TW202214270A - Method of prophylaxis of coronavirus and/or respiratory syncytial virus infection - Google Patents

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 a symptom associated with a CoV and/or RSV infection in an individual, reducing the severity and/or duration of a CoV and/or RSV infection in an individual, or treating a CoV and/or RSV infection in an individual, preventing or reducing viral shedding in an individual infected with a CoV and/or RSV infection, or reducing transmission of a CoV and/or RSV in a population comprising administering to the individual an effective amount of a macromolecule. The present invention also relates to a device for delivering a composition comprising a macromolecule.

Description

Methods of preventing coronavirus and/or respiratory syncytial virus infection

Field of Invention

The present invention pertains to methods and compositions for preventing or reducing the likelihood, prevention or reduction of infection by coronavirus (CoV) and/or respiratory syncytial virus (RSV) in an individual. The likelihood or severity of a symptom associated with a CoV and/or RSV infection in an individual, reducing the severity and/or duration of a CoV and/or RSV infection in an individual, or treating the A CoV and/or RSV infection, preventing or reducing viral shedding in an individual infected with a CoV and/or RSV infection, or reducing transmission of a CoV and/or RSV in a population, including The subject is administered an effective amount of a macromolecule. The present invention also relates to a device for delivering a composition comprising a macromolecule.

Background of the Invention

Viral respiratory tract infections (VRTIs) are one of the most common infections worldwide and a major public health problem. Respiratory viruses cause infections in all age groups and are a major contributor to morbidity and mortality. Severity of illness can range from mild, common cold-like illness to severe, life-threatening respiratory infections. The burden of VRTIs is generally more pronounced in individuals with chronic comorbidities or clinical risk factors.

In the past, a substantial proportion of respiratory diseases could not be attributed to a specific pathogen. The advent of molecular testing and genotyping technologies has led to a dramatic increase in the identification of several newly discovered non-influenza respiratory viruses associated with disease.

Such potential pathogens include coronaviruses, adenoviruses, rhinovirus species, human respiratory syncytial virus, and human Polkavirus. Coronaviruses (CoVs) are ubiquitous throughout the world and are associated with relatively mild respiratory illnesses (eg, the common cold) until severe acute respiratory syndrome (SARS).

Coronaviruses are enveloped viruses with a positive, single-stranded RNA genome. CoV infection poses a serious threat to both humans and animals; it can cause endemic animal infections and, in humans, SARS by SARS-CoV, Middle-East Respiratory Syndrome by MERS-CoV East respiratory syndrome, MERS), and the outbreak of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. COVID-19 is a disease caused by the newly discovered SARS-CoV-2. Some people with SARS-CoV-2 infection are asymptomatic, while in others the infection causes mild to moderate COVID-19 disease and COVID-19 pneumonia, leading to the need for intensive care support in some patients, And in some cases it can lead to death, especially in the elderly. Symptoms such as fever, cough, and loss of taste, and signs such as oxygen saturation or lung auscultation are the first and most readily available diagnostic information.

In humans, CoVs typically cause acute respiratory infections. Symptoms and severity can range from mild upper respiratory tract infections (eg, the common cold) to more severe acute respiratory distress syndrome (ARDS), pneumonia, to single and multiple organ failure. Part of the pathogenicity of human CoVs is due to the long incubation period and the fact that infected and infected persons do not show or often show mild symptoms, which means that many people do not realize they are infected and go about their daily routines activities, thereby spreading infection.

CoV is usually transmitted via airborne droplets to the nasal mucosa, where the virus then invades the respiratory tract. Contaminated droplets on hands may also spread to the oral and/or nasal mucosa. Currently, hygiene practices are recommended to prevent transmission, and symptom management to treat the disease. Mild symptoms such as the common cold are usually treated with nonsteroidal anti-inflammatory drugs. Since the filing of the provisional application filing this application, the vaccine has been marketed and distribution has begun. Although a number of potential drugs have been proposed based on previous SARS-CoV studies, and some initial clinical testing has been conducted, no drug has yet been shown to be highly effective in the treatment of SARS-CoV-2 infection. The SARS-CoV-2 spike S protein binds to this ACE2 receptor for viral entry, and PIKfyve, TPC2, and cathepsin L are also believed to be critical for viral entry. In a recent study at UCSD, 332 high-confidence SARS-CoV-2-human protein-protein interactions and 66 druggable human proteins or host factors were identified among 69 existing FDA-approved drugs , clinical trial drugs and/or preclinical compounds targeted. In addition, there are various drugs under development and testing, or are being tested against SARS-CoV-2, such as neutralizing antibodies against GM-CSF, IL-6R, CCR5, the S protein of MERS, and drugs including the following: Remdesivir, ribavirin, tilorone, favipiravir, Kaletra (lopinavir/ritonavir) ), Prezcobix (darunavir/cobicistat), nelfinavir, mycophenolic acid, Galidesvir, Antingen Actemra, OYA1, BPI-002, Ifenprodil, APN01, EIDD-2801, baricitinib, camostat mesylate, lycorine ), Brilacidin, BX-25, and interferon, more specifically IFNβ. Several antiviral compounds have been used to treat COVID-19 and may reduce disease duration and infection index; however, these drugs are not administered due to poor efficacy (Solidarity Trial, WHO), cost and side effects widely used or approved by institutions.

Respiratory syncytial virus (RSV) is a respiratory virus that is a member of the Pneumoviridae family and infects most humans after the age of 2 years. Symptoms are mild in healthy adults, but in some individuals symptoms can be severe (especially in infants and the elderly) and lead to hospitalization. RSV is the cause of acute respiratory infections in more than 60% of children worldwide. The virus also makes individuals susceptible to secondary bacterial infections, such as pneumonia or otitis media. In the United States, an estimated 11,000 to 17,000 adults die each year from RSV infection, and the number of hospitalized patients is approximately 10 times that number each year. RSV infection in adults is usually not a primary infection and is predominantly mild to moderate in severity unless the patient has an underlying risk factor such as being immunocompromised, suffering from an underlying chronic pulmonary or circulatory disease sick, living in a long-term care facility, or frail. Due to RSV infection in solid organs and in bone marrow transplant recipients, the mortality rate from RSV is as high as 30% to 100%, especially when infection occurs within a few days of transplant surgery. Those who are immunosuppressed or immunocompromised are at increased risk of severe RSV infection. RSV is the third leading cause of human influenza disease in the elderly. However, it is the second leading cause of hospitalization.

After years of research, current treatments to lower the virus are limited to treating symptoms, and an effective vaccine has yet to be developed. One of the challenges is that many candidate cellular receptors for RSV entry have been described, including annexin II, CX3 chemokine receptor 1, epidermal growth factor receptor (EGF), calcium-dependent lectins , Duo-like receptor 4, intercellular adhesion molecule 1 (intercellular adhesion molecule 1, ICAM-1), and nucleolin. Some receptors such as EGF are claimed to be used only by certain strains of RSV. Furthermore, RSV is a rapidly evolving virus that makes vaccine development difficult, particularly because RSV evades or suppresses B cell memory in humans.

Antiviral dendrimers with activity against HIV, HPV and HSV have been developed in selected animal models, see eg WO02/079299 and WO2007/045009. However, antiviral agents are generally selective for their antiviral effects, primarily because of receptor specificity and mode of action. There are currently no approved broad-spectrum antiviral agents for a broad class of viral agents, such as enveloped RNA viruses or negative stranded RNA viruses. Even within the same family, such as the Herpesviridae, agents that are effective against one virus are often not necessarily effective against other viruses, eg, treatments against chickenpox, EBV or HSV are not mutually effective.

Therefore, there is a need for agents capable of preventing or reducing the transmission of VRTIs, particularly CoVs and/or RSVs. There is also a need to reduce the severity and duration of disease with VRTIs, particularly COVs and /RSVs.

Summary of Invention

The inventors have found that the dendrimer SPL7013 has activity against CoVs and RSVs in vitro. Therefore, SPL7013 and structurally related compounds will be used to reduce the transmission of CoVs and/or RSVs, and to prevent or reduce the incidence, severity and duration of related conditions. In one aspect, the present invention provides a method of preventing or reducing the likelihood of coronavirus (CoV) and/or respiratory syncytial virus (RSV) infection in an individual, comprising: administering to the individual an effective amount of A macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation A dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of preventing or reducing the likelihood or severity of a symptom associated with a coronavirus (CoV) and/or respiratory syncytial virus (RSV) infection in an individual, comprising: Administer an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof to the individual, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the The macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of preventing or reducing the likelihood of a coronavirus (CoV) infection in an individual, comprising: administering to the individual an effective amount of a macromolecule or a pharmaceutically acceptable amount thereof The accepted salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having a or A plurality of sulfonic acid- or sulfonate-containing moieties are attached to one or more surface groups of the dendrimer.

In one aspect, the invention provides a method of preventing or reducing the likelihood or severity of a symptom associated with a coronavirus (CoV) infection in an individual, comprising: administering to the individual an effective amount of A macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation A dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of reducing the severity and/or duration of a coronavirus (CoV) infection in an individual, comprising: administering to the individual an effective amount of a macromolecule or a A pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer, It has one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of treating a coronavirus (CoV) infection in an individual, comprising: administering to the individual an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, Or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid-containing An acid or sulfonate-containing moiety is attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of preventing or reducing viral shedding in an individual infected with a coronavirus (CoV), comprising: administering to the individual an effective amount of a macromolecule or a pharmaceutically acceptable amount thereof acceptable salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having a Attached to one or more surface groups of the dendrimer are sulfonic acid- or sulfonate-containing moieties.

In one aspect, the present invention provides a method of reducing the spread of a coronavirus (CoV) in a population comprising: administering to a portion of the respiratory tract of the population an effective amount of a macromolecule or a pharmaceutically acceptable molecule thereof The accepted salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 1 to 8 generation dendrimer having an or A plurality of sulfonic acid- or sulfonate-containing moieties are attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of preventing or reducing the likelihood of respiratory syncytial virus (RSV) infection in an individual, comprising: administering an effective amount of a macromolecule or a macromolecule thereof to the respiratory tract of the individual A pharmaceutically acceptable salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer, which One or more surface groups having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer.

In one aspect, the invention provides a method of preventing or reducing the likelihood or severity of a symptom associated with a respiratory syncytial virus (RSV) infection in an individual, comprising: administering to the airway of the individual a An effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to Generation 5 dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of reducing the severity and/or duration of a respiratory syncytial virus (RSV) infection in an individual, comprising: administering to the respiratory tract of the individual an effective amount of a large molecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer A polymer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of treating a respiratory syncytial virus (RSV) infection in an individual comprising: administering to the respiratory tract of the individual an effective amount of a macromolecule or a pharmaceutically acceptable amount thereof salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more A sulfonic acid- or sulfonate-containing moiety is attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a method of preventing or reducing viral shedding in an individual infected with a respiratory syncytial virus (RSV), comprising: administering an effective amount of a macromolecule or a macromolecule to the respiratory tract of the individual A pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer, It has one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the invention provides a method of reducing transmission of a respiratory syncytial virus (RSV) in a population comprising: administering an effective amount of a macromolecule or a pharmaceutically acceptable amount thereof to the respiratory tract of a portion of the population acceptable salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a dendrimer of generation 1 to 8 having a Attached to one or more surface groups of the dendrimer are sulfonic acid- or sulfonate-containing moieties.

In some embodiments, the CoV is selected from an alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. In some embodiments, the CoV is a betacoronavirus.

In some embodiments, the CoV is a subtype of SARS-CoV-2 or a variant thereof. In some embodiments, the CoV is SARS-CoV-2.

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.

其中至少50%的R係

，且其中該醫藥上可接受之鹽係一鈉鹽。 In some embodiments, the dendrimer is

At least 50% of which are R series

, and wherein the pharmaceutically acceptable salt is a sodium salt.

In one aspect, the present invention provides a composition for: preventing or reducing the likelihood of a coronavirus (CoV) infection in a subject, or treating a coronavirus (CoV) infection in a subject; reducing the severity and/or duration of CoV infection in an individual; preventing or reducing viral shedding in an individual infected with a COV; or reducing transmission of a CoV in a population, wherein the composition comprises: an effective Amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 A generated dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer.

In one aspect, the present invention provides a device for delivering a nasal, oral or pulmonary composition comprising a macromolecule or a pharmaceutically acceptable salt thereof, or a A composition of a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached. One or more surface groups attached to the dendrimer.

In one aspect, the present invention provides a composition comprising: an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutical The composition of the above acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer and the card One or more surface groups of Carbopol 974 or Carbopol 971, wherein the composition comprises a ratio of Carbopol 974 or Carbopol 971 to the macromolecule of about 1:20 to about 1:10 w/w ratio.

In one aspect, the present invention provides a composition comprising: an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutical The composition of the above acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer and the card The one or more surface groups of Pop 974, wherein the composition comprises about 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 0.05% w/w Carbopol 974.

In one aspect, the present invention provides a composition comprising: an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutical The composition of the above acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer and the card The one or more surface groups of Pop 971, wherein the composition comprises about 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% w/w Carbopol 971.

In one aspect, the present invention provides a nasal moisture barrier dressing comprising a macromolecule or a pharmaceutically acceptable salt thereof, or a macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable salt thereof The composition of the received carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer surface groups.

Unless specifically stated otherwise, any implementation aspect herein should be deemed to apply mutatis mutandis to any other implementation aspect. For example, as those skilled in the present invention will appreciate, the examples of macromolecules outlined above for the methods of the present invention are equally applicable to the compositions of the present invention.

The scope of the present invention is not limited by the specific embodiments described herein, which are provided for illustrative purposes only. As described herein, functionally equivalent products, compositions and methods are clearly within the scope of the present invention.

In this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of composition of matter shall be deemed to encompass one and more (ie, one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter.

In the following the invention will be described by means of the following non-limiting examples with reference to the figures.

Description of the invention definition

The articles "a (a)" and "an (an)" as used herein refer to one or more (ie, at least one) grammatical objects of the article. For example, "an element" refers to one element or more than one element.

In the scope of this specification and subsequent claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply that The recited integers, or steps, or groups of integers or steps are included but not excluded from any other integers, or steps, or groups of integers or steps.

As used herein, the term "about" means relative to a reference quantity, level, value, dimension, size or amount that varies by as much as 30%, 25%, 20%, 15%, 10%, 5% or 1% of a quantity, level, value, dimension, size or quantity.

As used herein, the term "individual" refers to any individual susceptible to infection by a CoV virus and/or an RSV virus. In a specific embodiment, the system is a human, including fetuses, infants, children, young adults, and adults. In some embodiments, the system is a human adult. In one embodiment, the system is an animal. In one embodiment, the child is one or more of the following: less than 16 years old, less than 14 years old, less than 12 years old, less than 10 years old, less than 5 years old, less than 3 years old, less than 2 years old age, age less than 1 year, age less than 6 months, age less than 3 months, and age less than 1 month. In one embodiment, the child is 12 years old or older. In one embodiment, the infant is a premature infant. In one embodiment, the adult is an elderly person. In one embodiment, the adult is one or more of the following: age greater than 60, age greater than 65, age greater than 70, age greater than 75, age greater than 80, age greater than 85, age greater than 90 years old. In some implementations, the system is a human. In some embodiments, the system is immunocompromised. In some aspects, the individual has recently undergone surgery. In some embodiments, the system is 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 1.5 weeks, or 2 weeks, or 3 weeks after surgery . In some embodiments, the system may be a transplant recipient. In some embodiments, the system will either be 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, hydropleura, pertussis, COVID-19, asbestosis, bronchiectasis, pneumothorax, silicosis, and tuberculosis.

As used herein, the term "prevention" or "prophylaxis" refers to reducing the likelihood of developing or developing an infection or a symptom thereof. Prevention need not be complete, and does not imply that a subject will not eventually develop or develop the infection or a symptom thereof.

As used herein, the term "treating" or "treatment" refers to achieving, at least in part, a desired therapeutic result. In one embodiment, treating comprises preventing or delaying the onset of one or more symptoms of a CoV and/or RSV infection. In one embodiment, treating comprises preventing or reducing the development of one or more symptoms of a CoV and/or RSV infection.

As used herein, the phrase "reducing the severity of an infection" or similar phrases includes reducing in an individual one or more of: the titer of a virus, the duration of infection by the virus, in an individual a The severity or duration of one or more symptoms of a viral infection. In one embodiment, the viral infection is a CoV and/or an RSV viral infection.

As used herein, the term "duration of a CoV and/or RSV infection" refers to the time during which an individual has a CoV and/or RSV infection or a symptom caused by a CoV and/or RSV infection.

As used herein, the term "macromolecule and pharmaceutically acceptable salts thereof" is used interchangeably with "macromolecule" as the context requires.

As used herein, "SPL7013" refers to astronomer sodium (INN, USAN), CAS No. 676271-69-5. SPL7013 is also known as 2,6-bis-{(1-naphthyl-3,6-disulfonic acid)-oxyacetamido}-2,6-bis-2,6-bis-2,6- Bis-(2,6-Diamino-hexamido)-2,6-diamino-hexanoic acid (diphenylmethyl)-amide polysodium salt (2,6-Bis-{(1 -napthalenyl-3,6-disulfonic acid)-oxyacetamido}-2,6-bis-2,6-bis-2,6-bis-(2,6-diamino-hexanoylamino)-2,6-diamino-hexanoic acid (diphenylmethyl)-amide, polysodium salt); or 64 sodium N2,N6-bis{N2,N6-bis[N2,N6-bis(N2,N6-bis{N2,N6-bis[(3,6-bissulfonate) Acido-naphthalen-1-yloxy) acetyl]-1-lyamido}-1-lyamido)-1-lyamido]-1-lyamido}-N1-( Diphenylmethyl)-l-lysine acid amide (Tetrahexacontasodium N2,N6-bis{N2,N6-bis[N2,N6-bis(N2,N6-bis{N2,N6-bis[(3,6 -disulfonatonaphthalen-1-yloxy)acetyl]-l-lysyl}-l-lysyl)-l-lysyl]-l-lysyl}-N1-(diphenylmethyl)-l-lysinamide).

As used herein, "astodrimer" refers to CAS number 1379746-42-5. Also known as 2,6-bis-{(1-naphthyl-3,6-disulfonic acid)-oxyacetamido}-2,6-bis-2,6-bis-2,6-bis -(2,6-Diamino-hexamido)-2,6-diamino-hexanoic acid (diphenylmethyl)-amide; or N2,N6-bis{N2,N6-bis[ N2,N6-bis(N2,N6-bis{N2,N6-bis[(3,6-disulfonaphthalen-1-yloxy)ethanoyl]-1-lyamido}-1- lysinyl)-1-lysinyl]-1-lysinyl}-N1-(diphenylmethyl)-1-lysinamide. Macromolecules and their pharmaceutically acceptable salts

The present disclosure relates to the use of macromolecules and/or pharmaceutically acceptable salts thereof. Given that the macromolecule may contain multiple sulfonate groups, the pharmaceutically acceptable salt may contain multiple cations.

The pharmaceutically acceptable salt can be of any suitable species. Examples of suitable salts include, but are not limited to, metal salts (eg, aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc), organic salts (eg, organic amines such as N,NI-di Benzylethylenediamine, chloroprocaine, diethanolamine, ethylenediamine, dicyclohexylamine, cyclohexylamine, meglumine, (N-methylglucamine) and procaine), quaternary amines (eg, choline), peronium salts, and phosphonium salts. In particular embodiments, the salt is selected from sodium and potassium, especially sodium. In one embodiment, the salt is a monosodium salt (eg, it may be a polysodium salt).

Those skilled in the art will appreciate that many organic compounds can form complexes in solvents, in which they react or are precipitated or crystallized therefrom. These complexes are referred to as "solvates". For example, a complex with water is called a "hydrate". Solvates such as hydrates exist when the compound contains a solvent. It will be appreciated that the macromolecules and salts thereof of the present invention may exist in the form of solvates. Solvates of suitable macromolecules are those in which the relevant solvent is pharmaceutically acceptable.

Macromolecules used in the present invention comprise 3 to 5 generation dendrimers having one or more surface groups attached to the dendrimer with one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer group. The dendrimers useful in the present invention can be any suitable 3 to 5 generation dendrimers capable of presenting one or more sulfonic acid- or sulfonate-containing moieties on their surface. In some embodiments, the dendrimer is selected from the group consisting of polylysine, polyglutamic acid, polyaspartic acid, polyamidoamine (PAMAM), polyamides having 3 to 5 generations. (ether hydroxylamine), polyether, polyester, or poly(propyleneimide) (PPI) dendrimer. In some embodiments, the dendrimer has 2 to 6 generations. In some embodiments, the dendrimer has 3 to 4 generations. In some embodiments, the dendrimer has 4 generations. In some embodiments, the dendrimer is an amino acid dendrimer selected from the group consisting of polylysine, polyglutamic acid, and polyaspartic acid.

The macromolecule also includes one or more surface functional groups with one or more sulfonic acid- or sulfonate-containing moieties attached to the outermost generation of the dendrimer. For example, when the dendrimer is a polylysine, polyamidoamine, poly(etherhydroxylamine), or poly(acrylimide) dendrimer, the surface functional groups are amines groups, and when the dendrimer is a polyglutamic acid or polyaspartic acid dendrimer, the surface functional groups are carboxylic acids.

Dendrimers are branched polymeric macromolecules composed of a plurality of branched monomers radiating from a central core. The number of branch points increases as it moves from the dendrimer core to its surface and is defined by successive layers or "generations" of monomers (or building blocks). Each generation of building blocks is numbered to indicate the distance from the core. For example, generation 1 (G1) is the layer attached to the building block of the core, generation 2 (G2) is the layer attached to the building block of generation 1, and generation 3 (G3) is the layer attached to the building block of the core. Layers of the building blocks of Generation 2, etc.

The outermost generations of building blocks provide the surface of the dendrimer and present functional groups to which the at least one sulfonic acid- or sulfonate-containing moiety is covalently bound. The sulfonic acid- or sulfonate-containing group can be bound directly to the surface functional group, or can be attached to the surface functional group through a linker.

Dendrimers contemplated herein can be prepared by methods known in the art. For example, they may be in a convergent manner (in which, effectively, the branches are preformed and then attached to the core) or in a divergent manner (in which the layers or generations are constructed continuously outwards from the core). Those skilled in the art will understand both of these methods.

For example, in the case of lysine dendrimers, a divergent synthesis might involve the reaction of the amine groups of a layer of lysine residues with the carboxyl groups of amine protected lysine acid , using amidation chemistry to "build" the dendrimer and form the next generation of building blocks. The protecting groups can then be removed, revealing the amine groups of a new generation of lysine building blocks.

The dendrimers may contain any suitable core. As used herein, "core" refers to the portion on which the generations of monomers or building blocks are built (either by a divergent method or a convergent method), and can be any one having at least one reactive or The portion of a functional site from which monomers or building blocks are continuously generated (or a pre-formed "branch" is attached to the site).

The core may be formed from a core precursor having reactive groups suitable for reaction with building blocks, eg the core may be formed from a core having 1, 2, 3 or 4 reactive groups formed from the core precursors. Some exemplary suitable cores contemplated herein include those formed from core precursors having 1, 2, 3, or 4 reactive groups independently selected from amines group, carboxyl, thiol, alkyl, alkynyl, nitrile, halogen, azide, hydroxylamine, carbonyl, maleimide, acrylate or hydroxyl group in which a layer of units or monomers is constructed or Generations can be attached to these reactive groups.

In some embodiments, the core is covalently attached to two building blocks via an amide bond, each amide bond being at a nitrogen atom present in the core and present in a building block formed between the carbon atoms of an acyl group. Thus, the core can be formed, for example, from a core precursor containing two amine groups.

A core part can be the same as or different from a building block.

Exemplary cores include polyaminohydrocarbons, disulfide-containing polyamines, poly(glycidyl ether), aminoethanol, ammonia, arylmethyl halides, piperazine, aminoethylpiperazine , poly(ethyleneimine), alkylene/arylenedithiol, 4,4-dithiobutyric acid, mercaptoalkylamine, thioether alkylamine, isocyanurate, heterocyclic compounds, Macrocycles, Polyglycidyl Methacrylate, Phosphine, Porphine, Ethylene Oxide, Ethylene Sulfide, Butylene Oxide, Aziridine, Azetidine, Polyazido Functional Groups, Silicones Alkane, oxazoline, carbamate or caprolactone.

。 Some non-limiting examples of core moieties contemplated herein include ammonia and diamine C2 _{- Ci2} alkanes such as ethylenediamine, 1,4-diaminobutane, and 1,6-diamine base hexane. However, it will be understood that the core is not necessarily a linear moiety having a single reactive group at each end. The present invention also contemplates non-linear, cyclic or branched core moieties. For example, arylmethylamines such as benzhydrylamine (BHA) are suitable cores. In some embodiments, the core system either comprises a monobenzylamine (BHA) group:

.

並且，在該等樹枝狀聚合物中，係透過兩個氮原子共價地附接至建構單元。一BHALys核心可以係例如從一核心前驅物所形成：

具有兩個反應性胺基氮原子。 In some preferred embodiments, the core is a benzyhydrylamine-lysine (BHALys). A BHALys core has the following structure:

And, in these dendrimers, it is covalently attached to the building block through two nitrogen atoms. A BHALys core can be formed, for example, from a core precursor:

Has two reactive amine nitrogen atoms.

In some preferred embodiments, the core is a BHALys core comprising an L-lysine residue.

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

； 醯胺基胺建構單元：

； 醚羥基胺建構單元：

； 丙烯亞胺建構單元：

； 麩胺酸建構單元：

； 天冬胺酸建構單元：

； 聚酯建構單元：

；以及 聚醚建構單元：

。 The dendrimers also contain one or more building blocks. In some embodiments, the building blocks of the dendrimer are selected from: lysine building blocks:

; amidoamine building block:

; ether hydroxylamine building block:

; Propylene imine building block:

; Glutamic acid building block:

; Aspartic acid building block:

; Polyester building blocks:

; and polyether building blocks:

.

。 In some preferred embodiments, the building block is a lysine residue, such as:

.

。 In some preferred embodiments, the building block is an L-lysine residue, such as:

.

其中K係不存在或者係選自於-C _1-6伸烷基-、-C _1-6伸烷基NHC(O)-、-C _1-6伸烷基C(O)-、-C _1-3伸烷基-O-C _1-3伸烷基-、-C _1-3伸烷基-O-C _1-3伸烷基NHC(O)-、及-C _1-3伸烷基-O-C _1-3伸烷基C(O)-；J係選自於CH或N；L以及M係獨立地不存在或者係選自於-C _1-6伸烷基-或者-C _1-3伸烷基OC _1-3伸烷基；前提係當 L及/或M不存在時，J係CH；**表明在該離胺酸或離胺酸類似物與該樹枝狀聚合物之核心或建構單元之先前世代之間的鍵結；以及***表明在該離胺酸或離胺酸類似物與離胺酸或離胺酸類似物之後續世代之間的鍵結，或者形成該樹枝狀聚合物的表面胺基基團。 In some embodiments, the building block or the building blocks of the dendrimer are lysine or a lysine analog, wherein the lysine analog is selected from a compound having the formula:

Wherein K does not exist or is selected from -C _1-6 alkylene-, -C _1-6 alkylene NHC(O)-, -C _1-6 alkylene C(O)-, -C _1-3 alkylene-OC _1-3 alkylene-, -C _1-3 alkylene-OC _1-3 alkylene NHC(O)-, and -C _1-3 alkylene-OC _{1 -3} alkylene C(O)-; J is selected from CH or N; L and M are independently absent or selected from -C _1-6 alkylene- or -C _1-3 alkylene group OC _1-3 alkylene; provided that when L and/or M are absent, J is CH; ** indicates the core or building block of the lysine or lysine analog and the dendrimer and *** indicates the bonding between the lysine or lysine analog and the subsequent generation of lysine or lysine analog, or the formation of the dendrimer surface amine groups.

； 具有以下結構的類似物2，其中a係一整數1或2；且b及c係獨立地為整數1、2、3或4：

； 具有以下結構的類似物3，其中a係一整數0、1或2；且b及c係獨立地為整數2、3、4、5或6：

；以及 具有以下結構的類似物4，其中a係一整數0、1、2、3、4或5；且b及c係獨立地為整數1、2、3、4或5：

； 其中每個#代表該羧基基團的羰基殘基，其與該核心的一氮原子或建構單元之一先前世代的一氮原子形成一醯胺鍵結；且其中該等建構單元的任何伸甲基基團可被一伸甲氧基(CH ₂-O)或伸乙氧基(CH ₂-CH ₂-O)基團替代，前提係此不會導致在該建構單元內形成一碳酸酯(-OC(O)-O-)或胺甲酸酯(-OC(O)-N-)部分。 Exemplary lysine analog building blocks include the following: Glyamido-lysine 1 having the following structure:

; Analog 2 having the following structure, wherein a is an integer 1 or 2; and b and c are independently integers 1, 2, 3, or 4:

; Analog 3 having the following structure, wherein a is an integer 0, 1 or 2; and b and c are independently an integer 2, 3, 4, 5 or 6:

and analog 4 having the following structure, wherein a is an integer 0, 1, 2, 3, 4, or 5; and b and c are independently an integer 1, 2, 3, 4, or 5:

; wherein each # represents the carbonyl residue of the carboxyl group, which forms an amide bond with a nitrogen atom of the core or a nitrogen atom of a previous generation of one of the building blocks; and wherein any extension of these building blocks The methyl group may be replaced by a methoxy (CH ₂ -O) or ethoxy (CH ₂ -CH ₂ -O) group, provided this does not result in the formation of a monocarbonate ( -OC(O)-O-) or urethane (-OC(O)-N-) moiety.

； 具有以下結構的類似物6，其中a係0至2的一整數；b及c係相同或不同，且係2至6的整數；A ₁及A ₂係相同或不同，且係選自於NH ₂、CO ₂H、OH、SH、X、烯丙基-X、環氧化物、氮丙啶、N ₃或炔烴，其中X係F、Cl、Br或I，

；以及 具有以下結構的類似物7，其中a係0至5的一整數；b及c係相同或不同，且係1至5的整數；A ₁及A ₂係相同或不同，且係選自於NH ₂、CO ₂H、OH、SH、X、烯丙基-X、環氧化物、氮丙啶、N ₃或炔烴，其中X係F、Cl、Br或I，

； 其中每個#代表該羧基基團的羰基殘基，其與該核心的一氮原子或建構單元之一先前世代的一氮原子形成一醯胺鍵結；且其中該等建構單元的任何伸甲基基團可被一伸甲氧基(CH ₂-O)或伸乙氧基(CH ₂-CH ₂-O)基團替代，前提係此不會導致在該建構單元內形成一碳酸酯(-OC(O)-O-)或胺甲酸酯(-OC(O)-N-)部分。 Other suitable building blocks/building block precursors include: Analog 5 having the following structure, wherein a is an integer from 0 to 2; b and c are the same or different and are integers from 1 to 4; A ₁ and A ₂ are the same or different, and are selected from _NH2 , _CO2H , OH, SH, X, allyl-X, epoxide, aziridine, N3 or alkyne, wherein X is F, _Cl , Br or I,

; Analog 6 having the following structure, wherein a is an integer from 0 to 2; b and c are the same or different, and are integers from 2 to 6; A ₁ and A ₂ 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,

and analog 7 having the following structure, wherein a is an integer from 0 to 5; b and c are the same or different, and are integers from 1 to 5; A ₁ and A ₂ are the same or different, and are selected from in _NH2 , _CO2H , OH, SH, X, allyl-X, epoxide, aziridine, N3 or alkyne, wherein X is F, _Cl , Br or I,

; wherein each # represents the carbonyl residue of the carboxyl group, which forms an amide bond with a nitrogen atom of the core or a nitrogen atom of a previous generation of one of the building blocks; and wherein any extension of these building blocks The methyl group may be replaced by a methoxy (CH ₂ -O) or ethoxy (CH ₂ -CH ₂ -O) group, provided this does not result in the formation of a monocarbonate ( -OC(O)-O-) or urethane (-OC(O)-N-) moiety.

；

；或者

； 其中

。 In some embodiments, the macromolecule is a polylysine dendrimer having lysine building blocks, especially a polylysine dendrimer having a diphenylmethylamine group, such as A dendrimer shown below:

;

;or

; in

.

In some aspects, the dendrimer contains 3 to 5 generations of building blocks, for example, in some embodiments, it contains a core and 3 to 5 generations of building blocks. In some embodiments, the dendrimer comprises a BHALys core and 3 to 5 generations of lysine building blocks. In some aspects, the dendrimer provides 16, 32, or 64 nitrogen atoms on the surface layer of the building block for attachment to the sulfonic acid- or sulfonate-containing moiety (directly or via a linker) son). In some embodiments, the dendrimer provides 32 nitrogen atoms on the surface layer of the building block for attachment to the sulfonic acid- or sulfonate-containing moiety (either directly or via a linker).

The sulfonic acid- or sulfonate-containing moiety is a moiety capable of presenting the sulfonic acid or sulfonate group on the surface of the dendrimer. In some embodiments, the sulfonic acid- or sulfonate-containing moiety has a sulfonic acid or sulfonate group. In other embodiments, the sulfonic acid- or sulfonate-containing moiety has more than one sulfonic acid or sulfonate group, such as 2 or 3 sulfonic acid or sulfonate groups, especially 2 sulfonate groups acid or sulfonate group. In some embodiments, the sulfonic acid- or sulfonate-containing moiety comprises an aryl group, such as a phenyl group or a naphthyl group, especially a naphthyl group. In some embodiments, the sulfonic acid- or sulfonate-containing moiety comprises a naphthyl group (also referred to as a mononaphthyldisulfonate moiety) substituted with two sulfonic acid or sulfonate moieties, eg A 3,6-disulfonate naphthyl moiety. In some embodiments, a 3,6-disulfonaphthyl moiety attached to the dendrimer through the 1-position of the naphthalene is used.

When the sulfonate-containing moiety is present, the moiety may be present in ionic form ( ^-SO3- ) or as a _monosalt , eg, the sodium salt (-SO3Na).

、

、

、

以及

， 其中n係0或1至20的一整數，m係1或2的一整數，且p係1至3的一整數。在一些實施態樣中，p＝2。 Examples of suitable sulfonic acid- or sulfonate-containing moieties include, but are not limited to, the following: -NH-( _CH2 ) _nSO3- ^, _- ( _{CH2 ) nSO3-} ^,

,

,

,

as well as

, where n is 0 or an integer from 1 to 20, m is an integer from 1 or 2, and p is an integer from 1 to 3. In some embodiments, p=2.

、

、

、

，以及

，尤其係

。 In some embodiments, the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of:

,

,

,

,as well as

, especially the

.

In some embodiments, more than one sulfonic acid- or sulfonate-containing moiety is present on the surface of the dendrimer. In some embodiments, at least 5, at least 15, or at least 30 sulfonic acid- or sulfonate-containing moieties are present on the surface of the dendrimer. In some embodiments, 32 sulfonic acid- or sulfonate-containing moieties are present on the surface of the dendrimer.

In some embodiments, the sulfonic acid- or sulfonate-containing moiety is directly bound to the surface amine groups of the dendrimer. In other embodiments, the sulfonic acid- or sulfonate-containing moiety is attached to the surface amine groups of the dendrimer through a linker group.

Suitable linker groups include straight-chain and branched alkylene or alkenylene groups in which one or more non-adjacent carbon atoms are optionally replaced with an oxygen or sulfur atom to provide an ether, thioether, polyether or polysulfide; or a group -X ₁ -(CH ₂ ) _q -X ₂ or -X ₁ -(CR ₁ R ₂ ) _q -X ₂ -, wherein X ₁ and X ₂ are independently selected From -NH-, -C(O)-, -O-, -S- and -C(S), R ₁ and R ₂ are independently selected from hydrogen or -C _1-6 alkyl, and q is an integer from 1 to 10, and, wherein the linker contains more than one _CH2 group, optionally one or more non-adjacent ( _CH2 ) groups may be replaced by -O- or -S- to Monoethers, sulfides, polyethers or polysulfides are formed.

In some embodiments, the linker is a group _-X1- ( _CH2 ) _q -C(O) _- , wherein X1 is the atom attached to the sulfonic acid- or sulfonate-containing moiety , and is 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 attached to the surface of the dendrimer amine group.

In some embodiments, the linker is a group -X ₁ -(CR ₁ R ₂ ) _qC (O)-, wherein X ₁ is the moiety attached to the sulfonic acid or sulfonate-containing moiety atom, and is selected from the group consisting of O, NH and S; R ₁ and R ₂ are independently selected from hydrogen or -C _1-6 alkyl, and q is an integer from 1 to 3; and The carbons of the -C(O)- groups are attached to the surface amine groups of the dendrimer.

In some embodiments, the linker is #-O-(CR ₁ R ₂ )-C(O)-*, wherein R ₁ is -C _1-6 alkyl (eg, methyl, ethyl, propyl , butyl, pentyl, or hexyl), R ₂ is hydrogen, and where # represents the attachment to the sulfonic acid-containing moiety, and * represents the surface amine group attached to the dendrimer.

In some embodiments, the linker is #-O-( _CH2 ) _q -C(O)-* wherein q is an integer from 1 to 6, and wherein # represents attachment to the sulfonic acid-containing moiety , and * represents the surface amine groups attached to the dendrimer.

In a particular embodiment, the linker is #-O- _CH2 -C(O)-* where # represents attachment to the sulfonic acid-containing moiety, and * represents attachment to the dendrimer surface amine groups.

； 或者一其醫藥上可接受之鹽。 In some embodiments, the sulfonic acid- or sulfonate-containing moiety is attached to the surface amine group of the dendrimer through a linker group, and the linker-sulfonic acid/sulfonate Part of the department:

; or a pharmaceutically acceptable salt thereof.

，且該連接子係 #-O-(CR ₁R ₂)-C(O)-*， 其中R ₁係-C _1-6烷基(例如甲基、乙基、丙基、丁基、戊基或己基)，R ₂係氫，且其中#代表附接至該含磺酸部分，且*代表附接至該樹枝狀聚合物的表面胺基基團。 In some embodiments, the sulfonic acid- or sulfonate-containing moiety is

, and the linker is #-O-(CR ₁ R ₂ )-C(O)-*, wherein R ₁ is -C _1-6 alkyl (eg methyl, ethyl, propyl, butyl, pentyl group or hexyl), R ₂ is hydrogen, and where # represents attachment to the sulfonic acid-containing moiety, and * represents surface amine groups attached to the dendrimer.

，且該連接子係 #-O-(CH2) _q-C(O)-* 其中q係從1至6的一整數，且其中#代表附接至該含磺酸部分，且*代表附接至該樹枝狀聚合物的表面胺基基團。 In some embodiments, the sulfonic acid- or sulfonate-containing moiety is

, and the linker is #-O-(CH2) _q -C(O)-* where q is an integer from 1 to 6, and where # represents attachment to the sulfonic acid-containing moiety, and * represents attachment to the surface amine groups of the dendrimer.

；

；

； 其中每個R基團係由式IV之基團或者氫所表示：

IV； 前提係至少一個R基團係式IV之基團；或者一其醫藥上可接受之鹽。 Exemplary dendrimers useful in the present invention include those of formulae I, II and III:

;

;

; wherein each R group is represented by a group of formula IV or hydrogen:

IV; provided that at least one R group is a group of formula IV; or a pharmaceutically acceptable salt thereof.

In certain embodiments, more than one R group is a group of formula IV, for example, in some embodiments, at least 10 such R groups are groups of formula IV, and at least 15 such R groups are groups are of formula IV, at least 20 of these R groups are of formula IV, at least 25 of these R groups are of formula IV, or at least 30 of these R groups are of formula Group IV. In some embodiments, all of the R groups are groups of Formula IV.

其中至少25%的R係

，且其中該醫藥上可接受之鹽係一鈉鹽。 In some embodiments, the dendrimer is

At least 25% of which are R series

, and wherein the pharmaceutically acceptable salt is a sodium salt.

其中R係氫或者

，且其中至少25%、至少50%、至少75%，或者至少90%的R係

，且其中該醫藥上可接受之鹽係一鈉鹽。 In some embodiments, the dendrimer is

where R is hydrogen or

, and at least 25%, at least 50%, at least 75%, or at least 90% of which are R-series

, and wherein the pharmaceutically acceptable salt is a sodium salt.

其中R代表式IV之基團：

(IV)； 其中*代表附接點至該樹枝狀聚合物的表面胺基基團，且其中該醫藥上可接受之鹽係鈉。 In some embodiments, the macromolecule is a dendrimer of Formula I:

wherein R represents a group of formula IV:

(IV); wherein * represents the point of attachment to the surface amine group of the dendrimer, and wherein the pharmaceutically acceptable salt is sodium.

其中R係氫或者一基團R’， R’係一經連接之含磺酸或含磺酸鹽部分，其中該含磺酸或含磺酸鹽部分係

，且該連接子係 #-O-(CR ₁R ₂)-C(O)-*, 其中R ₁係-C _1-6烷基(例如甲基、乙基、丙基、丁基、戊基或己基)，R ₂係氫，且其中#代表附接至該含磺酸或含磺酸鹽部分，且*代表附接至該樹枝狀聚合物的表面胺基基團，且其中至少25%、至少50%、至少75%、或至少90%，或所有的R係R’，且其中該醫藥上可接受之鹽係一鈉鹽。 In some embodiments, the dendrimer is

wherein R is hydrogen or a group R', R' is a linked sulfonic acid- or sulfonate-containing moiety, wherein the sulfonic acid- or sulfonate-containing moiety is

, and the linker is #-O-(CR ₁ R ₂ )-C(O)-*, wherein R ₁ is -C _1-6 alkyl (such as methyl, ethyl, propyl, butyl, pentyl or hexyl), R is _hydrogen , and wherein # represents attachment to the sulfonic acid- or sulfonate-containing moiety, and * represents surface amine groups attached to the dendrimer, and wherein at least 25 %, at least 50%, at least 75%, or at least 90%, or all R is R', and wherein the pharmaceutically acceptable salt is a monosodium salt.

其中R係氫或者一基團R’， R’係一經連接之含磺酸或含磺酸鹽部分，其中該含磺酸或含磺酸鹽部分係

，且該連接子係 #-O-(CH ₂) _q-C(O)-* In some embodiments, the dendrimer is

wherein R is hydrogen or a group R', R' is a linked sulfonic acid- or sulfonate-containing moiety, wherein the sulfonic acid- or sulfonate-containing moiety is

, and the linker is #-O-(CH ₂ ) _q -C(O)-*

wherein q is an integer from 1 to 6, and wherein # represents attachment to the sulfonic acid- or sulfonate-containing moiety, and * represents surface amine groups attached to the dendrimer, and wherein at least 25%, at least 50%, at least 75%, or at least 90%, or all R is R', and wherein the pharmaceutically acceptable salt is a monosodium salt.

。 A particular dendrimer of formula I has all R groups such as those of formula IV (SPL7013). SPL7013, also known as astronomer sodium, has the following structure:

.

In some embodiments, the macromolecule is astronomer. In some embodiments, the macromolecule is a pharmaceutically acceptable salt of astronomer. In some embodiments, the pharmaceutically acceptable salt thereof is SPL7013 (astronomer sodium).

A particular dendrimer of formula II has all R groups such as those of formula IV (SPL7320). A particular dendrimer of formula III has all R groups such as those of formula IV (SPL7304).

The synthesis of dendrimers of formula I, II and III is described in WO02/079299.

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

As used herein, the "Coronaviridae" commonly known as "Coronavirus" or " CoV " are enveloped, positive, single-stranded RNA viruses. The coronavirus family has two subfamilies, Letovirinae and Orthocoronavirinae . The phylogenetic history of coronaviruses is outlined in the Coronaviridae Study Group (2020).

In one embodiment, the CoV is selected from the group consisting of Alphacoronavirus ( Alphacoronavirus , alphaCoV), Betacoronavirus ( Betacoronavirus , betaCoV), Gammacoronavirus ( Gammacoronavirus , gammaCoV), and deltacoronavirus Virus genus ( Deltacoronavirus , deltaCoV).

In one embodiment, the alphaCoV is selected from coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), and feline infectious peritonitis virus (feline infectious peritonitis virus, FIPV).

In one embodiment, the betaCoV 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 Related coronavirus-2 (SARS-Cov-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), murine hepatitis virus (MHV), and/or bovine coronavirus (BCoV).

In one embodiment, the CoV line is capable of infecting a human.

In one embodiment, the CoV capable of infecting a human is selected from: SARS-CoV-2, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, SARS-CoV, and MERS-CoV or 1. Subtypes of its variants.

In one embodiment, the CoV has a mortality rate of about 0.001 to about 10% in humans. In one embodiment, the CoV has a mortality rate of about 0.01 to about 9% in humans. In one embodiment, the CoV has a mortality rate of about 0.01 to about 9% in humans. In one embodiment, the CoV has a mortality rate of about 0.01 to about 7% in humans. In one embodiment, the CoV has a mortality rate of about 0.01 to about 6% in humans.

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

In one embodiment, the CoV is SARS-CoV-2 or a subtype or variant thereof. In one embodiment, the SARS-CoV-2 is SARS-CoV-2 subtype L as described in Tang et al., 2020. In one embodiment, the 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 comprises the sequence as described in the NCBI reference sequence: NC_045512.2 or a variant thereof. In one embodiment, SARS-CoV-2 comprises the sequence as described in GenBank: MN908947.3 or a variant thereof. In one embodiment, the SARS-CoV-2 is B.1.1.7 (also known as 20I/501Y.V1 or VOC 202012/01) or a variant thereof. In one embodiment, the SARS-CoV-2 is B.1.351 (also known as 20H/501Y.V2) or a variant thereof. In one embodiment, the SARS-CoV-2 is P1 (also known as 20J/501Y.V3) or a variant thereof. In one embodiment, the SARS-CoV-2 is B.1.526 or a variant thereof. In one embodiment, the SARS-CoV-2 is B.1.427 or a variant thereof. In one embodiment, the 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 variant lines are classified by CDC as variants of interest.

Examples of SARS-CoV-2 variants are described, for example, in Shen et al., 2020 and Tang et al., 2020. Foster et al (2020) have identified 3 variants, A, B and C, based on genome analysis. In some embodiments, the SARS-CoV-2 is SARS-CoV-2 variant A. In some embodiments, the SARS-CoV-2 is SARS-CoV-2 variant B. In some embodiments, the SARS-CoV-2 is SARS-CoV-2 variant C.

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

In different animal species including camels, cattle, cats, and bats, CoV infection can cause respiratory, intestinal, hepatic, and neurological diseases.

CoV 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 airways. Typically, the system is a human individual. In some embodiments, the system is a livestock or livestock. Typically, during infection, CoV can be found in the respiratory tract, eg, the upper nasal passages. In some embodiments, CoV can be found in the lower respiratory tract, eg, bronchi and/or alveoli.

In one embodiment, a CoV infection can cause one or more symptoms, wherein the symptoms are selected from one or more of the following: fever, cough, sore throat, shortness of breath, viral shedding, respiratory insufficiency, runny nose , nasal congestion, malaise, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS). In one embodiment, the 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 100 mmHg < PaO2/FiO2 ≤ 200 mmHg) is PaO2/FiO2 ≤ 100 mmHg).

In one embodiment, a SARS-CoV-2 infection can cause one or more symptoms, wherein the symptoms are selected from one or more of the following: fever, cough, sore throat, shortness of breath, viral shedding, respiratory function Insufficiency, runny nose, nasal congestion, malaise, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS).

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

In one embodiment, the macromolecule reduces the CoV antibody titer of the individual. In one embodiment, the IgA, IgG and/or IgM antibody titers are measured by ELISA and are reduced below detectable levels. In some embodiments, the antibody is directed against protein S or N. In some embodiments, the tested sample is obtained from a buccal swab, nasal swab, blood sample, throat swab, or lung fluid.

In some embodiments, the macromolecule is retained in the lungs and does not penetrate 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 pharmaceutically active agent from the lungs to the blood, either directly into the pulmonary microvessels via absorption or after absorption into the pulmonary microlymphatic vessels.

In one embodiment, the CoV is not SARS-CoV. In one embodiment, the CoV is not alphaCOV. In one embodiment, the CoV is not a canine coronavirus. respiratory syncytial virus

As used herein, " Orthopneumovirus ", commonly known as "respiratory fusion virus" or "RSV", is a negative-directed, single-stranded RNA virus. RSV is a member of the family Pneumoviridae . 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 subtypes can be determined based on the reactivity of the F and G surface proteins to monoclonal antibodies. RSV infection can cause symptoms in primates, humans, rats, mice, cows, guinea pigs, ferrets, and hamsters.

In one embodiment, the RSV is a human RSV (HRSV). In one embodiment, the HRSV is HRSV long.

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

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

In one embodiment, the RSVA is a member of one of the twenty-three genotypes identified in Ramaekers et al 2020. In one embodiment, the RSVA line 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 one embodiment, the RSVB is a member of one of the six RSVB genotypes identified in Ramaekers et al 2020. In one embodiment, the RSVB is selected from genotypes Bl, B2, B3, B4, B5 and B6.

In one embodiment, the RSV infection causes one or more of the following symptoms: nasal congestion or runny nose, decreased appetite, cough, mucus (yellow, green, or gray mucus) when coughing, sneezing, sore throat, mild headache, Fever, wheezing, shortness of breath or difficulty breathing, bluish skin (cyanosis), severe asthma symptoms in individuals with asthma, acute bronchitis, severe bronchitis, airway inflammation, airway obstruction, chronic obstructive pulmonary disease, heart Obstruction, bacteremia, pneumonia, acute otitis media, and recurrent otitis media.

RSV infection can lead to secondary infections such as, for example, bacteremia, pneumonia, acute otitis media, and recurrent otitis media.

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 airways. Typically, the system is a human individual. In some embodiments, the system is a livestock or livestock. In one embodiment, the livestock is a cow. Typically, during infection, RSV can be found in the respiratory tract, eg, the upper nasal passages. In some embodiments, RSV can be found, for example, in the lower airways of the bronchi and/or alveoli.

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

In one embodiment, the macromolecule reduces the RSV antibody titer of the individual. In one embodiment, the IgA, IgG, IgM and/or IgE antibody titers are measured by ELISA and are reduced below detectable levels. In some embodiments, the tested sample is obtained from a buccal swab, nasal swab, blood sample, throat swab, or lung fluid.

In some embodiments, the macromolecule is retained in the lungs and does not penetrate 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 pharmaceutically active agent from the lungs to the blood, either directly into the pulmonary microvessels via absorption or after absorption into the pulmonary microlymphatic vessels.

Treatment of RSV can include one or more of the following: hospitalization, intensive care treatment, ICU admission, intubation, and supplemental oxygen. Method and use

The present invention pertains to methods and uses for preventing or reducing the likelihood of, preventing or reducing, in a subject, one associated with, a CoV and/or an RSV infection in a subject Likelihood of symptoms, reducing the severity and/or duration of a CoV and/or an RSV infection in an individual, treating a CoV and/or an RSV infection in an individual, preventing or reducing an infection with a CoV and/or viral shedding in an RSV-infected individual, or reducing transmission of a CoV and/or an RSV in a population, comprising administering to the individual an effective amount of a macromolecule as described herein.

In one embodiment, the macromolecules as described herein are intended for administration to the respiratory tract. As used herein, the term "airway" refers to the passages formed by the mouth, nose, throat, and lungs through which air passes during breathing. Reference to the respiratory tract includes both the upper and/or lower respiratory tract. In one embodiment, the macromolecules as described herein are intended for administration to the upper respiratory tract. In one embodiment, the macromolecules as described herein are intended for administration to the lower respiratory tract. Those skilled in the art will understand that the upper respiratory tract includes one or more of the following: 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 following: vestibular area, olfactory area, superior turbinate, middle turbinate, inferior turbinate, and nasopharynx. Those skilled in the art will understand that the lower respiratory tract includes one or more of the following: trachea, main bronchi, and lungs. In some embodiments, the macromolecule is delivered nasally. In one embodiment, administering a macromolecule comprises administering to the mucosa of one or more regions of the respiratory tract. In one embodiment, the macromolecule is administered into the nasal cavity. In one embodiment, a macromolecule as described herein is administered to the nasal mucosa. In one embodiment, the macromolecule is administered to one or more of the turbinate, nasopharynx, and/or oropharynx. In one embodiment, the macromolecules as described herein are administered to the oral mucosa. In one embodiment, the macromolecules as described herein are administered to the mucosa of the main bronchus. In one embodiment, the macromolecules as described herein are administered to the mucosa of the lung.

The lungs are known to be a particularly harsh environment for the stability of active agents. Small molecules rapidly pass through the lung epithelium and are cleared into the vasculature. Particle size is important for reaching associated diseased structures in the lung. Another difficulty encountered in delivering large particles to the lungs is that ciliary action in the lungs tends to rapidly remove the agent delivered to the lungs and lead to excretion via feces. Thus, a particular advantage of some embodiments of the present invention is that the dendrimer is not degraded or rapidly excreted by cilia after administration to the pulmonary environment. In some embodiments, the macromolecule resides in the lung for an extended period of time. In some embodiments, the macromolecule remains in the lung for up to one month, one week, or one day.

In some embodiments, the macromolecule is administered topically. In one embodiment, the macromolecule is administered topically to the epidermis or eye. In some embodiments, local administration does not include administration to the respiratory tract.

In some embodiments, the macromolecule is administered transdermally. For example, the macromolecule can be administered percutaneously to one or more of the hand, wrist, forearm, face, and neck.

In some embodiments, the macromolecule is delivered via a parenteral route (eg, 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 by injection. In some embodiments, the macromolecule is delivered intravenously.

In some embodiments, the macromolecules are applied to a surface. In some embodiments, the macromolecules are applied to surfaces including metals, polymers such as paints, plastics and rubbers, fabrics, polymers, wood, ceramics, glass, concrete, skin, human tissue, mucous membranes, and bone . In some embodiments, the macromolecules are applied to personal protective equipment (PPE), including gloves, masks, gowns, and hospital gowns. In some embodiments, the macromolecules are applied to tissues and tissues. In some embodiments, the macromolecules are administered to the surgical/medical field, including patients, workstations, and equipment. This surgical/medical field can be used for human or veterinary use. composition

In some embodiments, a composition comprising the macromolecule and a pharmaceutically acceptable carrier is used. Compositions as described herein are suitable for, eg, nasal, pulmonary, respiratory tract administration, ocular administration, transdermal administration, and/or parenteral administration.

The pharmaceutical composition may also include polymeric excipients/additives or carriers such as polyvinylpyrrolidone, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose , microcrystalline cellulose/carboxymethyl cellulose, Ficolls (a polymeric sugar), hydroxyethylstarch (HES), dextran (e.g. cyclodextrins such as 2-hydroxypropyl- β-cyclodextrin and sulfobutyl ether-β-cyclodextrin), polydextrose, PVP, inulin, polyethylene glycol, and pectin. The pharmaceutical composition may also include amino acid or sugar carriers such as glycine, leucine, alanine, mannitol, and trehalose. Such compositions further include diluents, buffers, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), flavoring agents, taste-masking agents, inorganic salts (eg, sodium chloride), Antimicrobials (eg, xylalidium chloride), sweeteners, antistatic agents, sorbitol esters, lipids (eg, phospholipids, such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids such as cholesterol), and chelating agents such as EDTA, zinc and other such suitable cations. Other pharmaceutical excipients and/or additives suitable for use in the compositions of the present invention are listed in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and "Physician's Desk Reference", 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and "Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H. Kibbe, Pharmaceutical Press, 2000.

The carrier, excipient, or diluent can include any and all one or more of the following: conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, viscosity modifiers, isotonicity agents, and Absorption enhancers or delayers, activity enhancers or delayers, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art and 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, it is contemplated for use in the compositions of the present invention.

In some embodiments, the composition of macromolecules comprises a flow modifier, especially a polyacrylic acid (carbomer), eg, a Carbopol® such as Carbopol® 971P, 974P or 71G Polymer, or Noveon Polycarbophil from Lubrizol, or its equivalent. In some embodiments, the rheology modifier is Carbopol® 974P. These may be homopolymers of acrylic acid, or crosslinked with allyl ether of neotaerythritol, allyl ether of sucrose, or allyl ether of propylene. In one embodiment, it is a carbomer. In one embodiment, it is a carboxy polyalkylene. In one embodiment, it is an acrylic polymer. Those skilled in the art will understand that chains can be of different lengths, different degrees of cross-linking, molecular weights, etc., and can have different grades for specific uses (such as a P system for pharmaceuticals). In some embodiments, the carbopol polymer is a NF (national formulary) version. Those skilled in the art will know when it is appropriate to use pharmaceutical and non-pharmaceutical grades. The rheology modifier may be present in an amount of 1-10% w/w, especially about 2 to 5% w/w, or 0.01 to 0.1% w/w. In some embodiments, the rheology modifier is Carbopol. The Carbopol rheology modifier is present in an amount such as 0.01% to 1% w/w, or about 0.01% to 0.1%, especially 0.05% to 0.1%, especially 0.05% w/w. In some implementations, the Carbopol is Carbopol 974. In some embodiments, Carbopol 974 is formulated at a dosage such as from 0.05% w/w to about 5% w/w, or from about 0.05% w/w to about 3% w/w, or from about 0.05% w/w to Carbopol 974 is present in an amount of 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 implementations, the Carbopol is Carbopol 971. In some embodiments, Carbopol 971 is formulated at a dosage such as from 0.05% w/w to about 1% w/w, or from about 0.05% w/w to about 1.5% w/w, or from about 0.05% w/w to Carbopol 971 is present in an amount of about 1.8%. In some embodiments, the rheology modifier is a cellulose, eg, hydroxypropyl methylcellulose or microcrystalline cellulose/carboxymethyl cellulose. In some embodiments, the rheology modifier is hydroxypropyl methylcellulose. In some embodiments, hydroxypropyl methylcellulose is present in an amount such as 0.01% to 1% w/w, or about 0.05 to 0.5% w/w, especially about 0.1%. In some embodiments, the rheology modifier is microcrystalline cellulose/carboxymethyl cellulose. In some embodiments, the microcrystalline cellulose/carboxymethyl cellulose is in an amount such as 0.5% to 5% w/w, or about 1% to 3% w/w, especially about 2% w/w exist. The rheology modifier contributes to the bioadhesive/mucoadhesive properties of the composition.

The macromolecular composition may also include a chelating agent, such as a polyaminocarboxylic acid. A particularly useful chelating agent is ethylenediamine tetraacetic acid (EDTA) and its salts. Suitable amounts of chelating agents are in the range of 0.001% to 2% w/w, especially 0.005% to 1% w/w. In some embodiments, the chelating agent is present in a low amount, such as 0.001% to 0.1% w/w, especially about 0.005%. Other ingredients that may be included in the gel composition include preservatives such as parabens, eg methyl paraben and propyl paraben, or mixtures thereof, in amounts up to 1% w/w. Suitable amounts of parabens are in the range of 0.01% to 0.5% w/w, especially 0.01% to 0.2% w/w. In some embodiments, methylparaben is present in an amount such as 0.05% to 0.2% w/w, especially about 0.18%. In some embodiments, methylparaben is present in an amount such as 0.14% to 0.23% w/w. In some embodiments, propyl paraben is present in an amount such as 0.01% to 0.05% w/w, especially about 0.02%. In some embodiments, propyl paraben is present in an amount such as 0.015% to 0.0025% w/w. In some embodiments, the xylylene ammonium chloride is present in an amount ranging from 0.01% to 0.1% w/w, especially about 0.05%.

Other ingredients that may be included in the composition include, for example, solvents such as water, pH adjusters such as hydroxide and/or hydrochloric acid, and emollients and humectants such as glycerin and propylene glycol, in amounts up to 5% . In some embodiments, glycetin (glycerol) is present. In some embodiments, glycerol is present in an amount such as 0.1% to 5% w/w, 0.5% to 2% w/w, especially about 1% w/w. In some embodiments, propylene glycol is present. In some embodiments, propylene glycol is present in an amount such as 0.1% to 5% w/w, 0.5% to 2% w/w, especially about 1% w/w.

In one embodiment, the composition creates a moisturizing and protective barrier in the nasal cavity when delivered by a nasal spray device as described herein. In one embodiment, the composition creates a moisturizing and protective barrier on the nasal mucosa when delivered by a nasal spray device as described herein. Respiratory Compositions (Nasal and Oral)

In some embodiments, administering the macromolecule to the respiratory tract can include delivering the macromolecule to the diseased lung via an oral or nasal route; to the upper respiratory tract via the nasal route; or via the nasal route to the nasal cavity and/or nasal mucosa. For example, in some embodiments, the macromolecule can be delivered by inhalation, such as through the mouth and/or nose. In some embodiments, the macromolecule can be delivered by intratracheal instillation or insufflation. Thus, the macromolecule can be delivered to the respiratory tract without the need for a separate targeting agent that targets the pharmaceutically active agent to diseased tissues or cells.

For example, in some embodiments, the pharmaceutical composition can be an aerosol composition, a nebulized composition, a dry powder composition, an aqueous composition, or an insufflation composition. In some embodiments, the pharmaceutical compositions can be included in pressurized metered dose inhalers, dry powder inhalers, nebulizers, nebulizers, and the like. In one embodiment, the composition is suitable for administration in a nasal spray, an oral spray, an inhaler, or a nebulizer. See Zarogoulidis et al (2012) for more discussion.

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 turbinate, nasopharynx, and/or oropharynx.

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. Nasal spray compositions may include purified aqueous solutions of the active agent with a preservative and isotonicity agent. Such compositions can be adjusted to a pH and isotonic state compatible with the nasal mucosa. In some embodiments, the macromolecule is delivered as a powder, a gel, a liquid, an aerosol, or an emulsion. In some embodiments, the pH of the composition is from 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.

In some embodiments, the osmolality of the composition is from about 200 to about 700 Osmol/kg. In some embodiments, the osmolality of the composition is from about 300 to about 600 Osmol/kg. In some embodiments, the osmolality of the composition is from about 300 to about 700 Osmol/kg. In some embodiments, the osmolality of the composition is about 200 to about 400 Osmol/kg, more preferably about 280 Osmol/Kg. Osmolality modifiers include NaCl, lysine, _CaCl2 , sodium citrate, and pH modifiers include _H2SO4 , _NaOH , tromethamine, HCl. In some embodiments, the osmolality of the composition is from about 200 to about 400 mOsmol.

In some embodiments, the composition comprises about 0.14% to about 0.23% methylparaben.

In some embodiments, the composition comprises about 0.015% to about 0.025% propylparaben.

In one embodiment, the nasal spray composition has antiviral activity against CoVs. In one embodiment, the nasal spray composition inactivates more than 90%, or more than 92%, or more than 95%, or more than 99%, or more than 99.9% of the CoVs. In one embodiment, the nasal spray composition inactivates more than 90%, or more than 92%, or more than 95%, or more than 99%, or more than 99.9% of SARS-CoV-2. In one embodiment, the nasal spray composition inactivates more than 90%, or more than 92%, or more than 95%, or more than 99%, or more than 99.9% of the CoVs that cause COVID-19. In one embodiment, the nasal spray composition has antiviral activity against an RSV virus. In one embodiment, the nasal spray composition inactivates more than 90%, or more than 92%, or more than 95%, or more than 99%, or more than 99.9% of RSV. In one embodiment, the inactivated line is exposed to a composition as described herein for at least 1 minute. In one embodiment, the nasal spray composition provides a moisturizing layer to help retain moisture in nasal tissue. Hydration of nasal tissue prevents drying and damage, making it more difficult for viruses to penetrate.

In some embodiments, the macromolecule is formulated for delivery to the lung. Neutral pH and osmolarity are important factors for lower airway delivery to avoid bronchoconstriction in patients with breathing disorders due to poor lung buffering.

In some embodiments, the pharmaceutical composition can 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 5um and greater than 1um.

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

Examples of ingredients suitable for nasal or oral delivery are provided in Table 1 below.

Table 1. Suitable ingredients for nasal delivery. Element IIG for nasal route, %w/w Features Alcohol (ethanol), 200 degrees (proof) 2 cosolvent Anhydrous dextrose 0.5 Osmotic agent Sodium Tricitrate Anhydrous 0.0006 buffer Benzyl alcohol 0.0366 preservative Benzylammonium chloride 0.119 preservative Butyl Hydroxymethoxybenzene 0.0002 Antioxidants Cellulose crystallites 2 Suspending agent, stabilizer Chlorobutanol 0.5 preservative Carboxymethylcellulose Na 0.15 suspending agent Hydroxypropylmethylcellulose (4-local) Disodium etanerate 0.5 Chelating Agents, Antioxidants hydrochloric acid not reported pH adjustment Methylparaben 0.7 preservative Oleic acid 0.132 penetration enhancer PEG400 20 Surfactant, co-solvent PEG3500 1.5 Surfactant Phenylethanol 0.254 Preservatives, Masking Agents Macrogol 400 Stearate 15 Surfactant Polysorbate 20 2.5 Surfactant Polysorbate 80 10 Surfactant Propylene Glycol 20 cosolvent Propylparaben 0.3 preservative Sodium chloride 1.9 Osmotic agent sodium hydroxide 0.004 pH adjustment sulfuric acid 0.4 pH adjustment Succinic acid disodium succinate Zinc acetate sugar, or flavorings such as sodium saccharin

Rapid mucociliary clearance in the nasal cavity and the presence of nasal lysozyme and macrophages are challenges for mucosal delivery. Mucoadhesive excipients may be required. Depending on the intended mode of administration, the compositions may contain a bioadhesive. In one embodiment, the bioadhesive is a mucoadhesive polymer. A bioadhesive may alter viscosity, rheology, and/or ciliary beating frequency (CBF). Examples of mucoadhesive polymers include poly(acrylates), chitosan, cellulose and derivatives including carboxymethyl cellulose and hydroxypropyl cellulose, hyaluronic acid derivatives, pectin, tragacanth, starch , poly(ethylene glycol), sulfated polysaccharides, carrageenan, sodium alginate, polyvinyl alcohol, polyvinylpyrrolidone, acacia, alginic acid, and gelatin. In one embodiment, the composition may comprise a nasal mucoadhesive component.

However, the viscosity should not hinder airflow. In some embodiments, the viscosity of the composition is between 1 and 10000 cP, or between 1 and 1000 cP, or between 100 and 1000 cP, or between 100 and 500 cP, or at Between 100 and 400 cP, or between 150 and 300 cP, or between 150 and 250 cP, or between 1 and 200 cP, or between 1 and 100 cP, or between 1 and 50 cP between 1 and 25 cP, or between 1 and 10 cP. In a preferred embodiment, the composition has a viscosity of 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 below 1000, or below 500 mm ² s ⁻¹ .

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

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

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

Table 2. Provides examples of nasal or oral compositions comprising SPL7013 as described herein. Element % w/w SPL7013 0.5 to 5.0 purified water qs to 100 glycerin 0 to 1.0 Propylene Glycol 0 to 2.0 Methylparaben 0 to 0.5 Propylparaben 0 to 0.05 Disodium EDTA 0.005 to 0.1 citric acid 0 to 5.0 Carbomer 971P or 934P, 974P or other viscosity/rheology modifiers such as cellulose 0.1 to 5.0 and 0.05 to 0.1 Benzylammonium chloride 0 to 0.1 Sodium hydroxide or other pH modifier qs to pH 4.5 to 7

In some embodiments, the pharmaceutical composition may also include any other therapeutic ingredients, surfactants, propellants, stabilizers, and the like. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the composition and not unduly deleterious to the recipient.

In some embodiments, the pharmaceutical composition can 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.

In one embodiment, the average particle size is from about 0.21 to about -200 μm. In one embodiment, the average particle size is from about 1 to about 200 μm. In one embodiment, the average particle size is from about 1 to about 50 μm. In one embodiment, the average particle size is from about 1 to about 20 μm. In one embodiment, the average particle size is from about 1 to about 5 μm.

In some embodiments, particles with a diameter of 1 to about 5 μm are favorable for delivery to the lower airways; particles from 5 to 10 μm are mostly deposited in the trachea and bronchi, while particles > 10 μm in diameter are mostly deposited in the trachea and bronchi. deposited in the nose. Particles typically smaller than the median aerodynamic diameter of 10 µm can reach the lower airways during nasal breathing. The composition can be a liquid, gel or powder.

In some embodiments, the Dv90 suitable for lower airway delivery is about 5 to 20 μm. In some embodiments, the Dv50 suitable for lower airway delivery is about 5 to 10 μm. In some embodiments, the Dv10 suitable for lower airway delivery is about 1 to 5 μm. In some embodiments, Dv10 suitable for nasal delivery is greater than about 10, 15, or 20 μm.

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

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

The macromolecules of the present invention can be delivered in any composition suitable for application to the eye, eg, solutions, ointments, gels, lotions, in slow release polymers or coated, bound to or impregnated in contact lenses. In one embodiment, the composition can be delivered to the eye in an eyedrop. In one embodiment, the composition can be delivered to the eye in a spray.

"Suitable for administration to the eye" means that any component of the composition does not cause a long-term deleterious effect on the eye or the subject being treated. There may be transient effects such as mild irritation or "tingling" upon administration, but no long-term harmful effects. The macromolecule can be formulated into a simple aqueous solution. Alternatively, the macromolecule can be formulated to have one or more of physiologically compatible osmolality and pH, for example, by adding salts and buffers, as well as other components such as preservatives, gelling agents , viscosity control agents, ophthalmic lubricants, mucoadhesive polymers, surfactants, antioxidants, and the like are included in a solution, gel, lotion, or ointment.

The macromolecules of the present invention are retained on or in the epithelium for a period of time, allowing the macromolecules to diffuse out of the epithelium. This diffusion allows slow release of the drug into the ocular environment so that the antiviral activity of the macromolecule can be delivered over time and is not quickly washed away by ocular fluids and physical cleansing. The macromolecule can be released from the epithelium over a period of time in excess of 10 minutes, more specifically in a period of more than 1 hour, and more specifically in a period of more than 6 hours.

In some embodiments, the present invention provides a composition comprising a macromolecule as described herein and at least one pharmaceutically acceptable carrier, wherein the carrier provides a pH and osmolality that is compatible with the eye.

Suitable ophthalmically acceptable salts that can be used as osmolality include those with sodium, potassium or ammonium cations and chlorides, citrates, ascorbates, borates, phosphates, bicarbonates, sulfates, thiols Salts of sulfate or bisulfite ions. Examples of suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfate, and ammonium sulfate.

Suitable ophthalmically acceptable pH adjusting agents and/or buffers include acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; including, for example, sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, bases of sodium lactate and tris; and buffers such as citrate-dextrose, sodium bicarbonate, and ammonium chloride.

Suitable preservatives include stabilized ammonium compounds such as xylidine ammonium chloride, cetyltrimethylammonium chloride and cetylpyridinium chloride, mercury compounds such as phenylmercuric acetate, imidazolidinyl urea, such as Parabens of methylparaben, ethylparaben, propylparaben or butylparaben; phenoxyethanol, chlorophenoxyethanol, phenoxypropanol, chlorine Butanol, chlorocresol, phenethyl alcohol, EDTA, sorbic acid and their salts.

Suitable gelling agents or viscosity-controlling agents include those that increase viscosity when they come into contact with tear fluid, eg, lacrimation caused by blinking or tears. Such gelling agents can be used to reduce the loss of the macromolecules due to tear drainage and allow the macromolecules to have an increased residence time and thus absorption in the eye or in the epidermis of the eyelid. Suitable gelling agents include gellan gum, especially low acetylated gellan gum, alginate or chitosan. The viscosity modifier may also include a film-forming polymer such as an alkyl cellulose such as methyl cellulose or ethyl cellulose, a hydroxyalkyl cellulose such as hydroxyethyl cellulose or hydroxypropyl methyl cellulose Cellulose, hyaluronic acid or its salt, chondroitin sulfate or its salt, polydextrose, cyclodextrin, polydextrin, maltodextrin, dextrin, gelatin, collagen, polygalacturonic acid such as pectin Derivatives, natural gums such as xanthan gum, locust bean gum, gum arabic, tragacanth and carrageenan, agar, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, acrylamide, acrylic acid Polymers with polycyanoacrylates, and polymers of methyl methacrylate and 2-hydroxy-ethyl methacrylate. The viscosity control agent or gelling agent may be present in an amount of 0.1% to about 6.5% w/w of the composition, especially about 0.5% to 4.5% w/w of the composition.

Suitable lubricants include polyvinyl alcohol, methylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

Suitable mucoadhesive polymers include hydroxypropylmethylcellulose, carboxymethylcellulose, poly(methyl methacrylate), polyacrylamide, polycarbophil, polyethylene oxide, seaweed sodium, and dextrin.

Suitable ophthalmically acceptable surfactants include non-10mc surfactants such as polyoxyethylene fatty acid glycerides and vegetable oils including polyoxyethylene (60) hydrogenated castor oil, polyoxyethylene alkyl ethers, and polyoxyethylene polyethers such as octylphenol 10 and the alkyl phenyl ether of octylphenol polyether 40.

Suitable antioxidants include ascorbic acid and sodium metabisulfite.

Ophthalmic ointments may also include one or more thickening agents, such as liquid paraffin, yellow soft paraffin, hard paraffin, and/or lanolin.

In one embodiment, the ophthalmic compositions described herein are suitable for the treatment and or prevention of a CoV infection. In one embodiment, the ophthalmic compositions described herein are suitable for preventing, reducing or sequestering CoV viral shedding in an individual suffering from a CoV infection.

In one embodiment, the ophthalmic compositions described herein are suitable for use in the treatment and or prevention of an RSV infection. In one embodiment, the ophthalmic compositions described herein are suitable for preventing, reducing or sequestering RSV viral shedding in an individual suffering from an RSV infection.

As discussed above, while the compositions of the present invention may be formulated in topical ocular compositions with carriers, diluents and excipients commonly used in the art, it is well known that many commonly used preservatives are used in topical ocular compositions There are always disadvantages. For example, some preservatives can cause eye irritation and, if used for long-term treatment, may, among other things, cause eye damage. In addition, some preservatives are ineffective against some bacterial strains that can cause spoilage of these compositions. Due to the irritating nature of parabens, parabens and the like are generally considered unsuitable for use in ophthalmic compositions. In some cases, eye drop compositions are formulated without preservatives to reduce irritation. However, such compositions must be packaged for single use or must be refrigerated once opened.

In some embodiments, an ophthalmic composition as described herein consists of an aqueous solution of one of the macromolecules together with at least one pharmaceutically acceptable excipient, wherein the at least one excipient provides 7.0 to 7.6 pH and osmolality of 240 to 310 mOsm/kg, especially an osmolality isotonic with tears. In other embodiments, the composition comprises an aqueous solution of the macromolecule and at least one pharmaceutically acceptable excipient, wherein the at least one excipient provides a pH of 7.0 to 7.5 and 240 to 310 mOsm/kg osmolality, but excludes preservatives other than this macromolecule. other composition

In one embodiment, the compositions as described herein are suitable for transdermal administration and may be formulated in an aqueous, gel or emulsion composition.

In one embodiment, the composition as described herein is suitable for use as a topical spray, wash or paper towel, including hand lotion, surgical field preparation.

In one embodiment, a composition as described herein is embedded, or applied, or incorporated into personal protective equipment (PPE), such as a face mask, gloves, or surgical gown, or a filter for a face shield.

In some embodiments, the macromolecules as described herein are formulated in a composition suitable for parenteral delivery. For example, for intravenous delivery, the composition may be an aqueous composition such as ringers, saline, water or dextrose solution, or may be diluted in 0.9% saline or 5% dextrose as use.

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

The compositions as described herein may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a calculated predetermined quantity of active ingredient in association with the required pharmaceutical carrier and/or diluent. Can produce the desired preventive or therapeutic effect.

All methods include the step of combining the macromolecule with a carrier that constitutes one or more accessory ingredients. Generally, the compositions can be prepared by combining the macromolecule with a liquid carrier to form a solution or a suspension. Such dosage forms are contemplated for administration over a period of time (eg, from about a few seconds to about 2-6 hours for an inhaled dose, and from a few seconds to a bolus injection for a parenteral dose. 24 hours of an infusion). effective amount

The methods of the present disclosure require the administration of an effective amount of a macromolecule, or a composition comprising the macromolecule. An "effective amount" refers to an amount necessary to achieve, at least in part, the desired response, or delay the onset of infection, inhibit the progression of infection, or completely halt infection. An effective amount in a human patient may, for example, fall within the range of about 0.5 mg to about 5 mg. An effective amount for a human patient may, for example, fall within the range of about 0.5 mg to about 5 mg per actuation per nostril.

In some embodiments, the effective amount is in the range of about 0.04 mg to about 1 g, about 10 mg to about 500 mg, about 10 mg to about 100 mg, or about 100 mg to about 500 mg. In some embodiments, the effective amount is in the range of about 0.5 to 5 mg. In some embodiments, the effective amount is in the range of about 0.5 to 1.5 mg. In some embodiments, the effective amount is about 1 mg. In some embodiments, the effective amount is about 0.5 mg.

In some embodiments, the effective amount is about 0.1 mg to about 1 g/m ² , about 1 mg to about 100 mg/m ² , about 10 mg to about 100 mg/m ² , or about 10 mg to about 10 mg/m 2 in the range of about 500 g/m ² .

In some embodiments, the macromolecule is delivered at 0.1 to 10 mg/kg/day. In another embodiment, the macromolecule is delivered at 1 to 10 mg/kg/day. In another embodiment, the macromolecule is delivered at 0.1 to 1 mg/kg/day.

In some embodiments, the macromolecule is delivered via an infusion of one of 0.01 to 5 g/day. In another embodiment, the macromolecule is delivered via an infusion of one of 0.1 to 2 g/day. In some embodiments, the macromolecule is delivered via one infusion of 1 to 2 g/day. In some embodiments, the macromolecule is delivered via one infusion of 0.5 to 1 g/day.

In some embodiments, the composition containing the macromolecule is formulated to contain an amount of the macromolecule effective to establish a bulk concentration of the macromolecule in the range of from about 0.050 to about 25 μM Inside. An in vivo concentration is a plasma concentration or a lung fluid concentration, or a tissue concentration such as a lung tissue concentration. In some embodiments, the composition containing the macromolecule is formulated to contain an amount of the macromolecule effective to establish 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 composition containing the macromolecule is formulated to contain an amount of the macromolecule effective to establish 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. Each of these values can be combined to form a range having an upper limit of about 20 μM, about 17 μM, or about 15 μM. In some embodiments, the composition containing the macromolecule is formulated to contain an amount of the macromolecule effective to establish a bulk concentration of the macromolecule in a range of from about 0.1 to about 100 μM, from In the range of about 0.5 to about 50 μM, or from about 1 to about 25 μM.

In some embodiments, a single dose of 10 to 50 mg/kg can achieve an effective concentration. In another embodiment, a single dose of 20 to 40 mg/kg can achieve an effective concentration. In another embodiment, a single dose of 30 mg/kg achieves an effective concentration.

Typically, when infused, the macromolecule will be infused at a rate that is greater than the _EC50 , preferably greater than the _EC90 , in order to establish and/or maintain an in vivo concentration of the macromolecule, and which may be Avoid excessive side effects.

In some embodiments, the macromolecule is infused at a rate to establish and/or maintain an in vivo concentration of the macromolecule, wherein the in vivo concentration of the macromolecule is 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 infused at a rate to establish and/or maintain an in vivo concentration of the macromolecule, wherein the in vivo concentration of the macromolecule is 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, at least 0.3 mg/ml.

In some embodiments, the macromolecule is infused at a rate to establish and/or maintain an in vivo concentration of the macromolecule, wherein the in vivo concentration of the macromolecule is from about 0.01 to about 100 μM, from about 0.5 to about 50 μM, from about 1 to about 50 μM, from about 2 to about 50 μM, from about 5 to about 50 μM, or from about 10 to about 50 μM.

In some embodiments, the macromolecule is infused at a rate to establish and/or maintain an in vivo concentration of the macromolecule, wherein the in vivo concentration of the macromolecule is from about 0.001 mg/ml to about 2 mg /ml, ranging from about 0.01 mg/ml to about 1 mg/ml, or from about 0.05 to about 0.5 mg/ml.

In some embodiments, the macromolecule is infused at a desired rate in order to establish and/or maintain the in vivo concentration of the macromolecule at a desired concentration. For example, if a concentration of about 400 mg/L is targeted, in some embodiments, the infusion rate can be set at from about 500 to about 3000 mg/hr, from about 1000 to about 2000 mg/hr, or from In the range of about 1500 to about 1600 mg/hr (eg 1584 mg/hr) (eg 400 mg/L x 3.96 L/hr). For example, if a concentration of about 200 mg/L is targeted, in some embodiments, the infusion rate can be set at from about 250 to about 1500 mg/hr, from about 500 to about 1000 mg/hr, or from In the range of about 750 to about 800 mg/hr (eg 792 mg/hr) (eg 200 mg/L x 3.96 L/hr).

In some embodiments, the effective amount is formulated at about 0.1% to about 10% w/w, or about 0.5% to about 10% w/w, or about 0.5% to 5% w/w of the macromolecule , or about 0.5% to 3% w/w, or about 1% to 3% w/w. In some embodiments, the effective amount is formulated at about 0.5% w/w, at about 1% w/w, at about 2% w/w, at about 3% w/w, at about 3% w/w of the macromolecule 4% w/w, or about 5% w/w. In some embodiments, the composition comprises 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, about 30 mg/ml macromolecules.

In some embodiments, the composition is administered in a volume 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 0.2 ml, or about 5 ml . In some embodiments, the composition is administered in a volume of about 0.025 ml, 0.05 ml, 0.1 ml, or about 0.2 ml.

When used in a delivery system, the amount of antiviral composition included in a delivery system according to the present disclosure can be, for example, from about 0.10 g to about 2 g, or from about 0.1 g to about 0.5 g, or from About 0.1 g to about 0.25 g.

In some embodiments, when the composition is for nasal delivery, the dose can be administered in two actuations (sprays), once in each nostril. In some embodiments, when the composition is for nasal delivery, the dose can be about 5 μL to about 200 μL, about 5 μL to about 150 μL, about 5 μL to about 100 μL per nostril , 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 are administered in volumes. In a preferred embodiment, the dose is administered in a volume of about 100 μL per nostril. The macromolecule can be administered according to a dosage regimen that provides the desired effect. For example, the dose, macromolecule, or composition. In one embodiment, the dose, macromolecule or composition may be administered from 1 to 4 times per day. In one embodiment, the macromolecule, or composition, is administered to each nostril (eg, 4 times a day, including 4 times a day in each nostril). In some embodiments, the dose, composition or macromolecule is administered for about 1 to 2 weeks, about 1 month, about 3 months, or about 6 months. In some embodiments, the dose, composition or macromolecule is administered once daily, 4 times daily, 6 times daily, or 8 times daily. In one embodiment, the dose, macromolecule or composition may be administered up to 4 times per day. In one embodiment, the dose, macromolecule or composition may be administered up to 8 times per day. In some embodiments, the dose, composition or macromolecule is administered for up to 10 consecutive days. In some embodiments, the dose, composition or macromolecule is administered for up to 20 consecutive days. In some embodiments, the dose, composition or macromolecule is administered for up to 30 consecutive days. delivery device

In one aspect, the present invention provides a device for delivering a nasal, oral or pulmonary composition comprising a macromolecule as described herein. Devices as described herein can deliver the macromolecules to the upper and/or lower airways. In one embodiment, the device can deliver the macromolecule to the nasal cavity. In one embodiment, the device can deliver one or more doses. In one embodiment, the device is reusable.

In some aspects, a device as described herein comprises a composition as described herein.

In one embodiment, the device is a nasal delivery device. In one embodiment, the device is an oral delivery device. In one embodiment, the nasal delivery device is selected from a nebulizer, inhaler, nebulizer, or nasal wash.

In one embodiment, the device is a nasal spray. In one embodiment, the nasal spray is a pump spray. Such pumps may contain an actuating member. In one embodiment, the nasal spray as described herein is a displacement pump. In one embodiment, the pump is actuated by pressing the actuating member against the bottle, a piston moving downward in the metering chamber. A valve mechanism at the bottom of the metering chamber will prevent backflow into the dip tube. The downward movement of the piston will therefore create pressure within the metering chamber, which forces air (before priming) or liquid out through the actuator and creates a spray. When the actuating pressure is removed, a spring will force the piston and actuator back to their original positions. The metering chamber ensures the correct dose and an open vortex chamber at the tip of the actuator will aerosolize the metered dose. In these pumps, no measures are taken to prevent microbial contamination when used, so the composition usually contains a preservative, in most cases benzalkonium chloride (BAC) or parabens . In some embodiments, the device uses silver as a preservative. In one embodiment, the device uses a silver wire, a silver plated spring and ball at the tip of the actuator. These systems can prevent microorganisms from contaminating the composition between longer dosing intervals. Another approach is to use tip sealing techniques to prevent backflow into the device. In some embodiments, the total volume expelled per actuation of the device ranges from about 25 to about 200 μL per actuation. In some embodiments, the volume expelled per actuation is about 50 to about 150 μL per actuation. In one embodiment, the volume expelled per actuation is about 150 μL per actuation. In one embodiment, the volume expelled per actuation is about 100 μL per actuation. In one embodiment, the volume expelled per actuation is about 50 μL per actuation.

In some embodiments, each actuation produces an average particle size of about 10 to about 200 μm. In some embodiments, each actuation produces an average particle size of about 20 to about 180 μm. In some embodiments, each actuation produces an average particle size of about 40 to about 160 μm. In some embodiments, each actuation produces an average particle size of about 60 to about 110 μm.

In some embodiments, particle size is measured at an actuation speed of about 60 mm/s to about 110 mm/s. In some embodiments, particle size is measured at an actuation speed of about 60 mm/s to about 90 mm/s. In some embodiments, particle size is measured at an actuation speed of about 60 mm/s to about 80 mm/s. In some embodiments, particle size is measured at an actuation speed of about 60 mm/s. In some embodiments, particle size is measured at an actuation speed of about 80 mm/s. In some embodiments, particle size is measured at a distance of about 30 mm to about 80 mm from the point of dispersion to the vertical laser path. In some embodiments, particle size is measured at a distance of about 40 mm to about 80 mm. In some embodiments, particle size is measured at a distance of about 50 mm to about 70 mm. In some embodiments, particle size is measured at a distance of about 55 mm to about 65 mm. In some implementations, particle size is measured using an actuation of 60 mm/s and a distance of 40 to 70 mm.

In some embodiments, at a distance of 40 to 70 nm and an actuation speed of 60 mm/s, each actuation produces a droplet size distribution Dv10 of at least 10 μm (ie, 10% of the particles have less than 10 μm in diameter), or a droplet size distribution Dv10 of at least 15 μm (ie 10% of the particles have a diameter less than 15 μm). In some embodiments, at a distance of 40 to 70 nm and an actuation speed of 60 mm/s, each actuation produces a droplet size distribution Dv50 (median) of at least 50 μm or at least 70 μm. In some embodiments, less than 5% or less than 10% of particles smaller than 10 μm are produced per actuation.

In some implementations, the distance is measured from the actuation member. In some aspects, the distance is measured from a dispensing opening in the actuation configuration.

In one embodiment, the device is an oral delivery device. Those skilled in the art will understand that the oral delivery device may be a pulmonary oral delivery device, eg as described in Ibrahim et al (2015) or Chandel et al (2019). In one embodiment, the oral delivery device is selected from a nebulizer, inhaler, nebulizer, or mouth rinse. In one embodiment, the device can deliver one or more doses. In one embodiment, the device is reusable. In one embodiment, the nebulizer is a multi-dose nebulizer.

In one embodiment, the oral device is an oral sprayer. In one embodiment, the oral sprayer is a pump sprayer.

In one embodiment, the device is an inhaler. In one embodiment, the inhaler is a metered dose inhaler. In one embodiment, the inhaler is a multi-dose inhaler. In one embodiment, the inhaler is a dry powder inhaler. An example of an inhaler can be found in Chandel et al (2019).

In some embodiments, the total volume expelled per actuation of the inhaler ranges from about 5 to about 150 μL per actuation. In some embodiments, the total volume expelled per actuation of the inhaler is about 10 to about 110 μL per actuation. In one embodiment, the total volume expelled per actuation of the inhaler ranges from about 20 μL to about 100 μL per actuation. In one embodiment, the total volume expelled per actuation of the inhaler is about 100 μL per actuation. In one embodiment, the total volume expelled per actuation of the inhaler ranges from about 40 μL to about 80 μL per actuation.

In one embodiment, each inhaler actuation produces an average particle size of about 0.01 to about 7 μm. In one embodiment, each nebulizer actuation produces an average particle size of about 0.01 to about 5 μm. In one embodiment, each nebulizer actuation produces an average particle size of about 0.5 to about 5 μm. In one embodiment, each nebulizer actuation produces an average particle size of about 1 to about 5 μm. In one embodiment, each nebulizer actuation produces an average particle size of about 2 to about 4 μm.

In one embodiment, the atomizer is a jet atomizer. In one embodiment, the nebulizer is an ultrasonic nebulizer. In one embodiment, the atomizer is a vibrating mesh atomizer. In one embodiment, the nebulizer is a breath-actuated nebulizer. In one embodiment, the nebulizer is a breath-enhancing nebulizer. In one embodiment, the nebulizer is selected from: Spiriva Respimat®, the 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 a AKITA® Inhalation System (Activaero ).

In some embodiments, the total volume delivered by the nebulizer ranges from about 5 to about 150 μL per actuation. In some embodiments, the total volume delivered is about 10 to about 110 μL per actuation. In one embodiment, the total volume delivered is about 20 μL to about 100 μL per actuation. In one embodiment, the total volume delivered is about 100 μL per actuation. In one embodiment, the total volume delivered is about 40 μL to about 80 μL per actuation.

In one embodiment, the nebulizer produces an average particle size of about 0.01 to about 7 μm. In one embodiment, the nebulizer produces an average particle size of about 0.01 to about 5 μm. In one embodiment, the nebulizer produces an average particle size of about 0.5 to about 5 μm. In one embodiment, the nebulizer produces an average particle size of about 1 to about 5 μm. In one embodiment, the nebulizer produces an average particle size of about 2 to about 4 μm. nasal spray

In some embodiments, a macromolecule or composition as described herein is 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. Actuation of a nasal spray device as described herein comprising a composition as described herein delivers a moisturizing protective barrier to nasal mucus that helps keep nasal mucus moist and acts as a physical barrier to respiratory viruses .

In one embodiment, a composition as described herein is packaged in a container closure system, wherein the container closure system includes an integral spray pump unit that, upon actuation, delivers a precisely metered amount of a spray in the form of a spray amount of composition. In one embodiment, dispensing as a spray is accomplished by forcing the composition through the nasal actuator and its orifice.

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

In one embodiment, the nasal spray device comprises about 20 to about 120 sprays of the composition. In one embodiment, the nasal spray device comprises about 40 to about 100 sprays of the composition. In one embodiment, the nasal spray device comprises about 60 to about 80 sprays of the composition. In one embodiment, the nebulizer contains about 80 sprays of the composition. In a preferred embodiment, the metered amount of the composition is about 100 μL.

The non-sterile, pre-filled nasal spray device consists of SPL7013, wherein the SPL7013 is formulated into a mucoadhesive formulation containing a small amount of preservative, which adheres to at least the turbinates, nasopharynx, and/or oropharynx. The mucoadhesive composition adheres to the nasal cavity, where the respiratory viruses that cause colds, flu, and more severe respiratory diseases such as COVID-19, attach first and begin to multiply. As shown by the experiments described herein, SPL7013 has antiviral activity against CoV and RSV, and thus can act as a physical barrier to respiratory viruses such as CoV and RSV, helping to reduce exposure to respiratory viral pathogens and Reduce viral infection load. Reducing the infectious viral load can help prevent the acquisition or spread of infection. Due to its physical size and negative charge, SPL7013 is not absorbed into the blood after topical nasal administration. The inactivated virus is naturally eliminated through nasal mucus.

In one embodiment, the nasal spray device comprises a composition comprising a formulation as described herein. In one embodiment, the formulation is Variant 1 as described herein. In one embodiment, the formulation is Variant 2 as described herein. In one embodiment, the formulation is Variant 3 as described herein. In one embodiment, the formulation is Variant 4 as described herein. In one embodiment, the formulation is Variant 5 as described herein.

In one embodiment, a nasal spray device as described herein comprising a composition as described herein delivers a nasal moisture barrier dressing upon actuation. As used herein, a "nasal moisture barrier dressing" refers to a substance applied to the nasal passages (nostrils) that provides a protective moisture barrier to the external environment, and hydrates and soothes the nasal mucosa. In one embodiment, the nasal moisture barrier dressing has a Global Medical Device Nomenclature code 47679.

In one embodiment, a nasal moisture barrier dressing as described herein comprises one or more of the following features: i) moisturizing the nasal mucosa, ii) inactivating CoV, iii) reducing viral load of CoV.

In one embodiment, a nasal moisture barrier dressing as described herein comprises one or more of the following features: i) moisturizing the nasal mucosa, ii) inactivating SARS-CoV-2, iii) reducing SARS-CoV-2 viral load.

In one embodiment, a nasal moisture barrier dressing as described herein comprises one or more of the following features: i) moisturizing the nasal mucosa, ii) inactivating an RSV, iii) reducing the viral load of an RSV. co-invest

Although in some embodiments of the present disclosure, the macromolecules or salts thereof may be the only active ingredients used, in other embodiments, the macromolecules used are used in combination with one or more other active ingredients, For example an other active ingredient used to prevent, treat or reduce the likelihood of being infected by a virus. In one embodiment, the virus can infect an individual via the respiratory tract. In one embodiment, the virus line that can infect an individual via the respiratory tract is selected from the group consisting of: coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, syncytial virus, parainfluenza, adenovirus, interstitial pneumonia virus, and enteric Virus. In one embodiment, the virus is a CoV. In one embodiment, the virus is RSV.

In one embodiment, the active agent is selected from one or more of the following: an antiviral active agent, a vaccine, an immunomodulatory agent, a bronchodilator, an antibacterial agent, a neuraminidase inhibitor Agents, Cap-dependent endonuclease inhibitors, adamantanes, anticoagulants, drugs that enhance platelet formation, angiotensin-converting enzyme inhibitors, vitamins, convalescent plasma therapy and/or primary anti-inflammatory agents.

As used herein, the term "antiviral active agent" refers to a compound that is effective, directly or indirectly, to specifically interfere with at least one viral action selected from one or more of the following: a eukaryotic Viral penetration of cells, viral replication in a eukaryotic cell, viral assembly, viral release from infected eukaryotic cells, or effective nonspecific inhibition of viral titers increase, or effective nonspecific reduction in a eukaryotic cell or viral titer levels in mammalian host systems. It also refers to an agent that prevents or reduces the likelihood of getting a viral infection.

In one embodiment, the antiviral agent is selected from the primary antiviral agents described in Gordon et al., 2020 or Ghareeb et al (2021). In one embodiment, the antiviral active agent is selected from one or more of the following: carrageenan, GM-CSF, IL-6R, CCR5, the S protein of MERS, and drugs, including ribavirin , Tilorone, Favipiravir, Kualijia (lopinavir / ritonavir), Puzeli (darunavir / cobicita), nelfinavir, mycophenolic acid, Galide Wei, Antingle, OYA1, BPI-002, Ifendil, APN01, EIDD-2801, Baricitinib, Camostat mesylate, Lycorine, Briracetine, BX-25, Amostat, umifenovir, lopinavir, ritonavir, pleconaril, and favipiravir, an interferon (eg, IFN beta), antimalarial The drug chloroquine and the antibiotic azithromycin. In one embodiment, the anti-inflammatory agent is selected from one or more of the following: indomethacin, tocilizumab, a JAK inhibitor, and ruxolitinib .

In one embodiment, the active agent is selected from one or more of the following: acetaminophen, motavizumab, albuterol, epinephrine, ribavirin, and palivizumab Anti-(palivizumab).

Examples of carrageenans are described, for example, in CA2696009. In one embodiment, the carrageenan is selected from the group consisting of iota-carrageenan, kappa-carrageenan, and lambda-carrageenan. In one embodiment, the carrageenan is iota-carrageenan.

In one embodiment, the active agent reduces symptoms of one or more RSVs. In one embodiment, when the virus is an RSV, the active agent is selected from one or more of the following: acetaminophen, motavizumab, salbutamol, epinephrine, ribavirin, and pariway beadzumab.

In one embodiment, the antibacterial agent is an antibiotic. In one embodiment, the antibiotic is a broad-spectrum antibiotic.

In one embodiment, the immunomodulatory agent is an immunosuppressant, a cytokine inhibitor, an antibody, or an immunostimulatory agent. The immunomodulatory agent inhibits inflammation of the airways.

These macromolecules or their salts can also be used in combination with nonsteroidal anti-inflammatory drugs (NSAIDs). For example, the NSAID can be used to treat the symptoms of a CoV and/or RSV infection, and the macromolecule or salt thereof can be used to prevent transmission of the virus to another individual.

The present invention will now be described more fully with reference to the accompanying examples. It should be understood, however, that the following description is illustrative only and should not be construed in any way as limiting the generality of the invention described above. Example Example 1: SPL7013 based on CPE antiviral assay method:

Strain: SARS-CoV-2 hCoV-19/Australia/VIC01/2020 was a gift from the Peter Doherty Institute for Infection and Immunity, Melbourne (Melbourne, Australia). Documentation received with the parent stock indicates that prior to receipt the virus had been passaged as follows: two passages in Vero cells. A working stock was generated by two further passages in Vero cells in a viral growth medium containing L-glutamic acid-free, supplemented with 1% (w /v) Minimum essential medium of L-glutamic acid, 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 derived from BEI Resources (NR-52281). Virus lines were derived from African green monkey kidney Vero E6 cells or lung homogenates from human angiotensin converting enzyme 2 (hACE2) transgenic mice.

Cells: African green monkey kidney (Vero) cell line (ATCC-CCL81) was subcultured in cell growth medium containing L-glutamine free to generate cell bank stocks , minimum essential medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (w/v) L-glutamic acid. Cell stocks were frozen at -80°C overnight and then transferred to liquid nitrogen for long-term storage. Vero cells were passaged up to 13 passages, after which a new working cell bank stock was removed from liquid nitrogen for further use.

Test and Control Compound Preparation: SPL7013 was dissolved in water at 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.

Preparation of cells for assay: Vero cells (ATCC-CCL81) were seeded in 96-well plates for ²⁴ hours at 2x104 cells/well in 100 μL seeding medium (supplemented with 1% (w/v) L-gluten amino acid, 1% ITS, 0.2% BSA minimum essential medium). Plates were incubated overnight at 37°C, 5% CO ₂ .

Add test and controls to Assay Plate: Add a volume of 1400 μL of viral growth medium (supplemented with 1% (w/v) L-glutamic acid, 1% ITS, 0.2% BSA, 1 μg/ mL TPCK-trypsin, 1x Pen/Strep's minimum essential medium) was added to column A, rows 3-11 of a V-substrate rim PCR plate. Compound (40 mg/mL) was added to row 2 (1300 μL). A 1:3 serial dilution was made by transferring 700 μL of compound from row 2 to row 3, row 3 to row 4 and continuing to row 10 and then discarding. A 50 μL volume from each compound dilution series was added to columns B-G of an assay plate. SPL7013 was added to the assay plate 1 hour before infection or 1 hour after infection.

Addition of virus: A 50 μL volume of SARS-CoV-2 diluted in virus growth medium to give a moi of 0.05 was added to the plate. This moi has previously been determined to provide 100% CPE within 4 days. Virus was added to columns B, C and D to assess antiviral activity, and virus-free viral growth medium was added to columns E, F and G to assess cytotoxicity. Plates were incubated at 37°C, 5% CO for ₄ days before evaluating CPE.

Cytopathic Effect (CPE) assay: After four days of incubation, viable cells were assayed by using MTT staining. A 100 μL volume of 3 mg/mL MTT solution was added to the dish and incubated for 4 hours at 37°C in a 5% _CO2 incubator. Wells were blotted dry using a multi-channel manifold attached to a vacuum chamber, and formazan crystals were dissolved by adding 200 μL of 100% 2-propanol for 30 minutes at room temperature. Absorbance was measured at 540 - 650 nm on a disk reader.

縮寫：x，測試或對照品濃度；y，細胞保護百分比；Min，最小值；Max，最大值；D，斜率係數。 Determination of 50% Effective Concentration (EC ₅₀ ): The percentage of cytoprotection achieved by positive controls and test articles in virus-infected cells was calculated by the following formula: % cytoprotection = ([ODt] virus – [ODc] Virus / [ODc] Mock – [ODc] Virus) x 100 where: [ODt] Virus = Optical Density Measured in a Well to examine the effect of a given concentration of Test Article or Positive Control on virus-infected cells . [ODc] virus = optical density measured in a well to examine the effect of this negative control on virus-infected cells. [ODc] Mock = Optical density measured in a well to examine the effect of this negative control on mock-infected cells.

Abbreviations: x, test or control concentration; y, percent cytoprotection; Min, minimum value; Max, maximum value; D, slope coefficient.

The ₅₀ % cytotoxic concentration (CC50) is defined as the concentration of the test compound that reduces the absorbance of the mock-infected cells by 50% of the control value. The CC50 value is calculated as the ratio of ( _ODt )simulated/(ODc)simulated. IDBS XLFit4 Excel Add-in (ID Business Solutions Inc., Alameda, CA) was used to perform the above calculations.

Pre-infection prophylaxis assay: Nine concentrations of SPL7013 (astromime sodium) and remdesivir controls were prepared by three-fold serial dilution in assay medium (Assay Media, AM) and added to Vero cells in triplicate share. After 1 hour, 50 μl of AM containing the lowest MOI of SARS-CoV-2 hCoV-19/Australia/VIC01/2020 (experimentally determined to provide 100% CPE four days post infection) was added to compound and virus only control wells [multiplicity of infection (MOI) = 0.05]. An equal volume of AM only was added to the cytotoxic and cell only wells. Remdesivir was used as this positive control.

Post-infection treatment assay: AMs containing the lowest MOI of SARS-CoV-2 hCoV-19/Australia/VIC01/2020 experimentally determined to provide 100% CPE four days post-infection were added to virus-only control wells. [Multiplicity of infection (MOI) = 0.05]. An equal volume of AM only was added to the cytotoxic and cell only wells. After 1 hour, nine concentrations of SPL7013 (asjunmo sodium) and remdesivir controls were prepared by three-fold serial dilution in assay medium (AM) and added to Vero cells in triplicate. result:

The experimental results are provided in Table 3. The data show that the _EC50 of SPL7013 [25 μM and 24 μM] is in the micromolar range, indicating that it is an effective antiviral agent for the prevention and treatment of viral infections. In addition, an SI of approximately 3.5 in pre- and post-infection assays indicates that SPL7013 is selective for SARS-CoV2. The cytotoxicity of the controls in this assay was greater than expected, and the SI would be expected to be equal to or greater than 5 when replicated.

In contrast, chloroquine is reported to have an _IC50 of 8 μM and a _CC50 of 261, resulting in an SI line 30 against SARS (Keyaerts et al 2004). In a recent study, the activity of chloroquine against BetaCoV/Wuhan/WIV04/2019 was EC ₅₀ = 1.13 μM; CC ₅₀ >100 μM, SI > 88.50, and remdesivir was reported to have EC ₅₀ = 0.77 μM; CC ₅₀ > 100 μM; SI > 129.87 (Cell Research volume 30, pages 269–271, 2020) and EC ₅₀ =0.137 μM (Gilead in EMA Compassionate Use application Procedure No. EMEA/H/K/5622/CU). These assays have fewer replication rounds and shorter incubation times, so direct comparisons are not possible.

Table 3. SPL7013 based on CPE antiviral assay EC ₅₀ and CC ₅₀ data. compound EC ₅₀ CC ₅₀ 1 hour before SPL7013 infection 0.42 mg/mL 1.47 mg/mL 1 hour after SPL7013 infection 0.40 mg/mL 1.29 mg/mL remdesivir 5.05 μM >20 μM Example 2: SPL7013 virucidal assay

25 mg/mL of _SPL7013 (astronomer sodium) was incubated with an equal volume of 105 TCID50/mL units of SARS-CoV- ² . The virus-compound mixture was incubated at 37°C for 60 minutes and immediately titrated into Vero cells pre-seeded in 96-well plates to quantify infectious virus titers by TCID ₅₀ assay. Plates were incubated for three days at 37°C in a humidified 5% _CO2 environment. Visual scoring of virus-induced CPE. The TCID50 of the virus suspension was determined using the method of Reed and _Muench (1938). The virucidal effect was quantified as a percent and log reduction in viral titer compared to assay media titers for SARS-CoV-2 alone. Controls were: AM, AM+virus and sodium citrate 60 mM as the positive control.

SPL7013 showed virucidal activity at 25 mg/mL in this assay. The assay indicated that the compound stopped all viral growth. Example 3: Astronomer sodium (SPL7013) inhibits the replication of SARS-CoV-2 in vitro

This study evaluates the antiviral activity of astronomer sodium against SARS-CoV-2 in vitro. It was found that astronomer sodium was added to cells 1 hour before infection or 1 hour after infection to inhibit SARS-CoV-2 replication in Vero E6 cells, which was effective in reducing virus-induced cytopathic effects by 50% Concentrations ( _EC50 ) ranged from 0.090 to 0.742 µM (0.002 to 0.012 mg/mL). The selectivity index (SI) in these assays is as high as 2197. Asjuna was also effective in a virucidal assessment (EC ₅₀ 1.83 µM [0.030 mg/mL]) when mixed with virus 1 hour prior to cell infection. The results of a time of addition study showed that infectious virus was below the lower limit of detection at all time points tested, results consistent with compounds inhibiting early viral entry steps. The data for all studies were similar and consistent with the potent antiviral activity of astronomer sodium due to inhibition of virus-host cell interactions. method:

Virus, cell culture, astronomer sodium and controls: SARS-CoV-2 hCoV-19/Australia/VIC01/2020 was a gift from the Peter Doherty Institute for Infection and Immunity, Melbourne (Melbourne, Australia). Virus stocks were generated at 360 Biolabs (Melbourne, Australia) by two passages in Vero cells in virus growth medium containing L-glutamine free, supplemented with 1 % (w/v) L-glutamic acid, 1.0 µg/mL of L-(tosylamino-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin (US Worthington Biochemical, NJ), 0.2% bovine serum albumin (BSA), and 1% insulin-transferrin-selenium (ITS) in Minimal Essential Medium (MEM).

The SARS-CoV-2 2019-nCoV/USA-WA1/2020 strain was isolated in Washington State, USA in January 2020 from an oropharyngeal swab of a patient with a respiratory disease and developing clinical disease (COVID-19) out and derived from BEI Resources (NR-52281). Virus lines were derived from African green monkey kidney Vero E6 cells or lung homogenates from hACE2 human angiotensin-converting enzyme 2 (hACE2) transgenic mice. African green monkey kidney Vero E6 cells or lung homogenates were derived from human angiotensin-converting enzyme 2 (hACE2) transgenic mice.

Vero E6 and human Calu-3 cell lines were grown in minimal essential medium (MEM) without L-glutamine supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (w/v) L-glutamic acid. Vero E6 and Calu-3 cells were passaged up to 10 passages for antiviral and virucidal studies. Infections were performed using Hank's balanced salt solution (HBBS) containing 2% fetal bovine serum (FBS). The 2019-nCoV/USA-WA1/2020 strain antiviral assay was performed at a multiplicity of infection (moi) of 0.1. Viral inoculum for virucidal assays was ¹⁰⁴ , 105 and ¹⁰⁶ pfu/mL, ^1.5 mL was added to 2.5 x ¹⁰⁴ cells/well.

The viral inoculum for the virucidal assay was ¹⁰⁴ , ¹⁰⁵ and ¹⁰⁶ pfu/mL. After a defined incubation period, the solution was pelleted through a 20% sucrose pad (Beckman SW40 Ti rotor) and resuspended in 1.5 mL of MEM, which was then added to 2.5 x ¹⁰⁴ cells/well.

Astrolimer sodium is prepared at 86.29 mg/mL or 100 mg/mL in water and stored at 4°C. Sodium astronomer has a molecular weight of 16581.57 g/mol. The purity of the compound used in these studies was assessed to be 98.79% by ultra performance liquid chromatography (UPLC). Remdesivir (MedChemExpress, NJ, USA) was used as a positive control in the virus-induced cytopathic effect (CPE) inhibition assay as well as in the added time plaque assay.

Assay for Inhibition of Virus-Induced Cytopathic Effects: African Green Monkey Kidney (Vero E6, ATCC-CRL1586) cell stock lines were generated in cell growth medium containing L-glutamic acid-free, supplemented with MEM with 10% (v/v) heat-inactivated FBS and 1% (w/v) L-glutamic acid. Vero E6 cell monolayers were seeded in 96 ^- well dishes at 2x10 cells/well in 100 µL of growth medium (MEM supplemented with 1% (w/v) L-glutamic acid, 2% FBS) and were Incubate overnight at 37°C in 5% _CO . SARS-CoV-2 infection was established by infecting cell monolayers with an MOI of 0.05. Astrolimer sodium or remdesivir were serially diluted 1:3 for 9 times, and the antiviral efficacy and cytotoxicity of each compound concentration were assessed in triplicate. Astronomer sodium was added to Vero E6 cells 1 hour before infection with SARS-CoV-2 or 1 hour after infection with SARS-CoV-2. Cell cultures were incubated at 37°C in 5% CO for ₄ days prior to assessment of CPE. This viral growth medium is MEM supplemented with 1% (w/v) L-glutamic acid, 2% FBS, and 4 µg/mL TPCK-treated trypsin. On day 4, virus-induced CPE for the compound was determined by measuring the viable cells using the methylthiazolyldiphenyltetrazolium bromide (MTT) assay (MP Biomedicals, NSW, Australia) and Cytotoxicity. Absorbance was measured at 540-650 nm on a disk reader.

Antiviral Plaque Assay Assessment and Nucleoprotein Capsid ELISA: For this antiviral assessment, sodium astronomer was added to cells 1 hour before, during, and 1 hour after exposure to virus. For the antiviral and virucidal assays, 6 hours after infection, cells were washed to remove astronomer sodium and/or any virus remaining in the supernatant, in such a way that after initial infection, the cells were incubated The cells were cultured and the supernatant was recovered after 16 hours or 4 days. The amount of virus in the supernatant was determined by plaque assay (plaque forming units [pfu]) and by capsidase coupled immunosorbent assay (ELISA). The plaque assay used was as described in van den Worm et al (2012) using a 2% sodium carboxymethylcellulose overlay, cells were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. The nucleocapsid ELISA assay is as described in Bioss Antibodies, USA (BSKV0001). Assessment of astronomer sodium cytotoxicity occurred on day 4 by measuring lactate dehydrogenase (LDH) activity in the cytoplasm using an LDH assay kit (Cayman Chemical) and using 0.5% saponin as The positive cytotoxicity control.

Virucidal Assay: Astronomer sodium was serially diluted 1:3 9 times and tested in triplicate wells. SARS-CoV-2 with an MOI of 0.05 was mixed with diluted astronomer sodium and incubated for 1 hour at 37°C in 5% CO ₂ . Virus and compound mixtures were added to Vero E6 cell monolayers in 96-well dishes and incubated for 4 days at 37°C in 5% CO ₂ . On day 4, the virus-induced CPE was measured by the MTT assay as described above. For this virucidal assessment, concentrations of astronomer sodium (0.0046 to 30 mg/mL) were incubated with SARS-CoV-2 2019-nCoV/USA-WA1/2020 for a time range from 5 seconds to 2 hours. To neutralize the effect of astronomer sodium, unbound compound was isolated from the astronomer:virus mixture by precipitating the preincubation mixture through a 20% sucrose pad (Beckman SW40 Ti rotor). The astronomer sodium-containing supernatant was removed (ie, neutralized the effect of SPL7013), and the pelleted virus line was then gently resuspended and added to Vero E6 or Calu-3 cell cultures thing. Viral infection, cell culture, and cytotoxicity assessment were as described in the plaque assay, as described above in the antiviral plaque assay paragraph.

Determination of Effective Concentration ( _EC50 and EC90) and Cytotoxicity (CC50): The concentration of compound resulting in a ₅₀ % or ₉₀ % reduction in virus-induced CPE (respectively _EC50 ) was calculated using the formula described in Example 1 or EC ₉₀ ). The compound concentration that resulted in a 50% reduction in cell viability after 4 days of culture (CC ₅₀ ) was also calculated by the formula described in Example 1 .

Time of addition assay (TOA): Vero E6 cell monolayers were grown in MEM supplemented with 1% (w/v) L-glutamic acid, 2% FBS. To ensure strong infection, cell cultures were infected with SARS-CoV-2 with an MOI of 1. The virus line was adsorbed for 1 hr at 4°C and then paralleled before the addition of 0.345 mg/mL astronomer sodium, 15 µM hydroxychloroquine, 5 µM remdesivir, or a negative control (assay medium only) Cultures were heated to 37°C for 0 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours, 6 hours. For this 0 minute time point, test or control lines were added immediately after virus preadsorption. Eight hours after virus infection (the duration of one replication cycle), virus was harvested from the cells for each time point. The virus-containing supernatant was retained and virus titers at each time point were determined via virus yield assays.

Viral Yield Reduction Assay: Virus titers from this TOA study were quantified as a median tissue culture infective dose ( _TCID50 ) value. TCID ₅₀ is a measure of viral titer and represents the titer at which a virus can produce infection in 50% of tissue culture samples. Vero E6 cell monolayers were grown in MEM supplemented with 1% (w/v) L-glutamic acid, 2% FBS. Virus lines collected from each time point were added to three wells, and lines were serially diluted three-fold across the plate to obtain a total of nine different virus concentrations. Six of the wells contained assay medium only (ie, no virus) and served as controls. The discs were incubated for three days and the cell monolayers were then microscopically observed, and visual scoring of virus-induced CPE was used as an endpoint. The TCID50 of the virus suspension was determined using the method of Reed and _Muench (1938). For each time point, virus yield is expressed as a percentage relative to virus growth when no compound was added. result:

Virus-induced cytopathic effect inhibition assay: In two independent virus-induced CPE inhibition assays, astronomer sodium inhibited SARS-CoV-2 (hCoV-19/ SARS-CoV-2) in a dose-dependent manner. Australia/VIC01/2020) replication in Vero E6 cells (Figure 2; Figure 3). Virus replication was inhibited when added 1 hour before infection with SARS-CoV-2 or 1 hour after infection with SARS-CoV-2. It was initially tested for astronomer sodium in the range of 0.0013 to 8.63 mg/mL (0.078 to 520.4 µM). Astromer sodium was tested in the range of 0.0001 to 0.86 mg/mL (0.008 to 52.0 µM) in these replicate assays to help further characterize the lower bound of the dose-response curve. The effective and cytotoxic concentrations, and selectivity indices from these assays determined for CPE are individually shown in Figure 2, and are shown as averages. In these CPE studies, the selectivity index (SI) of astronomer sodium against SARS-CoV-2 ranged from 793 to 2197 in these initial assays, where the compounds were 1 before infection hours and 1 hour post-infection were added and were >70 to >80 in these replicate assays, with no cytotoxicity observed at the highest concentration tested (0.86 mg/mL). The positive control remdesivir was also active in the CPE inhibition assay with an SI of >33.

Antiviral efficacy: To determine the ability of astronomer sodium to inhibit globally diverse SARS-CoV-2 strains, the compound was evaluated against 2019-nCoV/USA-WA1/2020 in Vero E6 cells and human Calu-3 cells Virus. Antiviral readouts are based on the virological endpoint of the infectious virus or viral nucleic acid protein capsids released into the supernatant after infection. As shown in Table 4 and Figure 5, the EC ₅₀ of astronomer for inhibiting the release of infectious virus from the 2019-nCoV/USA-WA1/2020 strain ranged from 0.019 to 0.032 mg/mL and 0.0320 to 0.037 mg/mL, respectively. It was determined by plaque assay in Vero E6 and Calu-3 cells. These data are consistent with Astronomer inhibiting the replication of this Australian SARS-CoV-2 isolate in vitro. The ELISA dose-response data for the nucleic acid protein capsids released into the supernatant were similar to the infectious virus release data in each cell line (data not shown). The positive control Remdesivir was also active in the plaque assay.

Table 4: Antiviral efficacy against SARS-CoV-2 (2019-nCoV/USA-WA1/ 2020) were measured for selectivity. Compound/Assay Type cell line _EC50 (mg/mL) CC ₅₀ (mg/mL) SI Astronomer sodium added 1 hour before infection Vero E6 0.032 15.09 472 Calu-3 0.037 21.76 588 astronomer sodium added during infection Vero E6 0.020 15.09 755 Calu-3 0.035 21.76 622 Astronomer sodium added 1 hour after infection Vero E6 0.019 15.09 794 Calu-3 0.030 21.76 725 Remdesivir added 1 hour after infection Vero E6 0.791 µM N/A N/A Calu-3 0.589 µM N/A N/A _EC50 =50% effective concentration; CC50= ₅₀ % cytotoxic concentration; SI=selectivity index ( _CC50 / _EC50 ); N/A=not applicable

Viricidal efficacy: A study was conducted to determine whether astronomer sodium could reduce viral infectivity by irreversibly inactivating SARS-CoV-2 prior to infection of Vero E6 cells. Treatment with astronomer sodium showed similar levels of antiviral efficacy as these CPE studies (Figure 2; Figure 4A), with an EC ₅₀ of 1.83 μM (0.030 mg/mL) and an SI of 130; n=1. Antiviral activity at early, mid and late stages of viral replication was assessed by adding compounds at different times (0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 6 hours) post-infection. The amount of virus secreted into the supernatant _at 8 hours post infection was determined by TCID50. A virucidal assay was conducted to investigate whether astronomer sodium can reduce viral infectivity by irreversibly inactivating SARS-CoV-2 prior to infection in Vero E6 cells and human airway Calu-3 cells. After incubation of virus with astronomer for up to 2 hours and following neutralization of astronomer, the virus exposed to astronomer was added to the cell culture. After 16 hours or 96 hours (day 4), the cell culture supernatant was collected for assessment of progeny virus infectivity as determined by the amount of secreted infectious virus and nucleic acid protein coat. The SARS-CoV-2 replicative life cycle was completed in approximately 8 hours (Ogando et al., 2020), and in these studies, we performed either 16 hours (2 life cycles) or day 4 (12 life cycle) sampling. Possible 12 rounds of infection were initiated, and the sampling time point on day 4 (96 hours) determined that exposure of 106 pfu/mL SARS-CoV-2 to astronomer sodium for 1 to 2 hours resulted in a dose-dependent reduction in viral infectivity , compared to untreated virus (data not shown), use of 10 to 30 mg/mL astronomer sodium resulted in up to >99.999% (>5 log10) reduction in infectivity in Vero E6 cells, and in Vero E6 cells A >99.9% (>3 log10) reduction in infectivity was achieved in Calu-3 cells. SARS-CoV-2 infectivity in Vero E6 cells was also reduced by up to 99.999% when the incubation time of astronomer (10 to 30 mg/mL) with 106 pfu/mL virus was reduced to 15 to 30 minutes (not shown). Display Data).

Incubation of astronomer sodium (1 to 30 mg/mL) with viral inoculum of 10 ⁴ , 10 ⁵ , and 10 ⁶ pfu/mL produced evidence of reduced infectivity for as little as 5 seconds, and 10 to 15 minutes of exposure Sufficient to achieve >99.9% reduction in viral infectivity, and lower viral inoculums achieve greater reductions (>99.999%, 104 pfu/mL viral inoculum, ¹⁰ to 30 mg/mL astronomer sodium, and 10 to 15 minutes incubation time) (Table 5, Figure 6). When cells were assessed 16 hours after exposure to astronomer virus, exposure to ≥10 mg/mL astronomer sodium in as little as 1 minute resulted in >99.9% SARS-CoV-2 (104 pfu /mL) inactivated (Table 6, Figure 7). Exposure of astronomer sodium to the virus for 30 seconds had no detectable virucidal effect.

Table 5: Viricidal efficacy of 10 mg/mL astronomer sodium against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) by reduction in mean infectious virus at 96 hours post-infection (Log ₁₀ pfu/mL). Viral load (pfu/mL) Virus: Astronomer's Breeding Time Reduction vs. virus control (Log ₁₀ ± SD) Reduced vs. Virus Control (%) 10 ⁶ 5 seconds 0.10 ± 0.20 20.567 10 seconds 0.03 ± 0.06 7.388 30 seconds 0.10 ± 0.10 20.567 1 point 0.33 ± 0.12 53.584 10 points 2.20 ± 0.10 99.369 15 marks 3.67 ± 0.23 99.979 10 ⁵ 5 seconds 0.33 ± 0.21 53.584 10 seconds 0.23 ± 0.06 41.566 30 seconds 0.30 ± 0.17 49.881 1 point 0.47 ± 0.21 65.855 10 points 3.70 ± 0.26 99.980 15 marks 4.60 ± 0.10 99.998 10 ⁴ 5 seconds -0.13 ± 0.21 -35.936 10 seconds 0.07 ± 0.29 14.230 30 seconds 0.10 ± 0.10 20.567 1 point 0.10 ± 0.00 20.567 10 points 5.07 ± 0.25 >99.999 15 marks 5.83 ± 0.12 >99.999 Shading indicates data points with >99.9% virucidal efficacy (3 log ₁₀ reduction) relative to virus control; virus control = untreated virus, 0 mg/mL astronomer sodium; SD = standard deviation

Table 6: Viricidal efficacy of 10 mg/mL astronomer sodium against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) by reduction in mean infectious virus at 16 hours post-infection (Log ₁₀ pfu/mL). Viral load (pfu/mL) Virus: Astronomer's Breeding Time Reduction vs. virus control (Log ₁₀ ± SD) Reduced vs. Virus Control (%) 10 ⁵ 30 seconds 0.00 ± 0.36 0.000 1 point 2.63 ± 0.15 99.767 5 points 4.63 ± 0.31 99.998 15 marks 4.60 ± 0.10 99.998 10 ⁴ 30 seconds 0.20 ± 0.20 36.904 1 point 3.17 ± 0.12 99.932 5 points 3.67 ± 0.21 99.979 15 marks 4.00 ± 0.10 99.990 Shading indicates data points with >99.9% virucidal efficacy (3 log ₁₀ reduction) relative to virus control; virus control = untreated virus, 0 mg/mL astronomer sodium; SD = standard deviation

Time-of-Addition Analysis (TOA): To further investigate the mechanism of action of astronomer sodium, a TOA study was conducted. Compounds were added to virus-infected cells during the early, mid and late stages of the SARS-CoV-2 replication life cycle, which was completed in approximately 8 hours. Addition of 0.345 mg/mL sodium astronomer over the time range from 0 minutes to 6 hours post-infection resulted in viral levels below the lower limit of detection at each time point (Figure 4B). This finding is in contrast to the levels of detectable infectious virus at all the same time points in the positive controls (remdesivir and hydroxychloroquine sulfate) and virus-only cultures. Hydroxychloroquine sulfate at 15 µM had no apparent effect on viral replication at any time point. Remdesivir (5 µM, ~5-10-fold _EC50 ) inhibited viral replication by <1 log ₁₀ TCID ₅₀ when added within 15 or 30 minutes post-infection. discuss:

Astronomer sodium exhibits potent antiviral activity against a variety of SARS-CoV-2 cells in vitro. Antiviral activity is exhibited by reducing CPE, release of infectious virus, and release of viral nucleic acid protein coat protein. Antiviral activity was exhibited when astronomer sodium was added to cells prior to cell infection, and when the compound was added to cells that had been exposed to SARS-CoV-2. When mixed with virus for as little as 1 minute, astronomer sodium exhibits irreversible virucidal activity.

Notably, the SI of astronomer sodium was significantly higher in these antiviral assays compared to other antiviral compounds under investigation for their SARS-CoV-2 activity (Pizzorno et al., 2020) .

Remdesivir was used as the antiviral positive control for the CPE inhibition and antiviral assays, and the experimental EC ₅₀ was compared to published data using a different SARS-CoV-2 clinical isolate Consistent (Wang et al., 2020).

These antiviral data are consistent with astronomer sodium as a potent inhibitor of early events in the viral life cycle. The virucidal assay data indicate that astronomer sodium antiviral activity is consistent with binding to the virus, irreversibly inactivating the virus and blocking infection.

Complete elimination of viral infection at all time points in this TOA assay is also consistent with astronomer sodium being a potent antiviral agent that inhibits early viral infection and replication.

The virucidal activity of astronomer sodium demonstrates its irreversible inhibition of early viral infection and replication. These findings indicate that viral attachment, fusion and entry of viruses can be effectively inhibited, which prevents viral replication and release of infectious viral progeny. These findings indicate that viral attachment, fusion and entry of viruses can be effectively inhibited, which prevents viral replication and release of infectious viral progeny.

Data from the current study suggest that the compound exerts its antiviral activity against geographically distinct SARS-CoV-2 isolates by interfering with early virus-cell recognition events. Astronomer sodium is an effective virucidal agent that reduces the infectivity of SARS-CoV-2 by >99.9% after 1 minute exposure to the virus. These studies support the ability of astronomer sodium to prevent early viral entry steps, such as attachment, thereby reducing or preventing viral infection or cell-cell spread.

Primary antiviral agents that block the binding of viruses to target cells, such as astronomer sodium, can be used as primary prophylactic and/or therapeutic agents against SARS-CoV-2. These antiviral studies suggest that astronomer sodium reformulated for delivery to the respiratory tract may be an effective preventive strategy to block the spread of SARS-CoV-2 and enhance other protective and therapeutic strategies. Example 4: Evaluation of the virucidal properties of SPL7013 against three human coronaviruses (hCoV-229E, hCoV-NL63, and hCoV-OC43)

A virucidal suspension test (in vitro time kill method) was used to evaluate SPL7013 against hCoV-229E (ATCC #VR-740), hCoV-NL63 (ZeptoMetrix Corp. #0810228CF), and hCoV-OC43 (ZeptoMetrix Corp. # 0810024CF) for its virucidal properties. The test virus lines used in this study were derived from BSLI high titer virus stocks.

On the day of use, aliquots of stock virus were removed from the -70°C freezer and thawed before use in testing. After 60 seconds and 60 minutes of exposure to the test product, the percent and log ₁₀ reductions from the initial population of these strains were determined. The virus titer was determined using a 50% tissue culture infectious dose (TCID50) calculation (Quantal test). method:

Cell Culture: The cell lines used were human lung fibroblasts (MRC-5; ATCC#CCL-171), green monkey epithelial kidney cells (Vero; ATCC#CCL-81), and human colon adenocarcinoma, epithelial cells (HCT-8; ATCC# CCL-244). Cell lines were maintained as monolayers in disposable cell culture labware and were used for this virucidal suspension test. Host cell cultures were seeded onto 24-well cell culture dishes prior to testing. Before inoculation with coronavirus strain 229E, the MRC-5 cell line was approximately 90% confluent and the line was no more than 48 hours old. Before inoculation with coronavirus strain NL63, Vero cell lines were approximately 90% confluent and were no more than 48 hours old. At the time of inoculation with coronavirus strain OC43, the HCT-8 cell line was approximately 80% confluent and the line was no more than 48 hours old. The growth medium (GM) was replaced by a maintenance medium (MM) to support virus propagation.

Test product: SPL7013 aqueous solution 99.1 m/mL. An aliquot of 15.99 mL of the test product 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.

Viricidal Suspension Test: The Viricidal Suspension Test includes the parameters described in Table 7.

Test: A 0.5 mL aliquot of the test virus(s) was added to a vial containing 4.5 mL of the test product concentration. The test virus line(s) were exposed to the test product(s) for 60 seconds and 60 minutes. Immediately after the exposure(s), the test virus/product suspension(s) were neutralized in fetal bovine serum, mixed well, and serially diluted in MM. Each dilution was plated in quadruplicate.

Virus Control: A 0.5 mL aliquot of the test virus(s) was added to 4.5 mL of MM and exposed for 60 seconds and 60 minutes at ambient temperature. The subsequent test virus dilutions were made in MM and serially diluted in MM. Each dilution was plated in quadruplicate.

Cytotoxicity Control: A 0.5 mL aliquot of MM was added to a vial containing 4.5 mL of the test product concentration(s). The MM/product mixture was neutralized in fetal bovine serum, mixed well, and serially diluted in MM. Each dilution was plated in quadruplicate.

Neutralization Control: A 0.5 mL aliquot of MM was added to a vial containing 4.5 mL of undiluted test product. The MM/product mixture was diluted 1:10 in fetal bovine serum. An aliquot of the virus(s) is added to the neutralized product, mixed well, and exposed to the neutralized product for 10 to 20 minutes. Subsequent 10-fold dilutions of the neutralized test product/virus suspension were made in MM. Each dilution was plated in quadruplicate.

Neutralizer toxicity control: The effect of the neutralizer on viral infectivity was assessed by adding virus alone to the neutralizer (fetal bovine serum) followed by exposure for 10 to 20 minutes. Subsequent 10-fold dilutions of the neutralized test product/virus suspension were made in MM. Each dilution was plated in quadruplicate.

Cell Culture Controls: Intact cell culture lines served as controls for cell culture viability. This GM line was replaced by MM in all cell control wells.

The plates were incubated in a _CO2 incubator for ₁₀ to 14 days at a temperature appropriate for each virus. Cytopathic/cytotoxic effects were monitored using an inverted compound microscope.

Table 7: Parameters of the virucidal suspension test. parameter Summarize Plated Replicates Viricidal Suspension Test Virus + test product → exposure → neutralization → dilution → plate culture 4 reps per set virus control virus+diluent→exposure→dilution→plate 4 reps per set Neutralization control Test Product + Diluent → Neutralization → Dilution → Plate Culture 4 reps per set Cytotoxicity control Test product + diluent → neutralization → virus inoculation → dilution → plate culture 4 reps per set Neutralizer toxicity control Virus + Diluent → Neutralization → Dilution → Plate Culture 4 reps per set cell culture control maintenance medium 4 reps per set result:

Table 8, Table 9, and Table 10 provide virucidal data for SPL7013 against three human CoV strains. The SPL7013 aqueous solution 99.91 mg/mL reduced the infectivity of hCoV-229E by 0.75 log ₁₀ (82.22%) after 60 seconds and 60 minutes of exposure; it reduced the infectivity of hCoV-NL63 by 0.50 log ₁₀ (68.38%) after 60 seconds of exposure ), and reduced the infectivity of hCoV-NL63 by 0.75 log ₁₀ (82.22%) after 60 minutes of exposure; and reduced the infectivity of hCoV-OC43 by 0.50 log ₁₀ (68.38%) after 60 seconds and 60 minutes of exposure. These results show that SPL7013 is active against multiple human CoV strains.

Table 8: Aqueous virucidal activity of SPL7013 against coronavirus strain 229E (ATCC #VR-740). Dilution (- Log ₁₀ ) virus control test NTC NC CTC CC 60 seconds 60 points 60 seconds 60 points 0000 -2 NT NT ++++ ++++ NT NT 0000 N/A -3 ++++ ++++ ++++ ++++ ++++ ++++ 0000 -4 ++++ ++++ ++++ ++++ ++++ ++++ 0000 -5 ++++ ++++ ++++ +0+0 ++++ +++0 NT -6 +0+0 ++00 0000 000+ ++00 0++0 NT -7 000+ 0000 0000 0000 0+00 0000 NT TCID ₅₀ (log ₁₀ ) 6.25 6.00 5.50 5.25 6.25 5.75 1.50 Log ₁₀ lower N/A 0.75 0.75 N/A percentage decrease 82.22% 82.22% + CPE (cytopathic/cytotoxic effect) present; 0 CPE (cytopathic/cytotoxic effect) not detected CC cell control; CTC cytotoxicity control; NC neutralization control; NTC neutralizer toxicity control; NT not tested ; N/A Not applicable.

Table 9: SPL7013 aqueous solution virucidal activity against: coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF). Dilution (- Log ₁₀ ) virus control test NTC NC CTC CC 60 seconds 60 points 60 seconds 60 points 0000 -2 NT NT ++++ ++++ NT NT 0000 N/A -3 ++++ ++++ ++++ ++++ ++++ ++++ 0000 -4 ++++ ++++ ++++ ++++ ++++ ++++ 0000 -5 ++++ ++++ +++0 ++00 ++0+ ++++ NT -6 0+00 +000 0000 0000 0000 0000 NT -7 0000 0000 0000 0000 0000 0000 NT TCID ₅₀ (log ₁₀ ) 5.75 5.75 5.25 5.00 5.25 5.50 1.50 Log ₁₀ lower N/A 0.50 0.75 N/A percentage decrease 68.38% 82.22% + CPE (cytopathic/cytotoxic effect) present; 0 CPE (cytopathic/cytotoxic effect) not detected CC cell control; CTC cytotoxicity control; NC neutralization control; NTC neutralizer toxicity control; NT not tested ; N/A Not applicable.

Table 10: SPL7013 aqueous solution virucidal activity against: coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF). Dilution (- Log ₁₀ ) virus control test NTC NC CTC CC 60 seconds 60 points 60 seconds 60 points 0000 -2 NT NT ++++ ++++ NT NT 0000 N/A -3 ++++ ++++ ++++ ++++ ++++ ++++ 0000 -4 ++++ ++++ ++++ +++0 ++++ ++++ 0000 -5 ++++ ++0+ ++++ 0+++ +++0 ++++ NT -6 00++ +00+ 0000 +000 000+ 0000 NT -7 0000 0000 0000 0000 0000 0000 NT TCID ₅₀ (log ₁₀ ) 6.00 5.75 5.50 5.25 5.50 5.50 1.50 Log ₁₀ lower N/A 0.50 0.50 N/A lower percentage 68.38% 68.38% + CPE (cytopathic/cytotoxic effect) present; 0 CPE (cytopathic/cytotoxic effect) not detected CC cell control; CTC cytotoxicity control; NC neutralization control; NTC neutralizer toxicity control; NT not tested ; N/A Not applicable. Example 5: Evaluation of the virucidal properties of SPL7013 against the SARS-CoV-2 strain Slovakia/SK-BMC5/2020

To evaluate the virucidal properties of SPL7013 against this SARS-CoV-2 strain, Slovakia/SK-BMC5/2020. This strain was isolated in March 2020 from a COVID-19 patient from Slovakia. method:

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

Virus: Slovakia/SK-BMC5/2020 is provided through the European Virus Archive goes Global (Evag) platform. The SARS-Cov-2 line was amplified and titered on the Vero E6/TMPRSS2 cell line.

Cytotoxicity and virus quantification (Experiment 1): Cells were counted and their viability was assessed using a Vi-Cell robot. Cell lines were seeded at ~15,000 cells/well. Cell lines were pretreated for 1 hour at 37°C 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 culture medium. The reference compound (Apilimod) was prepared at three concentrations (1000, 300 and 100 nM). A volume of 10 µL of Slovakia/SK-BMC5/2020 was subsequently added to the pretreated cells at 1 MOI (~0.01) and incubated in a 37°C incubator for 48 hours. The supernatant was collected for viral load determination (RT-qPCR).

The CellTiter 96® Aqueous Nonradioactive Cell Proliferation Assay (MTS/PMS Assay) was performed on a plate control (no virus) and on treated and infected plates as described above. The assay (Promega ref #G5430) was performed according to the manufacturer's protocol. The supernatants were removed from the wells for PCR reactions, and a volume of 100 μL of fresh cell culture medium and a volume of 20 μL of MTS/PMS reagent were added to each well. Absorbance was recorded every hour for four hours.

Viral load was quantified by RTqPCR at the end of the experiment using the ORF1ab gene. RNA was extracted using this virus kit (Macherey-Nagel, #740709). RNA lines were frozen at -20°C until use. RT-PCR was performed with the SuperScript™ III One-Step QRT-PCR Systm kit (commercial kit #1732-020, Life Technologies) using a Bio-Rad CFX384™ instrument and associated software.

Microscopy (Experiment 2): Cell lines were seeded at ~15,000 cells/well. Cell lines were pretreated for 1 hour at 37°C 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 culture medium. The reference compound (apimod) was prepared at three concentrations (1000, 300 and 100 nM). Next, a volume of 10 µL of Slovakia/SK-BMC5/2020 was subsequently added to the cells at 1 MOI (~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, rabbit Mab primary antibody (Sino Biological, #40143-R019; 1:8000 dilution), and Operetta was used. FFU/mL imaging.

The results are shown in Figures 11 and 12. Example 6: In vivo evaluation of SPL7013 against SARS-CoV-2 infection in hACE2 transgenic mice after 7 days of nasal administration method:

Briefly, 4 groups of 5 animals approximately 6-8 weeks old were used. Human ACE2 transgenic mice K18-hACE2 (available from Jackson Laboratory, B6.Cg-Tg(K18-ACE2)2Prlmn/J, stock no. 034860) were used. obtained) to evaluate the effect of SPL7013 on viral load in vivo.

Animal lines in these groups were inoculated intranasally with 25 µL per nostril of a viral suspension containing 10 ⁴ PFU/µL of SARS-CoV-2 (total challenge: 5x10 ⁵ PFU) (2019- nCoV/USA-WA1/2020 strain). Animal lines were dosed continuously for 7 days with 25 µL/nostril (50 µL total) in PBS (phosphate buffered saline) or 1%, 3%, or 5% SPL7013 in PBS, resulting in a total daily dose of 0, 0.5, 1.5 and 2.5 mg of SPL7013 (on days 1 to 6). A first dose of SPL7013 was administered on day 0 5 minutes before virus inoculation, and a subsequent dose was administered on day 0 5 minutes after virus inoculation, and once daily at the same time on days 1 through 6 cast. Animals were euthanized on day 7. Infection status (day 7) was determined by measuring viral load from nasal swab samples by quantitative polymerase chain reaction (qPCR). result:

On day 7, there was a dose-dependent reduction (qPCR) in viral replicas in nasal swabs, which reached statistical significance compared to controls at the highest dose level (Figure 8A). This data indicates that SPL7013 can reduce nasally obtained SARS-CoV-2 viral load in a dose-dependent manner when administered nasally. The 2.5 mg/day dose was the most effective in reducing viral load. Example 7: Evaluation of the antiviral properties of SPL7013 against Severe Acute Respiratory Syndrome (SARS) Coronavirus and Middle East Respiratory Syndrome (MERS) Coronavirus method:

Cell Culture and Viruses: HEK-293T cells expressing hACE2 ⁺ and hTMPRSS2 ⁺ and Vero E6 cells (ATCC-CRL1586) were cultured in Minimum Essential Medium (MEM) L-glutamate-free, supplemented With 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (w/v) L-glutamic acid. Infections were performed using Hank's Balanced Salt Solution (HBBS) containing 2% FBS. Pseudotyped SARS-CoV-1 (Urbani), SARS-CoV-2 (Wuhan-Hu-1), MERS-CoV (HCoV-EMC) reporter virus particles (RVPs) were obtained by Integral Molecular (respectively) Generated for catalog numbers RVP-801, RVP-701, RVP-901). RVPs display the antigenically correct spike protein on a heterologous viral core and carry a modified genome that expresses a convenient optical reporter gene, green fluorescent protein (GFP), upon cell infection24 within hours. Recombinant Protein (mFc-Tag SARS-CoV-2 Spike RBD (318-541) Recombinant Protein (mFc-Tag) was used according to the manufacturer's instructions ) #41701, Cell Signalling Technology).

Analysis of SARS-CoV-1, SARS-CoV-2 and MERS-CoV pseudotyped GFP reporter lentiviral particles: Vero 6 cell line was seeded at 100,000 cells per well in a 96-well plate for analysis analyze. Cell lines were seeded and grown in DMEM/10% FBS. Before adding SARS-CoV-1, SARS-CoV-2 and MERS-CoV spike-pseudotyped GFP reporter lentiviral particles (RVP-801, RVP-701 and RVP-901, Integral Molecular) (50 µL) 1 24 hours, SPL7013 (0, 10 and 30 mg/mL in PBS) was added to Vero E6 cells. The percentage of GFP-positive or infected Vero E6 cells was determined by fluorescence-activated cell sorting (FACS) flow cytometry 48 hours after infection.

Conjugate focus microscopy study of SARS-CoV-2 spike binding to hACE2 ⁺ hTMPRSS2 ⁺ 293T cells: The hACE2 ⁺ hTMPRSS2 ⁺ 293T cell line was cultured on chamber slides. Cells were treated with SPL7013 (0, 1 mg/mL in PBS) for 1 hour prior to challenge with SARS-CoV-2 spike RBD recombinant protein with an mFc tag. After 1 hour, cells were washed twice and anti-mFc-PE IgG antibody (1 μg/mL) was added to cells to identify bound spike protein. After 30 minutes, cells were washed twice again, fixed, and analyzed by conjugation focus microscopy. result:

Analysis of SARS-CoV-1, SARS-CoV-2 and MERS-CoV pseudotyped GFP reporter lentiviral particles: SPL7013 was found to have a broad-spectrum antiviral effect, which is specific for inhibiting the attachment, fusion or both functions. Pseudotyped lentiviral particle lines were used to infect Vero E6 cells, wherein the pseudotyped lentiviral particles expressed antigenically correct spike proteins encoded by SARS-CoV-1, SARS-CoV-2 and MERS-CoV (Fig. 8B). SARS-CoV-1 and SARS-CoV-2 attach to Vero E6 cells via this ACE2 receptor. MERS-CoV attaches to this dipeptidyl peptidase 4 (DPP4). All three coronaviruses use the TMPRSS2 protease to cleave their S1/S2 regions. SPL7013 potently inhibits the binding of pseudotyped lentiviruses expressing the spike protein of SARS-CoV-1, SARS-CoV-2 and MERS-CoV to Vero E6 cells at concentrations of 10 and 30 mg/mL.

Conjugate focus microscopy studies of SARS-CoV-2 spike binding to hACE2 ⁺ hTMPRSS2 ⁺ 293T cells: In these conjugate focus microscopy studies, this negative control (no SPL7013 added) showed that in the absence of SPL7013, SARS- The CoV-2 spike protein binds to cells expressing the hACE2 receptor on the cell membrane. This is evidenced by an intense green immunofluorescence in the photomicrograph, which shows significant binding of the SARS-CoV-2 spike protein to host cells (photograph not shown). When SARS-CoV-2 was added in the presence of SPL7013, no detectable binding of the SARS-CoV-2 spike protein to the cells expressing the hACE2 receptor was observed. Green immunofluorescence was also completely absent in these photomicrographs (photograph not shown). These studies confirmed that SPL7013 works by blocking the SARS-CoV-2 spike protein. The SARS-CoV-2 spike protein is necessary to initiate the interaction of the virus with the target cell via the ACE2 receptor, which results in infection of the cell. In the absence of binding of the SARS-CoV-2 spike protein to cells, cell infection does not occur.

Current studies with SARS-CoV and MERS-CoV show that SPL7013 blocks the binding of SARS-CoV-2 spike proteins at concentrations that demonstrate efficacy against the binding and infection of the SARS-CoV-2 spike proteins. These studies show that, regardless of the cellular receptor involved, SPL7013 combats all these viruses through a common mechanism that blocks the interaction of the coronavirus spike protein with cells. Taken together, these data support that SPL-7013 has antiviral effects against human pathogenic coronaviruses. Example 8: Evaluation of antiviral properties of SPL7013 against respiratory syncytial virus (RSV)

The concentrations of the test product were prepared using approximately 1 :3 dilutions from the initial concentrations determined after the cytotoxicity test.

The host cell culture was washed with PBS, and 1.0 mL aliquots of test product dilutions were added to the cells. Cells were then incubated for 1 hour ± 15 minutes to reach equilibrium. After incubation, 1.0 mL aliquots of virus were added to the wells and incubated for 1 hour ± 15 minutes to adsorb virus. 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 recorded microscopically (approximately 5-10 days).

For the cytotoxicity control, the host cell culture was washed with PBS. The cell lines were then covered with 1.0 mL of the highest nontoxic test product concentration and incubated for 1 hour ± 15 minutes to equilibrate. After incubation, 1.0 μl aliquots of MM (mock infection) were added to the wells and incubated for 1 hour ± 15 minutes. After incubation, the mixture was removed and replaced with TM. The plates were then incubated in a _CO2 incubator.

For the virus control, the host cell culture was washed with PBS and a 1.0 mL aliquot of MM (surrogate test product) was added to the wells designated for the virus control and incubated for 1 hour ± 15 minutes to achieve balance. After incubation was complete, 1.0 mL aliquots of virus were added to the wells and incubated for 1 hour ± 15 minutes to adsorb virus. After incubation, virus was removed and replaced with TM. The plates were incubated in a _CO2 incubator.

Intact cell culture monolayers were used as controls for cell viability. The GM line in these cell culture control wells was replaced by TM.

After incubation, fixation and staining was performed by removing the TM, washing the plates with PBS, and fixing with 4% formaldehyde solution for 4 to 6 hours. Fixed cells were stained with crystal violet stain. Unstained cell lysates (viral plaques) were counted.

Antiviral Post-treatment Test - Determination of Product Cytotoxicity: The highest non-cytotoxic concentration of the test product was determined. The host cell culture was washed with PBS. A 1.0 mL aliquot of the test product was added to the cells and incubated in a _CO2 incubator at 37°C ± 2°C for 24 hours ± 1 hour. Toxicity was assessed using the CCK-8 assay and read at 450 nm using a VERSAmax™ adjustable microplate reader. The concentrations used in the antiviral test were determined in the cytotoxicity test. Results are expressed as a percentage of cell viability, where 100% cell viability is approximately equal to the mean of the cell control. The TC50 concentration of the test product was determined using GraphPad Prism 5.0 statistical software. The antiviral post-treatment test included the procedures listed in Table 11.

Table 11. Antiviral post-treatment test parameters. parameter Summarize repeat test Infect cells with ≤ 100 PFU/mL virus → inoculate with product dilution → incubate → fix → stain → count plaques 2 Cytotoxicity control Inoculation of a product onto cells → incubation → fixation → staining → visual assessment 2 virus control Infect cells with a virus → incubate → fix → stain → count plaques 2 cell culture control Maintenance medium → incubation → fixation → staining → visual assessment 2

The concentrations of the test product were prepared using approximately 1 :3 dilutions from the initial concentrations determined after the cytotoxicity test.

The host cell culture was washed with PBS and 1.0 mL aliquots of virus were added to the wells and incubated for 1 hour ± 15 minutes to adsorb virus. After incubation, the viral inoculum was removed and replaced with aliquots of these test product concentrations in TM. The plates were then incubated in a _CO2 incubator. The plates were then incubated until viral plaques in the viral control could be recorded microscopically (approximately 5-10 days).

For the cytotoxicity control, the host cell culture was washed with PBS. The cell lines were then covered with the highest nontoxic test product concentration in TM and incubated in a _CO2 incubator.

For the virus control, the host cell culture was washed with PBS. 1.0 mL aliquots of ≤ 100 PFU/mL virus will be added to the wells and incubated for 1 hour ± 15 minutes to adsorb virus. After incubation, the viral inoculum was removed and replaced with an aliquot of TM. The plates were incubated in a _CO2 incubator.

For this cell culture control, an intact cell culture monolayer was used as a control for cell viability. The GM line in these cell culture control wells was replaced by TM.

Following incubation, fixation and staining was performed by removing the TM, washing with PBS, and fixing with 4% formaldehyde solution for 4 to 6 hours. Fixed cells were stained with crystal violet stain. Unstained cell lysates (viral plaques) were counted.

Evaluation of antiviral properties: The antiviral properties EC50 and/or EC90 were determined using nonlinear regression analysis, GraphPad Prism 5.0 software.

Antiviral Test Acceptance Criteria: A valid test line as described in the Examples requires: 1) the plaques in the test and control samples are enumerable; 2) there is no significant cytotoxicity in the cytotoxicity control Effects; 3) Cell control wells were viable and attached to the bottom of the well; 4) The medium in all wells of the plate was free of contamination. result:

The results are summarized in Table 12 and Figure 9 .

Table 12. Summary of results of antiviral screening of SPL7013. virus name product application EC50, mg/mL EC90, mg/mL TC50, mg/mL Selectivity Index TC50/EC50 optimum value 95% CI optimum value 95% CI optimum value 95% CI human respiratory syncytial virus preprocessing 0.045 0.038 to 0.053 0.180 0.125 to 0.258 35.64 34.02 to 37.34 792 post-processing 0.107 0.064 to 0.181 1.159 0.368 to 3.649 30.90 28.69 to 33.28 289 EC = effective concentration; TC = toxic concentration; CI = confidence interval. Example 9: SPL7013 Nasal Spray

One embodiment of the device as described herein is a nasal spray. An implementation of the nasal spray is provided in this example and is referred to as "SPL7013 Nasal Spray." The SPL7013 nasal spray contains an aqueous nasal composition containing SPL7013, called "SPL7013 Nasal Spray Composition". SPL7013 is designed to inactivate viruses, including SARS-CoV-2 and/or RSV, and reduce exposure to viral load. Reducing viral load can reduce the acquisition or spread of infection. When including the SPL7013 nasal spray composition, the SPL7013 nasal spray can produce droplet sizes suitable for administration and delivery to the nasal cavity, wherein less than 5% of the droplets are 10 µM or smaller (10 µM particles are more suitable for delivery to the lungs).

Actuation from the SPL7013 nasal sprayer comprising the SPL7013 nasal spray composition can create a moisturizing and protective mucoadhesive barrier to the nasal mucosa, which can be used to inactivate and act as a barrier to respiratory viruses.

The SPL7013 nasal spray composition contains SPL7013 and a viscosity-modifying mucoadhesive substance. The SPL7013 nasal spray composition is as described in "Variant 4" listed in Table 14, or as described in "Variant 5" shown in Table 13 below. Variant 5 was the Variant 4 formulation additionally pH adjusted with hydrochloric acid. The formulation contains a Type B carbomer homopolymer to achieve a suitable viscosity that facilitates easy administration and facilitates retention of the product in the nasal cavity.

Table 13: SPL7013 nasal spray formulation (variant 5). component % w/w Quantity 1% w/w (kg / 100 kg batch) purified water qs 100% 98.51 ⁺ Asjun Moana (SPL7013) 1.0 ^1.0+ Propylene Glycol 1.0 1.0 glycerin 1.16 1.0 Methylparaben 0.18 0.18 Carbomer 974P (Carbomer® ^974P ) 0.05 0.05 Propylparaben 0.02 0.02 EDTA disodium salt dihydrate 0.005 0.005 Sodium Hydroxide ^{^} Adjust pH to 6.0 N/A Hydrochloric acid ^{^} Adjust pH to 6.0 N/A + The final amount of SPL7013 and water was determined by the water content of SPL7013 at the time of manufacture. ^ Prepared with purified water (EP) at 0.1 N.

The SPL7013 nasal spray device is provided as a 10 mL multi-dose, metered nasal spray device that delivers ~100 μL of the SPL7013 nasal spray composition per actuation. Other embodiments of the SPL7013 nasal spray may contain a slightly smaller or larger volume, or smaller or larger metered dose. The SPL7013 nasal spray composition can be self-administered by the user for up to 30 consecutive days, and/or up to 4 times a day (in each nostril), as desired, to inactivate the virus and reduce exposure to viral load.

With reference to the Global Medical Device Nomenclature (GMDN), the term applicable to the SPL7013 nasal spray is "Nasal Moisture Barrier Dressing", and its GMDN code is 47679. Due to the physical nature of the mechanism by which SPL7013 inactivates the virus, and the physical mode of action of the device, this product is considered a Class I medical device according to the European Medical Device Directive 93/42/EEC. device.

When comprising the SPL7013 nasal spray composition, the SPL7013 nasal spray upon actuation provides i) a moisturizing and protective mucoadhesive formulation that acts as a barrier to respiratory viruses when applied to the nasal mucosa; ii) Inactivate the virus and reduce exposure to viral load; iii) reduce viral load as a result of i) and/or ii). A reduction in viral load may help prevent the acquisition or spread of infection.

The acceptance criteria for the SPL7013 nasal spray composition are summarized below. The osmolality and pH of a nasal spray composition conform to the physiological conditions of <500 mOsmol and a pH of 5.5-6.5. The acceptance criteria for osmolality and pH of SPL7013 nasal spray were 200-400 mOsmol and 5.5-6.5, respectively. The SPL7013 nasal spray typically has a density of -1 g/mL, suitable for retention in the nasal cavity. The release and expiry acceptance limits for the SPL7013 assay were 0.80-1.20 % w/w. The acceptance criteria for methylparaben and propylparaben are 0.14%-0.23% and 0.015%-0.025%, respectively. Any microbial content in SPL7013 Nasal Spray was determined by a Microbial Limits Test according to Ph. Eur. 2.6.12 Microbiological Examination of Nonsterile Products: Microbial Enumeration Test and Ph. Eur. 2.6.13 Microbial Examination of Non-Sterile Products: test for Specified Micro-Organisms. The total number of viable bacteria present in the test material, as well as the total yeast and mold strains, were determined using standard pour plate methods. Specifications for microbial content follow the established limits described in Ph. Eur. 5.1.4 Microbiological Quality of Pharmaceutical Preparations. These designated biological systems are based on Ph. Eur 5.1.4, Microbiological Quality of Non-Sterile Pharmaceutical Preparations and Substances for Pharmaceutical Use.

The SPL7013 Nasal Spray is packaged as a non-pressurized, compact container closure system. The container closure system included a delivery system (pump with actuator) that administered a 100 μL spray of droplets of the SPL7013 nasal spray composition. The delivery device consists of a pump screwed onto a polyethylene (HDPE) bottle, and the pump's dip tube, housing, gasket and stem are made of polyethylene polymer. The ball is made of stainless steel 1.430 and is corrosion resistant. The inner liner is made of polyoxymethylene. Example 10: Biological evaluation of SPL7013 nasal spray

A comprehensive biological evaluation was performed on the SPL2013 nasal spray example described in Example 9.

The SPL7013 nasal spray is a surface device that can come into contact with mucous membranes (nose) and has an extended exposure time (>24 hours to 30 days). Tested according to ISO 10993 for SPL7013 nasal spray packaged in a container closed system as described in Example 9, including 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).

In vitro cytotoxicity: The results of an in vitro cytotoxicity study showed that SPL7013 nasal spray was not cytotoxic at 5,000 μg/mL. In a nasal irritation study, 100 μL of 1% SPL7013 nasal spray was administered into each nostril of rats four times a day for 14 consecutive days. The results of the in-life phase study and histopathological examination revealed no findings related to the product and indicated that SPL7013 nasal spray was not irritating.

Skin Sensitization: This skin sensitization study included the one-day Guinea Pig Maximization Test (GMPT) according to Magnusson and Kligman (1969). This test showed that 1% SPL7013 nasal spray was not a consistent sensitizer.

Nasal Tolerability and PK: SPL7013 nasal spray containing 1% or 3% SPL7013 was also administered nasally (50 μL per nostril) 4 times a day for 7 days to rats to test local toxicity and the systemic absorption potential of SPL7013 . The study showed that repeated nasal administration of SPL7013 nasal spray was well tolerated and did not result in any clinical signs of local or systemic toxicity. In addition, plasma samples from animals in this study were collected before the first administration of the product on study day 1, and 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours after the first administration , 4 hours, 5 hours, and 6 hours, and on Study Day 7 before the last product dose of the day, and 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours after the last dose of the day hours, 5 hours and 6 hours. Bioanalysis of pooled plasma samples by capillary electrophoresis showed that no samples at or above this level were detected in any samples from animals administered 1% or 3% SPL7013 nasal spray 4 times daily for 7 days. SPL7013 at the lower limit of quantitation (LLOQ, 0.5 μg/mL) of analysis, except for one sample from animals in the 1% cohort, which showed a 3-hour sample just above the LLOQ (0.635 μg/mL) (not shown). Display Data). These data show that SPL7013 is not systemically absorbed after administration to the nasal mucosa of rats following 4 times daily administration for 7 days, and the results for one sample in this lower dose cohort are a distortion. Example 11: SPL7013 formulation and testing

SPL7013 (Asjuna) formulations were prepared as described in Table 14.

Table 14. SPL7013 example formulations. Ingredients (%w/w) Variant 1 Variant 2 Variant 3 Variant 4 expected activity SPL7013 1.00 1.00 1.00 1.00 Active ingredient that physically inactivates the virus water 95.50 90.00 89.00 95.52 Solvent (will vary depending on pH adjustment) Carpop 974P - - - 0.05 Rheology Modifiers, Viscosity Modifiers, and Bioadhesives Hydroxypropylmethylcellulose 0.10 Rheology Modifiers, Viscosity Modifiers, and Bioadhesives Microcrystalline Cellulose/Carboxymethyl Cellulose 2.00 2.00 Rheology Modifiers, Viscosity Modifiers, and Bioadhesives Glycerin/glycerol 1.00 or 1.16 1.00 2.30 1.00 or 1.16 Moisturizer/helps dissolve/osmotic adjustment of parabens Propylene Glycol 1.00 1.00 1.00 Moisturizer/helps dissolve parabens Methylparaben 0.18 0.18 0.18 preservative Propylparaben 0.02 0.02 0.02 preservative Benzylammonium chloride 0.05 preservative EDTA 0.005 0.005 0.005 0.005 Antioxidant / Chelating Agent / Preservative sodium hydroxide 1.20 1.20 1.20 1.20 pH modifier, alkalizer Check/adjust pH to 6.0 6.0 6.0 6.0

Viscosity and osmolality were measured within hours of preparation. The results are provided in Table 15 below.

Table 15. SPL7013 Example Formulation Properties. Variant 1 Variant 2 Variant 3 Variant 4 Glycerin (%w/w) 1.16 1.00 2.30 1.16 Osmolality (mOsmol) 295 283 344 280 Viscosity (cP) 2.66 107.1 110.7 1.78

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. SPL7013 Example Formulations Droplet Size Distribution (DSD) Testing. formulation Dispenser 1 (100 ul) distance Dv10 Dv50 Dv90 %vol <10µm Variant 1 30mm 20.76 66.46 155.10 2.655 Variant 2 30mm 18.61 59.51 126.10 3.208 Variant 3 30mm 18.62 58.93 130.50 3.334 Variant 4 30mm 14.51 42.84 97.69 5.304 Variant 1 60mm 23.93 57.71 136.20 2.23 Variant 2 60mm 21.40 51.70 112.40 2.73 Variant 3 60mm 20.97 47.49 100.10 3.21 Variant 4 60mm 16.75 42.04 92.32 4.31 formulation Dispenser 2 (100 ul) distance Dv10 Dv50 Dv90 %vol <10µm Variant 1 30mm 11.96 30.57 76.34 7.28 Variant 2 30mm 10.25 27.81 69.81 9.55 Variant 3 30mm 11.79 31.59 81.75 7.27 Variant 4 30mm 10.36 28.22 72.13 9.41 Variant 1 60mm 14.13 33.47 66.63 6.04 Variant 2 60mm 13.79 31.45 62.85 5.86 Variant 3 60mm 14.70 33.00 69.31 5.15 Variant 4 60mm 13.61 30.50 59.36 6.21 formulation Dispenser 3 (100 ul) distance Dv10 Dv50 Dv90 %vol <10µm Variant 1 30mm 9.54 25.26 59.40 10.80 Variant 2 30mm 9.02 22.98 48.57 11.75 Variant 3 30mm 9.51 24.62 55.86 10.85 Variant 4 30mm 9.06 21.88 45.63 11.87 Variant 1 60mm 12.32 30.71 62.09 7.31 Variant 2 60mm 15.10 30.62 53.51 5.31 Variant 3 60mm 12.93 29.60 54.04 6.75 Variant 4 60mm 12.46 28.90 52.53 7.49

These results show that the formulations provide suitable particle sizes for intranasal delivery in various nasal pump delivery devices.

Further DSD studies were performed on this Variant 4 formulation to further investigate the particle size for delivery at suitable rates and are shown in Table 17 below. Experiments were performed using a Spraytec Open Spray with a 300 mm lens.

Table 17. Variant 4 formulation drop size distribution (DSD) testing. Actuation speed: 60 mm/s Actuation speed: 80 mm/s Dv10 Dv50 Dv90 %V＜10µm Dv10 Dv50 Dv90 %V＜10µm distance 30mm average 19 58.9 120.6 3.82 14.51 42.84 97.69 5.304 %RSD 10.7 11.4 9.5 - - - distance 40mm average 30.42 89.32 180.79 1.66 - - - - %RSD 15.4 13.7 10.7 - - - distance 60mm average - - - - 16.75 42.04 92.32 4.31 %RSD - - - - - - distance 70mm average 32.55 78.96 166.46 1.64 - - - - %RSD 13.3 13.8 10.8 - - - RSD = Relative Standard Deviation. Example 12: Evaluation of SPL7013 Hygroscopicity

A hygroscopicity assessment was performed as described in European Pharmacopoeia 5.11. Results from this analysis are interpreted as follows: Deliquescence - absorbs sufficient water to form a liquid; very hygroscopic - mass gain is equal to or greater than 15%; hygroscopic - mass gain is less than 15% and is Equal to or greater than 2%; Slightly hygroscopic - the mass increase is less than 2% and equal to or greater than 0.2%; and Non-hygroscopic - If the mass increase is less than 0.2%, the compound is not hygroscopic.

Therefore, SPL7013 was found to be very hygroscopic in nature, with an increase in mass of more than 15% (21.97%). These hygroscopic results were confirmed by water content analysis and a Karl Fischer test. Example 13: Activity of SPL7013 against SARS-CoV-2 in primary human airway cells

Primary human bronchial epithelial cells (HBEpC) (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 permissive for SARS-CoV-2 infection. These cell lines were used to determine the antiviral effect of astronomer sodium against SARS-CoV-2 in a primary human airway epithelial cell line.

Add 1 mL of ¹⁰³ pfu/mL of SARS-CoV-2 2019-nCoV/USA-WA1/2020 to ^2.5x104 cells/well to infect cells. SARS-CoV-2 spike protein antibody (pAb, T01KHuRb) (ThermoFisher, MA, USA) at 10 µg/mL was added to this positive control at the time of infection. Iota-carrageenan (Sigma-Aldrich, MO, USA) was used in the primary epithelial cell nucleic acid protein coat and plaque assays to compare the antiviral activity of this substance with astronomer sodium. The concentrations used were those reported to exhibit activity against SARS-CoV-2 (Bansal et al, 2020).

SPL7013 (0, 1.1, 3.3 and 10 mg/mL) or iota-carrageenan (0, 6, 60 and 600 µg/mL) were added to HBEpC cells 1 hour before infection with SARS-CoV-2. Cells were cultured for 4 days after infection and the amount of secreted SARS-CoV-2 nucleic acid protein coat in cell supernatants was analyzed by ELISA and infectious virus was quantified by plaque assay, as described in as described in Example 3.

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.

SPL7013 was found to reduce SARS-CoV-2 infection of HBEpC primary cells by up to 98% compared to viral controls by nucleoprotein capsid ELISA (Figure 10A), and up to 95% in this plaque assay (not shown). Display Data). In contrast, treatment with iota-carrageenan had very little antiviral effect against SARS-CoV-2 in this cell line, with the highest concentration tested reducing infection by only 17% by nucleoprotein shell ELISA (Fig. 10B) and only a 21% reduction in this plaque assay (data not shown). The maximum level of inhibition by astronomer sodium was comparable to that achieved by this SARS-CoV-2 spike protein antibody (pAb, T01KHuRb) positive control.

Astronomer sodium inhibits SARS-CoV-2 infection of primary human airway epithelial cells, while iota-carrageenan, a polyanionic compound in a commercially available nasal spray formulation, has been previously shown in Concentrations that reduced SARS-CoV-2 infection in Vero E6 cells failed to provide significant inhibition (Bansal et al, 2020). The unique structure of astronomer sodium, a sulfonated, roughly spherical molecule with a core and dense branches radiating outward from the core, appears to be more efficient than other polyanionic compounds such as iota-carrageenan and heparin. Can provide significant benefits, wherein iota-carrageenan and heparin are linear vulcanized molecules with a molecular weight distribution. The authors are unaware of data showing that iota-carrageenan is virucidal, whereas heparin has been shown to lack an irreversible, virucidal interaction with HSV virion components (Ghosh et al., 2009). Example 14: Biocompatibility study of rat SPL7013

Biocompatibility studies of SPL7013 in Formulation Variant 4 were performed in rats (data not shown).

The product was tested to evaluate its cytotoxic effect in Balb/c 3T3 cells. In conclusion, the solution of 5 mg/ml SPL7013 was not cytotoxic.

The product was tested in 10 albino guinea pigs to evaluate its sensitizing properties after intradermal and topical administration after challenge after 14 days. In conclusion, no macroscopic skin reactions attributable to sensitization were recorded after this challenge period, and the product was not classified as a skin sensitizer according to ISO 10993-10.

The product was administered to 3 female Sprague Dawley rats via the intranasal route at a dose of 0.1 ml per nostril four times a day for 14 days. No deaths were observed, no clinical symptoms associated with administration of the test product were observed, no erythema or edema was recorded on the treatment site, and body weight remained normal. There was no evidence of inflammatory changes or effects on epithelial cells. In conclusion, the test product was well tolerated and did not induce any evidence of irritation as assessed according to ISO 10993-10. Example 15: Clinical study

A clinical study was conducted on 40 patients who received 1% SPL7013 in Formulation Variant 4 or placebo (Formulation 4) administered in each nostril by a spray device four times a day for 14 days. Give 100 μl. There were no serious adverse events and the formulation was generally well tolerated with minimal irritation. Example 16: Summary

The experiments described herein have shown that SPL7013 exhibits potent antiviral activity against multiple SARS-CoV-2 strains and in different cell lines with very high SI. At the concentration of SPL7013 in this nasal spray (10 mg/mL), the reduction in infectious virus was >5 log ₁₀ (>99.999%) in Vero E6 cells and >3 in Calu-3 cells log ₁₀ (>99.9%). Studies examining the kinetics of virucidal activity revealed that dose-dependent inactivation of SARS-CoV-2 was observed upon exposure of SPL7013 to the virus for as little as 5 seconds.

Those skilled in the art will appreciate that various changes and/or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Accordingly, the present embodiments are to be considered in all respects to be illustrative and not restrictive.

All works discussed and/or cited herein are incorporated herein in their entirety.

This application claims that the Australian Provisional Application No. 2020901194 filed on April 15, 2020 and entitled "Coronavirus Infection Prevention Method" and the August 21, 2020 application entitled "Coronavirus Infection Prevention Method" ” of the Australian Provisional Application No. 2020902993, and the priority of the Australian Provisional Application No. 2020904246, filed on November 17, 2020, and entitled “Methods for the Prevention of Respiratory Syncytial Virus Infection”, the entire contents of which are hereby incorporated by reference Incorporated herein.

All works discussed and/or cited herein are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles, etc. that has been included in this specification is solely for the purpose of providing context for the present invention. It should not be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention simply because it existed before the priority date of each of the claims of this application.

The steps, features, integers, compositions and/or compounds disclosed herein or referred to in the specification of this application, individually or collectively, are in the form of two or more of said steps or features. any or all combinations. references

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Figure 1 provides the names and structures of the macromolecules SPL-7674, SPL-7615, SPL-7673, BAI-7021, BRI-2999, and BRI-2992.

Figure 2 shows the antiviral efficacy, as measured by the reduction in cytopathic effect (CPE) in virus-infected cells, and as SPL7013 against SARS-CoV-2 (hCoV) in Vero E6 cells -19/Australia/VIC01/2020) selectivity measure for infection. The markers are 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 provides a dose-response curve of the antiviral activity as measured by the reduction in CPE at day 4 of SPL7013 against SARS-CoV-2 (hCoV-19/Australia/VIC01/2020) replication in Vero E6 cells , and was measured as cell viability as a percentage of the cell control. A. Infected cell cultures one hour prior to infection - Assay 1 (left panel) and Assay 2 (right panel). B. Infected cell cultures one hour post infection - Assay 1 (left panel) and Assay 2 (right panel).

Figure 4: A. Virus and SPL7013 lines were mixed for one hour prior to infection of cell cultures. It indicates _EC50 value and _CC50 value and selectivity index. Dots and error bars represent the mean ± SD of triplicate readings. B. The amount of virus secreted into the supernatant at 8 hours post infection was determined by TCID ₅₀ . SPL7013 (0.345 mg/mL; squares), remdesivir (5 µM; grey triangles), hydroxychloroquine sulfate (15 µM; circles), and SARS-CoV-2 only (hCoV-19/Australia/VIC01/ 2020) (black triangles). Each point on the graph represents the viral titer that exists after one replication cycle following virus infection after compound addition at the indicated times. The infectious virus titers of SPL7013 were below the lower limit of detection (LLOD) at all time points.

Figure 5 shows the dose-response and cytotoxicity analysis of the SARS-CoV-2 (2019-nCoV/USA-WA1/2020) antiviral activity of SPL7013 in cells as measured by infectious virus release at day 4 post-infection ( A. was in Vero E6 cells and B. was in Calu-3 cells, as measured by Log ₁₀ pfu/mL). Dots and error bars represent the mean ± SD of triplicate readings.

Figure 6 provides the virucidal efficacy of SPL7013 against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) as a reduction in mean infectious virus at 96 hours post-infection in Vero E6 cells (Log ₁₀ pfu/ mL) measured.

Figure 7 provides the virucidal efficacy of SPL7013 against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) as a reduction in mean infectious virus at 16 hours post-infection in Vero E6 cells (Log ₁₀ pfu/ mL) measured. SPL7013 (0.0046 to 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. Treated virus was added to Vero E6 cells and the amount of infectious virus in the supernatant was determined by plaque assay 16 hours post infection. A. Dose response of ^SPL7013 virucidal activity using 104 pfu/mL viral inoculum. Dots and error bars represent the mean ± SD of triplicate readings. B. Log ₁₀ reduction in viral load (relative to baseline) with 10 mg/mL SPL7013. Bars and error bars represent the mean ± SD of triplicate readings.

Figure 8: A. Shows the evaluation of SPL7013 against SARS-CoV-2 infection in hACE2 transgenic mice after 7 days of nasal administration. B. Inhibition of SARS-CoV, MERS-CoV, and lentiviral infection expressing SARS-CoV-2 spikes in Vero E6 cells is shown by SPL7013.

Figure 9: A. Shows inhibition of human respiratory syncytial virus (HRSV) in Hep-2 cells after pre- and post-infection treatment with SPL7013. B. Shows the cytotoxicity of HRSV on Hep-2 cells before and after treatment with SPL7013.

Figure 10 shows the antiviral efficacy of SPL7013 and iota-carrageenan against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) as shown in human bronchial epithelial primary cells Measured by the reduction (ng/mL) of the nucleoprotein coat in primary cells, HBEpC) at day 4 post-infection. 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 before infection . A. Dose response showing antiviral activity of SPL7013. Dots and error bars represent the mean ± SD of triplicate readings. B. Dose response showing antiviral activity of carrageenan. A dot represents a repetition. The dotted line indicates the level of inhibition achieved by the SARS-CoV-2 pAb positive control.

Figure 11: A. Line RT-qPCR results in Vero E6 cells infected with SARS-CoV-2 Slovakia/SK-BMC5/2020 virus after treatment with SPL7013. All experiments were repeated independently (n=2). Results are expressed as a percentage of RNA expression compared to the infected, untreated control cells. B. Fluorescent foci of infection in Vero E6 cells infected with SARS-CoV-2 Slovakia/SK-BMC5/2020 virus after treatment with SPL7013. All experiments were repeated independently (n=2). Titers were determined using immunofluorescence focal analysis.

Figure 12: A. Viability of healthy Vero E6 cells following treatment with SPL7013. Cells were preincubated with SPL7013 for 1 hour. All experiments were repeated independently (n=2). Survival was assessed using the MTS survival assay. B. Viability of Vero E6 cells infected with SARS-COV-2 Slovakia/SK-BMC5/2020 after treatment with SPL7013. Cells were pre-incubated with SPL7013 for 1 hour prior to infection with the virus. The virus was incubated with the cells for 48 hours. All experiments were repeated independently (n=2). Survival was assessed using the MTS survival assay.

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Claims (46)

A use of an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in the manufacture of a medicament, the medicament For preventing or reducing the likelihood of coronavirus (Coronavirus, CoV) infection in a human individual, or preventing or reducing the likelihood or severity of a symptom of a CoV infection in a human individual, wherein the macromolecule comprises a 3 to 5 generations of polylysine dendrimers having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer, wherein the sulfonic acid-containing The acid or sulfonate-containing moiety is selected from the group consisting _of : -NH-( _CH2 ) _nSO3- ^, _- ( _CH2 ) _nSO3- ^,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w /w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. A use of an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in the manufacture of a medicament, the medicament For reducing the severity and/or duration of a coronavirus (CoV) infection in a human individual, or preventing or reducing viral shedding in a human individual infected with a CoV, wherein the macromolecule comprises A 3 to 5 generation polylysine dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surface groups of the dendrimer, wherein the containing The sulfonic acid or sulfonate-containing moiety is selected from the group consisting _of : -NH-( _CH2 ) _nSO3- ^, _- ( _CH2 ) _nSO3- ^,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. An effective amount of a macromolecule or a pharmaceutically acceptable salt thereof or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier is manufactured for the treatment of a human Use of a medicament for coronavirus (CoV) infection in an individual, wherein the macromolecule comprises a 3 to 5 generation polylysine dendrimer having one or more sulfonic acid- or sulfonate-containing moieties one or more surface groups attached to the dendrimer, wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting _of : -NH-( _CH2 ) _nSO3 ^- , -(CH ₂ ) _n SO ₃ ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. A use of an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in the manufacture of a medicament, the medicament For the prevention or reduction of the likelihood of a coronavirus (CoV) infection and/or a Respiratory syncytial virus (RSV) infection, or to prevent or reduce a symptom of a CoV and/or a RSV infection in a human subject The likelihood or severity of wherein the macromolecule comprises a 3 to 5 generation polylysine dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer wherein the sulfonic acid- or sulfonate _- containing moiety is selected from the group consisting of: -NH-( _CH2 ) _nSO3- , ^- ( _CH2 ) _n SO ₃ ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. The use of claim 1 or claim 4, wherein the symptoms of CoV infection are selected from one or more of the following: fever, cough, sore throat, shortness of breath, viral shedding, respiratory insufficiency, runny nose, nasal congestion, bronchial inflammation, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, and acute respiratory distress syndrome (ARDS); and Wherein the symptoms of RSV infection are selected from one or more of the following: nasal congestion or runny nose, decreased appetite, cough, mucus (yellow, green or gray mucus) when coughing, sneezing, sore throat, mild headache, fever , wheezing, shortness of breath or difficulty breathing, bluish skin (cyanosis), symptoms of severe asthma in individuals with asthma, acute bronchitis, severe bronchitis, airway inflammation, airway obstruction, chronic obstructive pulmonary disease, heart obstruction , bacteremia, pneumonia, acute otitis media, and recurrent otitis media. A use of an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in the manufacture of a medicament, the medicament For reducing the severity and/or duration of a coronavirus (CoV) infection and/or a respiratory fusion virus (RSV) infection in a human individual, or to prevent or reduce the risk of an infection with a CoV and/or a RSV Viral shedding in human subjects, wherein the macromolecule comprises a 3 to 5 generation polylysine dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer one or more surface groups of , wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of: -NH-(CH ₂ ) _n SO ₃ ^- , -(CH ₂ ) _n SO ₃ ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. An effective amount of a macromolecule or a pharmaceutically acceptable salt thereof or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier is manufactured for the treatment of a human Use of a medicament for a coronavirus (CoV) infection and/or a respiratory syncytial virus (RSV) in an individual, wherein the macromolecule comprises a 3 to 5 generation polylysine dendrimer having one or more A sulfonic acid- or sulfonate-containing moiety is attached to one or more surface groups of the dendrimer, wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of: -NH-(CH ₂ ) _n SO ₃ ^- , -(CH ₂ ) _n SO ₃ ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. The use of any one of claim 1 to claim 3 and claim 5 to claim 7, wherein the composition comprises about 0.01% w/w to about 0.1% w/w rheology modifier Carbopol 974 (Carbopol 974). The use of any one of claim 1 to claim 3 and claim 5 to claim 7, wherein the composition comprises about 0.05% w/w to about 0.1% w/w rheology modifier Carbopol 974. The use of any one of claims 1 to 3 and claims 5 to 7, wherein the composition comprises about 0.05% w/w of the rheology modifier Carbopol 974. The use of any one of claim 1 to claim 3 and claim 5 to claim 7, wherein the CoV is selected from one or more of the following: (a) an alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus; (b) Severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome-associated coronavirus (SARS-CoV), and Middle East respiratory syndrome-associated coronavirus (MERS-CoV), or a subtype or variant thereof; and (c) SARS-CoV-2 or a subtype or variant thereof. The use of any one of claims 1 to 3 and claims 5 to 7, wherein administering comprises administering locally or administering to the respiratory tract. The use of claim 12, wherein administration to the respiratory tract comprises administration to one or more of the following: upper respiratory tract, lower respiratory tract, nasal mucosa, nasal cavity, oral cavity, sinuses, throat, pharynx, larynx, turbinates, nasopharynx , oropharynx, trachea, main bronchi, and lungs. The use of any one of claim 1 to claim 3 and claim 5 to claim 7, wherein the composition comprises one or more of the following features: (a) about 0.5% to about 5% by weight of the macromolecule or a pharmaceutically acceptable salt thereof; (b) about 1% by weight of the macromolecule or a pharmaceutically acceptable salt thereof; and (c) the effective amount is about 0.1 to about 5 mg per dose; and (d) The effective amount is about 1 mg per dose. The use of any one of claims 1 to 3 and claims 5 to 7, wherein the macromolecule or a pharmaceutically acceptable salt thereof is administered in a nasal spray or an oral spray. The use of any one of claim 1 to claim 3 and claim 5 to claim 7, wherein the macromolecule or a pharmaceutically acceptable salt thereof is administered 1 to 8 times a day. The use of any one of claims 1 to 3 and claims 5 to 7, wherein the macromolecule or a pharmaceutically acceptable salt thereof is administered continuously for about 1 to about 2 weeks, or about 1 to About 3 weeks, or less than 30 days. A composition for preventing or reducing the likelihood of a coronavirus (CoV) infection in a subject, or treating a coronavirus (CoV) infection in a subject; reducing the severity of a CoV infection in a subject sex and/or duration; preventing or reducing viral shedding in an individual infected with a CoV; or reducing transmission of a CoV in a population, wherein the composition comprises: an effective amount of a macromolecule or a pharmaceutical acceptable salt, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having One or more sulfonic acid- or sulfonate-containing moieties are attached to one or more surface groups of the dendrimer, wherein the sulfonic acid- or sulfonate-containing moieties are selected from the group consisting of Groups: -NH-(CH ₂ ) _n SO ₃ ^- , -(CH ₂ ) _n SO ₃ ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w rheology modifier. A composition comprising: an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer and Carbopol 974 or Carbopol 971 one or more surface groups, wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of: -NH-( _CH2 ) _nSO3- ^, _- ( _CH2 ) _{nSO 3} ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises Carbopol 974 or Carbopol 971 and The w/w ratio of the macromolecules is from about 1:20 to about 1:10. A composition comprising: an effective amount of a macromolecule or a pharmaceutically acceptable salt thereof, or a composition comprising the macromolecule or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to one or more surfaces of the dendrimer and Carbopol 974 group, wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of: -NH-(CH ₂ ) _n SO ₃ ^- , -(CH ₂ ) _n SO ₃ ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w Carbopol 974. The composition of any one of claim 18 to claim 20, wherein the composition is a nasal bioadhesive composition. The composition of any one of claims 18 to 20, wherein the composition additionally comprises one or more pharmaceutical agents selected from one or more of the following: (a) an antiviral active agent, a vaccine, an immunomodulatory agent, an antibacterial agent, an anti-inflammatory agent and a nasal bioadhesive; and (b) an antiviral active agent selected from one or more of: i) an antibiotic or an additional dendrimer; and ii) a carrageenan, GM-CSF, IL-6R, CCR5, the S protein of MERS, and drugs including the following: ribavirin, tilorone, favipiravir, rapinavir (lopinavir/ritonavir) ( Kaletra (lopinavir/ritonavir), Prezcobix (darunavir/cobicistat), nelfinavir, mycophenolic acid, galidevir (Galidesivir), Actemra, OYA1, BPI-002, Ifenprodil, APN01, EIDD-2801, baricitinib, camostat mesylate , Lycorine (lycorine), Brilacidin (Brilacidin), BX-25, an interferon (eg IFNβ), chloroquine and azithromycin (azithromycin). The composition of any one of claim 18 to claim 20, wherein the composition comprises one or more of the following: glycerin, propylene glycol, methylparaben, propylparaben, chlorinated benzenediol Methylammonium, EDTA, and Sodium Hydroxide. The composition of any one of claim 18 to claim 20, wherein the composition comprises: (a) a preservative selected from one or more of the following: methylparaben, propylparaben, and benzalkonium chloride (b) an excipient selected from one or more of the following: glycerol, propylene glycol, EDTA; and (c) a pH modifier. The composition of any one of claim 18 to claim 20, wherein the composition comprises one or more of the following features: (a) a pH of about 3.5 to about 7.5, or a pH of about 5.5 to about 6.5; (b) a viscosity of about 1 to about 10 cP; (c) is suitable for administration in a device, wherein the device is selected from the group consisting of: a nasal spray, an oral spray, an inhaler, a nebulizer, a nasal wash or an oral cleaner; (d) is present in or applied to protective clothing or a cleaning article; and (e) The cleaning product of (d), wherein the cleaning product is selected from a paper towel, a surgical field preparation spray or a cleaning solution. The composition of claim 25, wherein the nebulizer, inhaler or nebulizer comprises a means for generating particles having a size of about 0.1 μm to about 100 μm, and/or wherein less than 6% of the particles have about 10 µm or smaller size. The composition of any one of claim 18 to claim 20, wherein the composition contains more than 90%, or more than 92%, or more than 95%, or more than 99%, or more than 99.9% of SARS-CoV2 or its A subtype or variant is inactive. The composition of claim 27, wherein exposure to the composition for about one minute results in more than 90%, or more than 92%, or more than 95%, or more than 99%, or more than 99.9% SARS-CoV2 or a subtype thereof or variant inactivation. The composition of any one of claim 18 to claim 20, wherein the macromolecule or a pharmaceutically acceptable salt thereof is a dendrimer comprising from 3 to 5 generations of lysine building blocks, and these sulfonic acid-containing or sulfonate-containing moieties are naphthyldisulfonate moieties. The composition of any one of claim 18 to claim 20, wherein the sulfonic acid-containing or sulfonate-containing moiety is selected from the group consisting of:

,

,

,

or

. The composition of claim 29 or claim 30, wherein the sulfonic acid-containing moiety is

. The composition of any one of claims 18 to 20, wherein the sulfonic acid- or sulfonate-containing moiety is attached to the dendrimer terminal amine group by a linker. The composition of claim 32, wherein the linker is an alkylene or alkenylene group, wherein one or more non-adjacent carbon atoms are optionally replaced by an oxygen or sulfur atom, or the linker is A group -X ₁ -(CH ₂ ) _q -X ₂ -, wherein X ₁ and X ₂ are independently selected from -NH-, -C(O)-, -O-, -S- and -C (S), and q is 0 or an integer from 1 to 10, and wherein one or more non-adjacent ( _CH2 ) groups may be replaced by -O- or -S-. The composition of claim 33, wherein the linker is #-O- _CH2 -C(O)-*, wherein # represents attachment to the sulfonic acid-containing moiety, and * represents attachment to the dendrimer the terminal amine group. The composition of any one of claims 18 to 20, wherein the dendrimer is

and at least 50% of which are R series

, and wherein the pharmaceutically acceptable salt is a sodium salt. The composition of any one of claims 18 to 20, wherein the composition is a nasal moisture barrier dressing. A device for delivering a nasal, oral or pulmonary composition comprising a macromolecule or a pharmaceutically acceptable salt thereof, or a medicament comprising the macromolecule or a pharmaceutically acceptable salt thereof A composition of an acceptable carrier above, wherein the macromolecule comprises a 3 to 5 generation dendrimer having one or more sulfonic acid- or sulfonate-containing moieties attached to the dendrimer or more surface groups, wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of: -NH-( _CH2 ) _nSO3- ^, _- ( _{CH2 ) nSO3} ^- ,

,

,

,

as well as

, wherein n is 0 or an integer from 1 to 20, m is an integer of 1 or 2, and p is an integer from 1 to 3, and wherein the composition comprises from about 0.01% w/w to about 1% w/w, or about 0.01% w/w to about 0.1% w/w, or about 0.05% w/w to about 0.1%, or about 0.05% w/w Carbopol 974. The device of claim 37, wherein the device is a nasal delivery device or an oral delivery device for delivering a spray. The device of claim 37, wherein the device is an inhaler or a nebulizer. The use of any one of claim 1 to claim 3 or claim 5 to claim 7, wherein the macromolecule or a pharmaceutically acceptable salt thereof is a dendrimer, wherein the dendrimer comprises from 3 to 5 generations of lysine building blocks, and the sulfonic acid- or sulfonate-containing moieties are naphthyldisulfonate moieties. The use of any one of claim 1 to claim 3 or claim 5 to claim 7, wherein the sulfonic acid- or sulfonate-containing moiety is selected from the group consisting of:

,

,

,

or

. The use of claim 40 or claim 41, wherein the sulfonic acid-containing moiety is

. The use of any one of claims 1 to 3 or 5 to 7, wherein the sulfonic acid- or sulfonate-containing moiety is attached to the dendrimer terminal amine via a linker base group. The use of claim 43, wherein the linker is an alkylene or alkenylene group, wherein one or more non-adjacent carbon atoms are optionally replaced by an oxygen or sulfur atom, or the linker is a The group -X ₁ -(CH ₂ ) _q -X ₂ - wherein X ₁ and X ₂ are independently selected from -NH-, -C(O)-, -O-, -S- and -C(S ), and q is 0 or an integer from 1 to 10, and wherein one or more non-adjacent ( _CH2 ) groups may be replaced by -O- or -S-. The use of claim 33, wherein the linker is #-O- _CH2 -C(O)-*, wherein # represents attachment to the sulfonic acid-containing moiety, and * represents attachment to the dendrimer Terminal amine group. The use of any one of claims 1 to 3 or claims 5 to 7, wherein the dendrimer is

and at least 50% of which are R series

, and wherein the pharmaceutically acceptable salt is a sodium salt.

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