of viral binding, fusion, and entry, preventing viral replication and the release of infectious viral progeny.

[0299] The data from this study demonstrate that the compound exerts antiviral activity against geographically diverse SARS-CoV-2 isolates by interfering with the initial viral cell recognition event. Astodrimer sodium is a potent virucidal agent that reduces the infectivity of SARS-CoV-2 by >99.9% one minute after exposure to the virus. These studies support the idea that astodrimer sodium can prevent early viral entry steps such as adhesion, thereby reducing or preventing viral infection or cell-to-cell spread.

[0300] Antiviral agents such as astodrimer sodium, which block the binding of the virus to target cells, may be useful as prophylactic and / or therapeutic agents against SARS-CoV-2. These antiviral studies suggest that the recombination of astodrimer sodium for respiratory delivery may be an effective prophylactic strategy that blocks SARS-CoV-2 transmission and enhances other prophylactic and therapeutic strategies.

[0301] Example 4: Evaluation of the virucidal properties of SPL7013 against three human coronaviruses (hCoV-229E, hCoV-NL63, and hCoV-OC43) The virucidal properties of SPL7013 against hCoV-229E (ATCC No. VR-740), hCoV-NL63 (ZeptoMetrix Corp. No. 0810228CF), and hCoV-OC43 (ZeptoMetrix Corp. No. 0810024CF) were evaluated using an in vitro time-kill suspension test. The test viruses used in this test were from the BSLI high-titer virus stock.

[0302] On the day of use, the stock virus packets were removed from the -70°C freezer and thawed before use in the test. Percentage and log of the virus strain from the initial population. 10 The decrease was determined after exposure to the test sample for 60 seconds and 60 minutes. Viral titers were determined using the 50% tissue culture infectious dose (TCID50) calculation (quantitative study).

[0303] method: Cell Culture: The cell lines used were human lung fibroblasts (MRC-5, ATCC No. CCL-171), green monkey epithelial kidney cells (Vero, ATCC No. CCL-81), and human colon adenocarcinoma epithelium (HCT-8, ATCC No. CCL-244). The cells were maintained as monolayers in disposable cell culture equipment and used for the antiviral suspension test. Prior to the test, host cell cultures were seeded in 24-well cell culture plates. MRC-5 cells were approximately 90% densitive and less than 48 hours before inoculation with coronavirus strain 229E. Vero cells were approximately 90% densitive and less than 48 hours before inoculation with coronavirus strain NL63. HCT-8 cells were approximately 80% densitive and less than 48 hours before inoculation with coronavirus strain OC43. Growth medium (GM) was replaced with maintenance medium (MM) to support viral growth.

[0304] Test sample: SPL7013 aqueous solution, 99.1 mg / mL. A 15.99 mL portion of the test sample was added to 34.01 mL of sterile water to obtain a concentration of 31.95 mg / mL. The final concentration tested was 28.76 mg / mL.

[0305] Viral suspension test: The viral suspension test included the parameters listed in Table 7.

[0306] Test: A 0.5 mL sample of the test virus was added to a vial containing 4.5 mL of the test sample. The test virus was exposed to the test sample for 60 seconds and 60 minutes. Immediately after exposure, the test virus / test sample suspension was neutralized with fetal bovine serum, thoroughly mixed, and serially diluted in MM. Each dilution was added to a plate by quadrupling.

[0307] Virus control: A 0.5 mL sample of the test virus was added to 4.5 mL of MM and exposed to ambient temperature for 60 seconds and 60 minutes. Subsequent dilutions of the test virus were performed in MM, followed by serial dilutions in MM. Each dilution was added to a plate using quadruple counting.

[0308] Cytotoxicity control: A 0.5 mL portion of MM was added to a vial containing 4.5 mL of the test sample. The MM / test sample mixture was neutralized with fetal bovine serum, thoroughly mixed, and serially diluted with MM. Each dilution was added to a plate in quadruple counts.

[0309] Neutralization control: A 0.5 mL portion of MM was added to a vial containing 4.5 mL of undiluted test sample. The MM / test sample mixture was diluted 1:10 with fetal bovine serum. The portion of virus was added to the neutralized test sample and thoroughly mixed, and the neutralized test sample was exposed for 10-20 minutes. Subsequent 10-fold dilutions of the neutralized test sample / virus suspension were performed with MM. Each dilution was added to a plate in quadruple counts.

[0310] Neutralizer toxicity control: The effect of the neutralizer on viral infectivity was evaluated by adding the virus to the neutralizer (fetal bovine serum) and exposing it for 10-20 minutes. Subsequently, a 10-fold dilution of the neutralized test product / virus suspension was performed using multipliers (MM). Each dilution was added to a plate in quadruple counts.

[0311] Cell culture control: The original cell culture served as a control for cell culture viability. GM was replaced with MM in all cell control wells.

[0312] The plates were incubated in a CO2 incubator for 10–14 days, adjusting the temperature to suit each virus. Cell necrotic / cytotoxic effects were monitored using an inverted combined microscope. [Table 7]

[0313] result: Virality data for SPL7013 against three human CoV strains are provided in Tables 8, 9, and 10. An aqueous solution of SPL7013 at 99.91 mg / mL reduced the infectivity of hCoV-229E to 0.75 log after 60 seconds and 60 minutes of exposure. 10 (82.22%) reduction, and 0.50 log of hCoV-NL63 infectivity after 60 seconds of exposure. 10 (68.38%) reduction, 0.75 log after 60 minutes of exposure. 10 (82.22%) Reduced the infectivity of hCoV-OC43 by 0.50 log after 60 seconds and 60 minutes. 10 (68.38%) reduction was achieved. These results indicate that SPL7013 is active against multiple human CoV strains. [Table 8] [Table 9] [Table 10]

[0314] Example 5: Evaluation of the virucidal properties of SPL7013 against SARS-CoV-2 strain Slovakia / SK-BMC5 / 2020 The antiviral properties of SPL7013 were evaluated against the SARS-CoV-2 strain Slovakia / SK-BMC5 / 2020. This strain was isolated from a COVID-19 patient in Slovakia in March 2020.

[0315] method: Cells: Vero E6 / TMPRSS2 non-human primate renal epithelial cells (National Institute for Biological Standards and Controls, UK).

[0316] The virus Slovakia / SK-BMC 5 / 2020 was supplied through the European Virus Archive goes Global (Evag) platform. SARS-CoV-2 was amplified and titrated in the Vero E6 / TMPRSS2 cell line.

[0317] Cytotoxicity and viral quantification (Experiment 1): Cells were counted and their viability was evaluated using a Vi-Cell automated system. Cells were seeded at approximately 15,000 cells / well. Cells were pretreated at 37°C for 1 hour as follows: Eight doses of SPL7013 (10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.0046 mg / mL) were prepared in cell medium. A reference compound (Apilimod) was prepared at three concentrations (1000, 300, and 100 nM). Subsequently, Slovakia / SK-BMC5 / 2020 was added to pretreated cells at one MOI (approximately 0.01) in a 10 μL volume and incubated in a 37°C incubator for 48 hours. The supernatant was collected for viral load determination (RT-qPCR).

[0318] The CellTiter96® AQueous non-radioactive cell proliferation assay (MTS / PMS assay) was performed on both plate controls (virus-free) and plates treated and infected as described above. The assay (Promega reference number G5430) was performed according to the manufacturer's protocol. The supernatant was removed from the wells for PCR reaction, and 100 μL of fresh cell medium and 20 μL of MTS / PMS reagent were added to each well. Absorbance was recorded every hour for 4 hours.

[0319] Viral load quantification by RTqPCR was performed at the end of the experiment using the ORF1ab gene. RNA was extracted using a virus kit (Macherey-Nagel, kit number 740709). The RNA was frozen at -20°C until use. RT-PCR was performed using a Bio-Rad CFX384™ instrument and connected software with the SuperScript™ III One-Step QRT-PCR System kit (commercial kit number 1732-020, Life Technologies).

[0320] Microscopic examination (Experiment 2): Cells were seeded at approximately 15,000 cells / well. The cells were pretreated at 37°C for 1 hour as follows: Eight doses of SPL7013 (10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.0046 mg / mL) were prepared in cell medium. The reference compound (Apilimod) was prepared at three concentrations (1000, 300, and 100 nM). Next, Slovakia / SK-BMC5 / 2020 was added to the cells in a volume of 10 μL at 1 MOI (approximately 0.5) and incubated at 37°C for 6 hours. Cells were fixed for immunofluorescence staining using SARS-CoV-2 (2019-nCoV) nucleoprotein / NP antibody and rabbit Mab primary antibody (Sino Biological, No. 40143-R019, 1:8000 dilution), and imaged using Operetta. FFU / mL.

[0321] The results are shown in Figures 11 and 12.

[0322] Example 6: In vivo evaluation of SPL7013 after 7 days of nasal administration for SARS-CoV-2 infection in hACE2 transgenic mice. method: In short, the effect of SPL7013 on in vivo viral load was evaluated using four groups of five human ACE2 transgenic mice K18-hACE2 (available from Jackson Laboratory, B6.Cg-Tg(K18-ACE2)2Prlmn / J, stock number 034860) aged approximately 6-8 weeks.

[0323] These groups of animals were 4 intranasally inoculated with 25 μL per nostril of a virus suspension containing 10 5 PFU / μL of SARS-CoV-2 (full load: 5×10

[0324] Results: There was a dose-dependent decrease in viral copies (qPCR) in nasal swabs on day 7, which reached statistical significance at the highest dose level compared to the control (Figure 8A). This data indicates that when administered nasally, SPL7013 can dose-dependently reduce the amount of SARS-CoV-2 virus acquired by the nose. A dose of 2.5 mg / day was the most effective in reducing the viral load.

[0325] Example 7: Evaluation of the antiviral properties of SPL7013 against severe acute respiratory syndrome coronavirus (SARS) and Middle East respiratory syndrome (MERS) coronavirus Methods: Cell culture and virus: hACE2 + and hTMPRSS2 +HEK-293T cells expressing [the specified virus], as well as Vero E6 cells (ATCC-CRL1586), were cultured in L-glutamine-free minimal essential medium (MEM) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (w / v) L-glutamine. Hanks equilibrium salt solution (HBBS) supplemented with 2% FBS was used for infection. Pseudotyped SARS-CoV-1 (Urbani), SARS-CoV-2 (Wuhan-Hu-1), and MERS-CoV (HCoV-EMC) reporter virus particles (RVPs) were generated using Integral Molecular (catalog numbers RVP-801, RVP-701, and RVP-901, respectively). RVP carries a modified genome that displays an antigenically accurate spike protein on a heterologous viral core and expresses green fluorescent protein (GFP), a convenient optical reporter gene, within 24 hours of cell infection. A recombinant SARS-CoV-2 spike receptor-binding domain (RBD) protein with an mFc tag (SARS-CoV-2 spike RBD (318-541) recombinant protein (mFc-Tag) number 41701, Cell Signalling Technology) was used according to the manufacturer's instructions.

[0326] Assays with SARS-CoV-1, SARS-CoV-2, and MERS-CoV pseudotyped GFP reporter lentivirus particles: 100,000 Vero 6 cells were seeded in each well of a 96-well plate for the assay. The seeded cells were cultured in DMEM / 10% FBS. SPL7013 (0, 10, and 30 mg / mL in PBS) was added to Vero E6 cells, and 1 hour later, SARS-CoV-1, SARS-CoV-2, and MERS-CoV spike pseudotyped GFP reporter lentivirus particles (RVP-801, RVP-701, and RVP-901, Integral Molecular) (50 μL) were added. The percentage of GFP-positive or infected Vero E6 cells was determined by fluorescence-activated cell sorting (FACS) flow cytometry 48 hours after infection.

[0327] Confocal microscopy of SARS-CoV-2 spike binding to hACE2+hTMPRSS2+293T cells: hACE2 + hTMPRSS2 + 293T cells were cultured on chamber slides. After treating the cells with SPL7013 (0, 1 mg / mL in PBS) for 1 hour, SARS-CoV-2 spike RBD recombinant protein with an mFc tag was loaded. After 1 hour, the cells were washed twice, and anti-mFc-PE IgG antibody (1 μg / mL) was added to the cells to identify the bound spike protein. After 30 minutes, the cells were washed twice again, fixed, and analyzed by confocal microscopy.

[0328] Results: Assays in SARS-CoV-1, SARS-CoV-2, and MERS-CoV pseudotyped GFP reporter lentiviral particles: SPL7013 was found to have a broad-spectrum antiviral effect specific to inhibition of spike protein function, binding, fusion, or both. Pseudotyped lentiviral particles expressing antigenically accurate spike proteins encoded by SARS-CoV-1, SARS-CoV-2, and MERS-CoV were used to infect Vero E6 cells (Figure 8B). SARS-CoV-1 and SARS-CoV-2 bind to Vero E6 cells via the ACE2 receptor. MERS-CoV binds to dipeptidyl peptidase 4 (DPP4). All three coronaviruses utilize the TMPRSS2 protease to cleave their S1 / S2 regions. SPL7013 potently inhibited the binding of pseudotyped lentiviruses expressing the spike proteins of SARS-CoV-1, SARS-CoV-2, and MERS-CoV to Vero E6 cells at concentrations of 10 and 30 mg / mL.

[0329] hACE2 + hTMPRSS2 +Confocal microscopy of SARS-CoV-2 spike binding to 293T cells: Confocal microscopy showed that the SARS-CoV-2 spike protein bound to cells expressing the hACE2 receptor on the cell membrane in the absence of SPL7013 in the negative control (without SPL7013). This was demonstrated by strong green immunofluorescence in micrographs showing significant binding between the SARS-CoV-2 spike protein and host cells (micrographs not shown). When SARS-CoV-2 was added in the presence of SPL7013, no detectable binding between cells expressing the hACE2 receptor and the SARS-CoV-2 spike protein was observed. This was indicated by the complete absence of green immunofluorescence in micrographs (micrographs not shown). These tests confirmed that SPL7013 acts by blocking the SARS-CoV-2 spike protein. The SARS-CoV-2 spike protein is essential for initiating the interaction between the virus and target cells via the ACE2 receptor, which leads to cell infection. If there is no binding between the SARS-CoV-2 spike protein and cells, cell infection cannot occur.

[0330] This study using SARS-CoV and MERS-CoV demonstrated that SPL7013 blocks the binding of the SARS-CoV-2 spike protein at concentrations that are effective against spike protein binding and infection. These studies show that SPL7013 acts against all of these viruses through a common mechanism that blocks the interaction of coronavirus spike proteins with cells, regardless of the cell receptor involved. In summary, these data support the antiviral effect of SPL-7013 against human pathogenic coronaviruses.

[0331] Example 8: Evaluation of the antiviral properties of SPL7013 against respiratory syncytial virus (RSV) The concentration of the test sample was prepared using a dilution of approximately 1:3 from the starting concentration determined according to the cytotoxicity test.

[0332] The host cell culture was washed with PBS, and 1.0 mL of diluted test solution was added to the cells. The cells were then incubated for 1 hour ± 15 minutes for equilibration. After incubation, 1.0 mL of virus was added to the wells, and the cells were incubated for 1 hour ± 15 minutes for viral adsorption. After incubation, the mixture was removed and replaced with TM. The plates were then incubated in a CO2 incubator. The plates were incubated until viral plaques in the viral control could be observed under a microscope (approximately 5–10 days).

[0333] For the cytotoxic control, host cell cultures were washed with PBS. Next, 1.0 mL of the highest concentration of the non-toxic test sample was added to the cells and incubated for 1 hour ± 15 minutes for equilibration. After incubation, 1.0 μl of MM (simulated infection) was added to the wells and incubated for 1 hour ± 15 minutes. After incubation, the mixture was removed and replaced with TM. Next, the plates were incubated in a CO2 incubator.

[0334] For the virus control, the host cell culture was washed with PBS, and 1.0 mL of MM (in place of the test sample) was added to the wells designated for the virus control. The wells were incubated for 1 hour ± 15 minutes for equilibration. After incubation was complete, 1.0 mL of virus was added to the wells and incubated for 1 hour ± 15 minutes for virus adsorption. After incubation, the virus was removed and replaced with TM. The plates were incubated in a CO2 incubator.

[0335] The unmodified cell culture monolayer was used as a control for cell viability. GM was replaced with TM in the cell culture control wells.

[0336] After incubation, fixation and staining were performed by removing TM, washing the plate with PBS, and fixing it with 4% formaldehyde solution for 4–6 hours. Fixed cells were stained using crystal violet stain. Unstained zones (viral plaques) of lysed cells were counted.

[0337] Post-Antiviral Treatment Test - Determination of Cytotoxicity of Test Substance: The maximum non-cytotoxic concentration of the test substance was determined. Host cell cultures were washed with PBS. 1.0 mL of the test substance was added to the cells and incubated in a CO2 incubator at 37°C ± 2°C for 24 hours ± 1 hour. Cytotoxicity was evaluated using the CCK-8 assay and read at 450 nm using a VERSAmax® adjustable microplate reader. The concentration used in the antiviral test was determined in the cytotoxicity test. Results are shown as a percentage of cell viability where 100% cell viability is approximately equal to the mean cell viability of the cell control. The TC50 concentration of the test substance was determined using GraphPad Prism 5.0 statistical software. The post-antiviral treatment test included the procedure outlined in Table 11. [Table 11]

[0338] The concentration of the test sample was prepared using a dilution of approximately 1:3 from the starting concentration determined according to the cytotoxicity test.

[0339] Host cell cultures were washed with PBS, and 1.0 mL of virus was added to each well. The cultures were incubated for 1 hour ± 15 minutes for viral adsorption. After incubation, the viral inoculum was removed and replaced with a fraction of the test sample concentration in TM. The plates were then incubated in a CO2 incubator. Subsequently, the plates were incubated until viral plaques in the viral control could be observed under a microscope (approximately 5–10 days).

[0340] For cytotoxic controls, host cell cultures were washed with PBS. The cells were then stacked with the highest non-toxicity test concentration in TM and incubated in a CO2 incubator.

[0341] For the viral control, host cell cultures were washed with PBS. 1.0 mL of ≤100 PFU / mL virus was added to the wells and incubated for 1 hour ± 15 minutes for viral adsorption. After incubation, the viral inoculum was removed and replaced with a fraction of TM. The plates were incubated in a CO2 incubator.

[0342] For the cell culture control, the unprocessed cell culture monolayer was used as a control for cell viability. GM was replaced with TM in the cell culture control wells.

[0343] After incubation, fixation and staining were performed by removing TM, washing with PBS, and fixing with 4% formaldehyde solution for 4–6 hours. Fixed cells were stained using crystal violet stain. Unstained zones (viral plaques) of lysed cells were counted.

[0344] Evaluation of antiviral properties: Antiviral properties EC50 and / or EC90 were determined using nonlinear regression analysis with GraphPad Prism 5.0 software.

[0345] Acceptance Criteria for Antiviral Testing: The validation tests described in the examples require that 1) plaques in the test and control samples are countable, 2) no significant cytotoxic effect is present in the cytotoxic control, 3) the cell control wells are viable and adhere to the bottom of the wells, and 4) the culture medium is not contaminated in any of the wells of the plate.

[0346] result: The results are summarized in Table 12 and Figure 9. [Table 12]

[0347] Example 9: SPL7013 Nasal Spray The embodiment of the device described herein is a nasal spray. An embodiment of the nasal spray is provided herein and referred to as “SPL7013 Nasal Spray”. SPL7013 Nasal Spray comprises an aqueous nasal composition containing SPL7013 and is referred to as “SPL7013 Nasal Spray Composition”. SPL7013 is intended to inactivate viruses, including SARS-CoV-2 and / or RSV, and reduce exposure to viral load. Reducing viral load can reduce the acquisition or transmission of infection. When SPL7013 Nasal Spray contains SPL7013 Nasal Spray Composition, it produces a droplet size suitable for administration and delivery to the nasal cavity, having less than 5% droplets of 10 μM or less (10 μM particles are more suitable for delivery to the lungs).

[0348] The action of SPL7013 nasal spray, including the SPL7013 nasal spray composition, can be used to create a moisturizing and protective mucosal adhesion barrier for the nasal mucosa and to inactivate respiratory viruses and act as a barrier against them.

[0349] The SPL7013 nasal spray composition comprises SPL7013 and a viscosity-modifying mucosal adhesive. The SPL7013 nasal spray composition is as described in “Variation 4” listed in Table 14 or “Variation 5” shown below in Table 13. Variation 5 is a Variation 4 formulation in which the pH is further adjusted with hydrochloric acid. The formulation contains carbomer homopolymer type B to achieve appropriate viscosity, aid in ease of administration, and promote retention of the product in the nasal cavity. [Table 13]

[0350] SPL7013 nasal spray is supplied as a 10 mL multi-dose, metered nasal spray device that delivers approximately 100 μL of SPL7013 nasal spray composition per action. Other embodiments of SPL7013 nasal spray may include slightly smaller or larger volumes, or smaller or larger metered doses. SPL7013 nasal spray composition can be self-administered by the user as needed for up to 30 consecutive days and / or up to four times a day (in each nostril) as needed for viral inactivation and reduction of exposure to viral load.

[0351] Referring to the Global Medical Device Nomenclature (GMDN), the applicable term for the SPL7013 nasal spray is "Nasal Moisture-Proof Covering," with GMDN code 47679. Considering the physical nature of the viral inactivation mechanism by SPL7013 and the physical mode of operation of the device, this product is considered a Class I medical device under the European Medical Device Directive 93 / 42 / EEC.

[0352] When SPL7013 nasal spray contains the SPL7013 nasal spray composition, it provides, when in operation, i) a moisturizing and protective mucosal adhesive formulation that acts as a barrier against respiratory viruses when applied to the nasal mucosa, ii) inactivating viruses and reducing exposure to viral load, and iii) reducing viral load as a result of i) and / or ii). A reduction in viral load may help prevent the acquisition or transmission of infections.

[0353] The acceptable limits for the SPL7013 nasal spray composition are summarized below. The osmotic pressure and pH of the nasal spray composition should be adjusted to a physiological state of <500 mOsmol at a pH of 5.5–6.5. The acceptable limits for SPL7013 nasal spray in terms of osmotic pressure and pH are 200–400 mOsmol and 5.5–6.5, respectively. SPL7013 nasal spray typically has a density of approximately 1 g / mL, which is suitable for retention in the nasal cavity. The acceptable limits for release and exhalation in the assay of SPL7013 are 0.80–1.20% w / w. The acceptable limits for methylparaben and propylparaben are 0.14%–0.23% and 0.015%–0.025%, respectively. The microbial content of SPL7013 nasal spray is determined by a Microbial Limits Test in accordance with Ph. Eur. 2.6.12 Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests and Ph. Eur. 2.6.13 Microbial Examination of Non-Sterile Products: test for Specified Micro-Organisms. The total aerobic number and total yeast and mold present in the test material are determined using the standard pour plate methodology. The specifications for microbial content adhere to the established limits described in Ph. Eur. 5.1.4 Microbiological Quality of Pharmaceutical Preparations. Identified organisms are based on Ph. Eur 5.1.4, Microbiological Quality of Non-Sterile Pharmaceutical Preparations and Substances for Pharmaceutical Use.

[0354] The SPL7013 nasal spray is packaged as a non-pressurized compact container closure system. The container closure system includes a delivery system (pump with actuator) that dispenses 100 μL of spray droplets of the SPL7013 nasal spray composition. The delivery device consists of a pump screwed into a polyethylene (HDPE) bottle, and the pump's dip tube, housing, gasket, and stem are made from polyethylene polymer. The ball is made from stainless steel 1.430 and is corrosion-resistant. The liner is made from polyoxymethylene.

[0355] Example 10: Biological evaluation of SPL7013 nasal spray A comprehensive biological evaluation was performed on the SPL2013 nasal spray described in Example 9.

[0356] SPL7013 nasal spray is a surface device that comes into contact with the mucous membrane (nose) during prolonged exposure (>24 hours to 30 days). In accordance with ISO 10993, tests for in vitro cytotoxicity (ISO 10993-5), nasal irritation after repeated administration in rats (ISO 10993-10), and skin sensitization in a guinea pig model (ISO 10993-10) were performed using SPL7013 nasal spray packaged in the container closure system described in Example 9.

[0357] In vitro cytotoxicity: In vitro cytotoxicity tests at 5,000 μg / mL showed that SPL7013 nasal spray was not cytotoxic. In nasal irritation tests, rats were administered 100 μL of 1% SPL7013 nasal spray into each nostril four times daily for 14 consecutive days. Results from survival stages and histopathological examinations showed no findings related to the product, indicating that SPL7013 nasal spray is not an irritant.

[0358] Skin sensitization: Skin sensitization testing consisted of the guinea pig maximization test (GMPT) by Magnusson and Kligman (1969). The test demonstrated that 1% SPL7013 nasal spray was not a sensitizer.

[0359] Nasal Tolerance and PK: SPL7013 nasal spray containing 1% or 3% SPL7013 was also tested for local toxicity and the potential for systemic absorption of SPL7013 by administering it nasally to rats four times a day (50 μL per nostril) for seven days. In this study, repeated nasal administration of SPL7013 nasal spray showed good tolerance and did not cause any clinical signs of local or systemic toxicity. In addition, plasma samples were collected from the animals in this study before the first administration of the product on day 1 of the study, and at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours after the first administration, and on day 7 of the study, before the last administration of the product on that day, and at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours after the last administration on that day. Bioanalysis of pooled plasma samples by capillary electrophoresis showed that SPL7013 was not detected above the lower limit of quantification (LLOQ, 0.5 μg / mL) in any sample from animals administered 1% or 3% SPL7013 nasal spray four times daily for seven days, except for one sample from the 1% group which showed a result slightly above LLOQ (0.635 μg / mL) at 3 hours (data not shown). The data indicate that SPL7013 is not systemically absorbed after application to the nasal mucosa of rats four times daily for seven days, and the result for one sample in the low-dose group is an aberration.

[0360] Example 11: SPL7013 formulation and test The SPL7013 (astodrimer sodium) preparation was prepared as shown in Table 14. [Table 14]

[0361] Viscosity and osmotic pressure were measured within a few hours of preparation. The results are shown in Table 15 below. [Table 15]

[0362] To evaluate the suitability of the formulations for nasal delivery, the formulations were screened using three nasal aerosol pumps, and particle sizes were measured at 30 mm and 60 mm, as shown in Table 16. [Table 16]

[0363] These results demonstrate that the formulation provides a particle size suitable for intranasal delivery in various nasal pump delivery devices.

[0364] Further DSD testing was performed on four modified formulations to further investigate particle size at a delivery rate suitable for optimal results, as shown in Table 17 below. The experiments were conducted using a Spraytec Open Spray equipped with a 300mm lens. [Table 17]

[0365] Example 12: Evaluation of SPL7013's hygroscopic properties Hygroscopicity was assessed as described in European Pharmacopoeia 5.11. The results of this assay are interpreted as follows: Deliquescent - sufficient water is absorbed to form a liquid; Highly hygroscopic - mass increase of 15% or more; Hygroscopic - mass increase of less than 15% and 2% or more; Slightly hygroscopic - mass increase of less than 2% and 0.2% or more; Not hygroscopic - if the mass increase is less than 0.2%, the compound is not hygroscopic.

[0366] Therefore, SPL7013 was found to have high hygroscopicity, with a mass increase exceeding 15% (21.97%). The hygroscopic result was confirmed by moisture content analysis using the Karl Fischer test.

[0367] Example 13: SPL7013 activity against SARS-CoV-2 in primary human airway cells Primary human bronchial epithelial cells (HBEpCs) (Sigma-Aldrich, MO, USA) were grown and maintained in HBEpC / HTEpC growth medium (Cell Applications, CA, USA). These primary cells express the ACE2 receptor and are tolerant to SARS-CoV-2 infection. Using these cells, the antiviral effect of astodrimer sodium against SARS-CoV-2 in primary human airway epithelial cell lines was determined.

[0368] The cells are 2.5 × 10 4 Each well contains 10 units of SARS-CoV-2 2019-nCoV / USA-WA1 / 2020. 3 Infection was induced by adding 1 mL of pfu / mL. Positive controls were treated with 10 μg / mL of SARS-CoV-2 spike protein antibody (pAb, T01KHuRb) (ThermoFisher, MA, USA) at the time of infection. Iota-carrageenan (Sigma-Aldrich, MO, USA) was used in primary epithelial cell nucleocapsid and plaque assays, and its antiviral activity was compared with that of astodrimer sodium. The concentrations used were those reported to exhibit activity against SARS-CoV-2 (Bansal et al., 2020).

[0369] SPL7013 (0, 1.1, 3.3, and 10 mg / mL) or iota-carrageenan (0, 6, 60, and 600 μg / mL) was added to HBEpC cells one hour before infection with SARS-CoV-2. The cells were cultured for 4 days after infection, and the amount of secreted SARS-CoV-2 nucleocapsid was analyzed from the cell supernatant by ELISA. Infectious virus was quantified by plaque assay as described in Example 3.

[0370] To determine the ability of SPL7013 to prevent SARS-CoV-2 infection in primary human epithelial cells, the compound was evaluated against the 2019-nCoV / USA-WA1 / 2020 strain in HBEpC cell cultures.

[0371] SPL7013 was found to reduce SARS-CoV-2 infection of HBEpC primary cells by up to 98% by nucleocapsid ELISA (Figure 10A) and up to 95% by plaque assay compared to a viral control (data not shown). In contrast, treatment with iota-carrageenan had minimal antiviral effect against SARS-CoV-2 in this cell line; the highest concentration tested reduced infection by only 17% by nucleocapsid ELISA (Figure 10B) and only 21% by plaque assay (data not shown). The maximum level of inhibition with astodrimer sodium was comparable to the inhibition achieved in SARS-CoV-2 spike protein antibody (pAb,T01KHuRb)-positive controls.

[0372] Astodrimer sodium inhibited SARS-CoV-2 infection of human respiratory tract primary epithelial cells, whereas iota-carrageenan, a polyanionic compound in commercially available nasal spray formulations, did not provide significant inhibition at concentrations previously shown to reduce SARS-CoV-2 infection in Vero E6 cells (Bansal et al., 2020). The unique structure of astodrimer sodium, a generally spherical sulfonated molecule with a core and densely branched radially from the core, appears to offer potential advantages over other polyanionic compounds such as iota-carrageenan and heparin, which are linear sulfonated molecules with a molecular weight distribution. We are not aware of any data showing that iota-carrageenan is virucidal, while heparin has been demonstrated to lack irreversible virucidal interactions with HSV virion components (Ghosh et al., 2009).

[0373] Example 14: Rat SPL7013 Biocompatibility Test Biocompatibility testing of SPL7013 in formulation variant 4 was conducted in rats (data not shown).

[0374] The product was tested to evaluate its cytotoxic effect in Balb / c3T3 cells. In conclusion, a 5 mg / ml solution of SPL7013 is not cytotoxic.

[0375] The product was tested to evaluate its sensitizing properties in 10 albino guinea pigs after intradermal and topical administration followed by a 14-day loading period. In conclusion, no macroscopic skin reactions due to allergies were recorded after the loading stage, and the product is not classified as a skin sensitizer according to ISO 10993-10.

[0376] The product was administered to three female Sprague Dawley rats via the intranasal route four times a day at a dose of 0.1 ml per nostril for 14 days. No deaths were observed, no clinical signs associated with the administration of the test product were observed, no erythema or edema was observed at the treatment site, and body weight remained normal. There was no evidence of inflammatory changes or effects in the epithelium. In conclusion, the test product was well-tolerated and did not induce any evidence of irritation as assessed according to ISO 10993-10.

[0377] Example 15: Clinical Trial The clinical trial involved 40 patients receiving either 1% SPL7013 in Formulation 4 or placebo (Formulation 4), administered via a spray device at a dose of 100 μl per nostril four times daily for 14 days. No serious adverse events were reported, and the formulations were generally well-tolerated with minimal irritation.

[0378] Example 16: Overview The experiments described herein demonstrate that SPL7013 exhibits potent antiviral activity with very high SIs against multiple strains of SARS-CoV-2 in different cell lines. At a concentration of SPL7013 in nasal spray (10 mg / mL), the reduction in infectious virus was >5 log in Vero E6 cells. 10(>9 9.999%), and >3log10 (>99.9%) in Calu-3 cells. Tests examining the dynamics of antiviral activity show that SARS-CoV-2 inactivation can be observed in a dose-dependent manner with just 5 seconds of exposure to the virus with SPL7013.

[0379] Those skilled in the art will understand that many variations and / or modifications can be made to the embodiments described above without departing from the broad general scope of this disclosure. Therefore, these embodiments should be considered in all respects as illustrative and not limiting.

[0380] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0381] This application claims priority to Australian Provisional Application No. 2020 / 901194, entitled “Method of Prophylaxis of Coronavirus infection,” filed on 15 April 2020; Australian Provisional Application No. 2020 / 902993, entitled “Method of Prophylaxis of Coronavirus infection,” filed on 21 August 2020; and Australian Provisional Application No. 2020 / 904246, entitled “Method of prophylaxis of respiratory syncytial virus infection,” filed on 17 November 2020, the entire contents of which are incorporated herein by reference.

[0382] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0383] Any considerations of documents, actions, materials, devices, articles, etc., included herein are solely for the purpose of providing context for the present invention. It should not be assumed that any or all of these matters constitute part of the foundation of the prior art or were general knowledge in the art related to the present invention that existed prior to the priority date of each claim of this application.

[0384] The processes, features, integers, compositions, and / or compounds disclosed herein or individually or collectively in the specification of this application, as well as any and all combinations of two or more such steps or features.

[0385] References Bansal et al (2020) bioRxiv doi:10.1101 / 2020.08.19.225854. Borchers et al (2013) Clin Rev Allergy Immunol 45(3):331-378 doi:10.1007 / s12016-013-8368-9. Chandel et al (2019) Biomedicine & Pharmaco doi:10.1016 / j.biopha.2019.108601. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses (2020) Nature microbiology doi:10.1038 / s41564-020-0695-z. Foster et al (2020) PNAS doi:10.1073 / pnas.2004999117. Ghareeb et al(2021)J Pharm Investig doi:10.1007 / s40005-021-00520-4. Ghosh et al(2009) Glycobiology 19(1), 2-15.doi:10.1093 / glycob / cwn092. Gordon et al (2020) BioRxiv doi:10.1101 / 2020.03.22.002386. Ibrahim et al(2015)With Devices (Auckl) doi:10.2147 / MDER.S48888. Keyaerts et al(2004)Biochem Biophys Res Commun 8;323(1):264-8. Kucharski et al(2020)Lance doi:10.1016 / S1473-3099(20)30144-4 Magnusson and Kilgman(1969)52(3):268-76.doi:10.1038 / jid.1969.42. Pizzorno et al(2020)Antiviral Res doi:10.1016 / antiviral.2020.104878. Melero et al(2013)Curr Top Microbiol Immunol 372:59–82.doi:10.1007 / 978-3-642-38919-1_3. Ramaekers(2020)Virus Evol 2Jul 24;6(2):veaa052.doi:10.1093 / ve / veaa0 Reed and Muench (1938) J Hyg 27(3), 493-497. Shen et al (2020) Clin Infect Dis doi:10.1093 / cid / ciaa2 Tang et al (2020) Nat Sci Rev doi:10.1093 / nsr / nwaa0 Int Res J of Pharm 7(4):9-16. van den Worm et al(2012)PLoS ONE 7(3):e32857 doi:10.1371 / journal.pone.0032857. Verma et al(2014)J der Pharmazie Forschung 2(1):1-10. Wang et al(2020) Cell Res 30(3), 269-271 doi:10.1038 / s41422-020-0282-0. Zarogoulidis et al(2012) Int J Nanomedicine 7:1551-72.

Claims

1. When administered to human subjects, To prevent or reduce the possibility of coronavirus (CoV) infection in the aforementioned human individual; To prevent or reduce the likelihood or severity of symptoms associated with coronavirus (CoV) infection in the aforementioned human individual; To prevent or reduce the severity and / or persistence of coronavirus (CoV) infection in the aforementioned human individual; To treat coronavirus (CoV) infection in the aforementioned human individual; or To prevent or reduce viral shedding in human individuals infected with coronavirus (CoV); or to administer to the respiratory system of a portion of a human population. To reduce the transmission of coronavirus (CoV) in the aforementioned human population. A pharmaceutical composition comprising a macromolecule or a pharmaceutically acceptable salt thereof, A composition in which the macromolecule is 2,6-bis-{(1-naphthalenyl-3,6-disulfonic acid)-oxyacetamide}-2,6-bis-2,6-bis-2,6-bis-(2,6-diamino-hexanoylamino)-2,6-diaminohexanoic acid (diphenylmethyl)-amide.

2. The composition according to claim 1, comprising a pharmaceutically acceptable salt of the macromolecule, 2,6-bis-{(1-naphthalenyl-3,6-disulfonic acid)-oxyacetamide}-2,6-bis-2,6-bis-2,6-bis-(2,6-diamino-hexanoylamino)-2,6-diamino-hexanoic acid (diphenylmethyl)-amide, polysodium salt.

3. The composition according to claim 1 or 2, wherein the symptoms associated with the CoV infection are selected from one or more of the following: fever, cough, sore throat, shortness of breath, viral shedding, respiratory failure, runny nose, nasal congestion, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, and acute respiratory distress syndrome (ARDS).

4. The composition according to any one of claims 1 to 3, wherein the CoV is selected from alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus.

5. The composition according to any one of claims 1 to 4, wherein administration includes local administration or administration to the respiratory system.

6. The composition according to any one of claims 1 to 5, wherein administration to the respiratory system includes administration to the upper respiratory tract and / or the lower respiratory tract.

7. The composition according to any one of claims 1 to 6, wherein the composition comprises 0.5% to 5% by weight of the macromolecule or a pharmaceutically acceptable salt thereof, or wherein the composition comprises 1% by weight of the macromolecule or a pharmaceutically acceptable salt thereof.

8. The composition according to any one of claims 1 to 7, wherein the effective amount is 0.1 to 5 mg per dose, or the effective amount is 1 mg per dose.

9. The composition according to any one of claims 1 to 8, wherein the macromolecule or a pharmaceutically acceptable salt thereof is administered 1 to 8 times a day, and / or the macromolecule or a pharmaceutically acceptable salt thereof is administered for 1 to 2 weeks, or 1 to 3 weeks, or less than 30 days.

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