Nitric oxide releasing nasal compositions and methods of using same

Abstract

The present invention relates generally to nitric oxide (NO) releasing nasal compositions and methods of use thereof.

Description

[Technical field]

[0001] The present invention relates generally to nitric oxide (NO)-releasing nasal compositions and methods of use thereof. [Background technology]

[0002] Highly pathogenic coronaviruses have been increasing in incidence over the past two decades. The first, severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in 2003, followed by Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012. The ongoing severe acute respiratory syndrome coronavirus two (SARS-CoV-2) epidemic is expanding the devastating global toll caused by highly pathogenic and infectious coronaviruses.

[0003] Classified as a betacoronavirus, SARS-CoV-2 is an enveloped, single, positive-stranded RNA virus that belongs to the Coronaviridae family. Coronaviruses typically encode four structural proteins, including the envelope (E) protein, membrane (M) protein, nucleocapsid (N) protein, and spike (S) protein. The E and M proteins are involved in virus assembly, and the N protein encapsulates the viral RNA into the virion particle, whereas the S protein is important for receptor binding, allowing the virion particle to enter the host cell and spread infection. The human angiotensin-converting enzyme 2 (ACE2) receptor has been identified as the primary host cell access point for both SARS-CoV and SARS-CoV-2. The nasal epithelium is speculated to be the primary target for viral entry of SARS-CoV-2. Specifically, ACE2 expression throughout the respiratory system has been shown to follow a gradient that is highest in nasal tissues and lower down the respiratory tract. Due to the high expression of ACE2, the nasal cavity is hypothesized to serve as an initial site of SARS-CoV-2 infection. Indeed, in vitro infection with SARS-CoV-2 is prevalent in nasal epithelial cells. Clinically, nasal and oropharyngeal samples from the upper respiratory tract have the highest viral loads in both asymptomatic and symptomatic individuals infected with SARS-CoV-2. Outside of the respiratory tract, ACE2 expression in numerous tissues reflects the various symptoms reported in association with COVID-19 (e.g., gastrointestinal, vascular). As the upper respiratory tract is a large reservoir of viral load, there is a high probability of transmission between individuals and of virus-laden droplets being aspirated into the lower respiratory tract within an individual, potentially resulting in lung damage (e.g., acute respiratory distress syndrome (ARDS)). Summary of the Invention [Problem to be solved by the invention]

[0004] A rapid increase in infectivity and associated increases in mortality and morbidity have accompanied each outbreak. The increasing frequency of such highly pathogenic coronaviruses highlights the need for treatments, such as antiviral treatments, to treat current and future emerging pathogens. [Means for solving the problem]

[0005] One aspect of the invention is directed to a composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and a buffer configured to maintain the pH of the composition in the range of from about 3, from about 3.5, from about 4 or 4.5, to about 5, to about 5.5, to about 6, to about 6.5, to about 7, to about 7.5, to about 8 or to about 8.5.

[0006] Another aspect of the present invention is directed to a composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and a buffering agent, wherein the buffering agent comprises a salt form of citric acid (e.g., a citrate salt) and / or citric acid in an amount of at least 100 mM.

[0007] An additional aspect of the invention is a kit comprising a first composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and a second composition comprising a buffering agent configured to maintain a pH of a composition comprising the first composition and the second composition in the range of from about 3, from about 3.5, from about 4 or from about 4.5 to about 5, to about 5.5, to about 6, to about 6.5, to about 7, to about 7.5, to about 8 or to about 8.5, wherein the first composition and the second composition are stored separately within the kit.

[0008] A further aspect of the invention is a kit comprising a first composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and a second composition comprising a salt form of citric acid (e.g., a citrate salt) and / or a buffer containing citric acid in an amount of at least 100 mM, wherein the first composition and the second composition are stored separately within the kit.

[0009] Another aspect of the invention is a method of treating and / or preventing an infection in a subject, the method comprising administering to said subject a composition of the invention. In some embodiments, the infection is a viral infection and / or an infection caused by SARS-CoV-2.

[0010] It should be noted that aspects described with respect to one embodiment may be incorporated within a different embodiment, even if not specifically described therein.

[0011] These and other aspects of the invention will now be described in more detail with respect to other embodiments described herein. It should be understood that the invention may be embodied in different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 is a graph showing the percent of initial body weight of 5-week-old Golden Syrian hamsters challenged with SARS-CoV-2 and treated with Berdazimer Sodium (n=10 for the hamster group and n=5 for the normal control group). Treatment with Berdazimer Sodium did not prevent weight loss after infection. Treatment with EIDD-2801 at a dose of 200 mg / kg / day prevented weight loss after infection (*P<0.01, compared to the placebo-treated group). [Diagram 2]Figure 2 is a graph showing lung viral titers in 5-week-old golden Syrian hamsters after challenge with SARS-CoV-2 and treatment with verdazimer sodium. Treatment with verdazimer sodium did not significantly reduce lung viral titers in animals infected with SARS-CoV-2. Treatment with EIDD-2801 reduced lung viral titers at day 6 post-infection (***P<0.0001, compared to the placebo-treated group). [Diagram 3] Figure 3 is a graph showing viral titers in nasal tissues of 5-week-old golden Syrian hamsters after challenge with SARS-CoV-2 and treatment with verdazimer sodium. Treatment with verdazimer sodium did not significantly reduce viral titers in nasal tissues in animals infected with SARS-CoV-2. Treatment with EIDD-2801 did not significantly reduce viral titers in the nasal cavity after infection. [Figure 4] Figure 4 is a graph showing viral titers in oropharyngeal swabs of 5-week-old golden Syrian hamsters challenged with SARS-CoV-2 and treated with verdazimer sodium. No significant differences in viral titers in oropharyngeal swabs were observed by one-way ANOVA. [Diagram 5] Figure 5 is a graph showing lung weights of 5-week-old golden Syrian hamsters challenged with SARS-CoV-2 and treated with verdazimer sodium. Lung weights were not statistically different between treatment groups when compared by one-way ANOVA. [Figure 6]Figure 6 is a graph showing percent initial body weight of 5-week-old Golden Syrian hamsters after being treated with verdazimer sodium and cohabiting with SARS-CoV-2-infected hamsters (n=2 hamsters per donor group and n=4 hamsters per naive group). Placebo-treated naive animals were protected from weight loss when donor animals were treated with 2 mg / mL verdazimer sodium. Treatment with 2 mg / mL verdazimer sodium prevented weight loss after exposure to SARS-CoV-2-infected hamsters when donor animals were also treated with 2 mg / mL verdazimer sodium (*P<0.05, ***P<0.001 compared to verdazimer sodium-treated donor animals). [Figure 7] Figure 7 is a graph showing lung viral titers on day 4 in hamsters treated with verdazimer sodium and exposed to SARS-CoV-2-infected hamsters. Animals were housed on days 1-3 of the study. Treatment with verdazimer sodium significantly reduced lung viral titers in naive animals housed with infected animals also treated with verdazimer sodium (****P<0.0001 compared to placebo-treated naive animals). [Figure 8] Figure 8 is a graph showing viral titers in nasal tissues of hamsters treated with verdazimer sodium and exposed to SARS-CoV-2-infected hamsters on day 4. Animals were housed on days 1-3 of the study. Treatment with verdazimer sodium did not significantly reduce nasal tissue viral titers in animals housed with SARS-CoV-2-infected hamsters. [Figure 9] Figure 9 is a graph showing the viral titers of oropharyngeal swaps in 5-week-old golden Syrian hamsters after being treated with verdazimer sodium and cohabiting with SARS-CoV-2-infected hamsters. Treatment with verdazimer sodium at a dose of 2 mg / mL did not significantly reduce oropharyngeal swap titers in hamsters cohabiting with SARS-CoV-2-infected hamsters. [Figure 10] Figure 10 is a graph showing lung weights of 5-year-old golden Syrian hamsters treated with verdazimer sodium and after cohabitation with SARS-CoV-2 infected animals. Lung weights were not statistically different between groups when compared by one-way ANOVA. [Figure 11] Figure 11 is a graph showing percent initial body weight of 16-week-old Golden Syrian hamsters after treatment with verdazimer sodium (8 mg / mL) and cohabitation with SARS-CoV-2 infected hamsters (n=2 hamsters per donor group and n=4 hamsters per naive group). No statistically significant difference in weight loss was observed when hamsters were treated with verdazimer sodium (8 mg / mL) and then cohabited with SARS-CoV-2 infected hamsters. [Figure 12] Figure 12 is a graph showing percent initial body weight of 16-week-old Golden Syrian hamsters after treatment with verdazimer sodium (2 mg / mL or 1 mg / mL) and cohabitation with SARS-CoV-2 infected hamsters (n=2 hamsters per donor group and n=4 hamsters per naive group). No statistically significant difference in weight loss was observed after treatment with verdazimer sodium (2 mg / mL or 1 mg / mL) and cohabitation with SARS-CoV-2 infected hamsters. [Figure 13] Figure 13 is a graph showing lung viral titers on day 4 in hamsters treated with verdazimer sodium (8 mg / mL) and exposed to SARS-CoV-2 infected hamsters. Animals were housed on study days 1-3. Treatment with verdazimer sodium (8 mg / mL) significantly reduced lung viral titers in naive animals housed with infected animals also treated with verdazimer sodium (8 mg / mL) (****P<0.0001 compared to placebo-treated naive animals). [Figure 14]FIG. 14 is a graph showing lung viral titers on day 4 in hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and exposed to hamsters infected with SARS-CoV-2. Cohabitation of animals occurred on days 1-3 of the study. Treatment with verdazimer sodium (2 mg / mL) significantly reduced lung viral titers in naive animals housed with infected animals also treated with verdazimer sodium (2 mg / mL). Treatment with verdazimer sodium (1 mg / mL) did not significantly reduce lung viral titers in naive animals housed with infected animals also treated with verdazimer sodium (1 mg / mL) (****P<0.0001 compared to placebo-treated naive animals). [Figure 15] Figure 15 is a graph showing viral titers in nasal tissues on day 4 of hamsters treated with verdazimer sodium (8 mg / mL) and exposed to SARS-CoV-2-infected hamsters. Cohabitation of animals occurred on days 1-3 of the study. Treatment with verdazimer sodium (8 mg / mL) did not significantly reduce viral titers in nasal tissues in animals cohabited with SARS-CoV-2-infected hamsters. [Figure 16] Figure 16 is a graph showing viral titers in nasal tissues on day 4 of hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and exposed to SARS-CoV-2-infected hamsters. Cohabitation of animals occurred on days 1-3 of the study. Treatment with verdazimer sodium (2 mg / mL or 1 mg / mL) did not significantly reduce viral titers in nasal tissues in animals cohabited with SARS-CoV-2-infected hamsters. [Figure 17] Figure 17 is a graph showing lung weights of 16-week-old golden Syrian hamsters treated with veratizomer sodium (8 mg / mL) and housed with SARS-CoV-2-infected animals. Lung weights were not statistically different between groups when compared by one-way ANOVA. [Figure 18]Figure 18 is a graph showing lung weights of 16-week-old golden Syrian hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and housed with SARS-CoV-2-infected animals. Lung weights were not statistically different between treatment groups when compared by one-way ANOVA. [Figure 19] FIG. 19 is a graph showing the percent of initial body weight in hamsters treated once daily with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. No statistically significant protection from weight loss was observed in hamsters treated once daily with verdazimer sodium at 2, 4 or 8 mg / mL. Although not statistically significant, hamsters treated with verdazimer sodium at 8 mg / mL lost less weight compared to placebo-treated animals. A similar trend was observed in hamsters treated with EIDD-2801 at a dose of 500 mg / kg / day. [Figure 20] Figure 20 is a graph showing lung viral titers on days 3 and 6 in hamsters treated once daily with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was started 24 hours prior to infection. Treatment with verdazimer sodium at a dose of 4 mg / mL significantly reduced lung viral titers on day 3 post-infection. Treatment with EIDD-2801 at a dose of 500 mg / kg / day significantly reduced lung viral titers on day 3 post-infection (*P<0.05, **P<0.01, compared to placebo-treated animals). [Figure 21]Figure 21 is a graph showing viral titers in nasal tissues on days 3 and 6 of hamsters treated once daily with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was started 24 hours prior to infection. Treatment with verdazimer sodium at a dose of 2, 4 or 8 mg / mL once daily did not significantly reduce viral titers in nasal tissues. Treatment with EIDD-2801 at a dose of 500 mg / kg / day did not significantly reduce viral titers in nasal tissues. [Figure 22] Figure 22 is a graph showing lung weights on days 3 and 6 of hamsters treated once daily with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Treatment with verdazimer sodium at doses of 2, 4 or 8 mg / mL once daily did not significantly reduce lung weights. Treatment with EIDD-2801 at a dose of 500 mg / kg / day did not significantly reduce lung weights. [Figure 23] Figure 23 is a graph showing the percent of initial body weight in hamsters treated with verdazimer sodium (2 mg / mL) twice daily and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Treatment with verdazimer sodium at a dose of 2 mg / mL twice daily did not prevent weight loss in SARS-CoV-2 infected hamsters. Although not statistically significant, hamsters treated with EIDD-2801 at a dose of 500 mg / kg / day lost less weight than the placebo-treated group. [Figure 24]Figure 24 is a graph showing lung viral titers on days 3 and 6 in hamsters treated twice daily with verdazimer sodium (2 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Treatment with verdazimer sodium at a dose of 2 mg / mL twice daily did not significantly reduce lung viral titers on days 3 or 6 post-infection. Treatment with EIDD-2801 at a dose of 500 mg / kg / day significantly reduced lung viral titers on day 3 post-infection (**P<0.01, compared to placebo-treated animals). [Diagram 25] Figure 25 is a graph showing viral titers in nasal tissues on days 3 and 6 of hamsters treated twice daily with verdazimer sodium (2 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Treatment with verdazimer sodium twice daily at a dose of 2 mg / mL significantly reduced viral titers in nasal tissues on day 3 post-infection. Treatment with EIDD-2801 at 500 mg / kg / day significantly reduced viral titers in nasal tissues on day 3 post-infection (**P<0.01, compared to placebo-treated animals). [Figure 26] Figure 26 is a graph showing lung weights on days 3 and 6 in hamsters treated twice daily with verdazimer sodium (2 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Lung weights were significantly affected on day 6 by twice daily treatment with verdazimer sodium and EIDD-2801 (**P<0.01, ***P<0.0001 compared to placebo-treated animals).

[0013] The invention will now be described herein below with reference to the accompanying drawings and examples in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented or all the features that may be added to the invention. For example, features illustrated with respect to one embodiment may be incorporated in other embodiments, and features illustrated with respect to a particular embodiment may be omitted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any one or combination of features described herein may be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, which do not depart from the invention. Thus, the following description is intended to illustrate some specific embodiments of the invention, and is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the present invention in this specification are for the purpose of describing particular embodiments only and are not intended to limit the present invention.

[0015] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is set forth.

[0016] Unless the context indicates otherwise, it is specifically intended that the various features of the present invention described herein can be used in any combination.Moreover, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be eliminated or omitted.For example, when the present specification states that a composition comprises component A, component B and component C, it is specifically intended that any of A, B or C, or combinations thereof, can be omitted and removed.

[0017] As used in the detailed description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0018] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0019] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to include not only the stated value, but also ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variation of the stated value. For example, "about X" is meant to include X, where X is a measurable value, as well as ±10%, ±5%, ±1%, ±0.5%, or ±0.1% variation of X. Ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​within the range.

[0020] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y", and phrases such as "about X to Y" mean "about X to about Y".

[0021] The recitation of ranges of values ​​herein, unless otherwise stated herein, is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if each separate value were individually set forth herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0022] As used in this specification, the words "comprises" and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0023] As used herein, the transitional phrase "consisting essentially of" as applied to the compositions of the present invention means that the claims should be interpreted to include the specific materials or steps recited in the claims, and which do not materially affect one or more of the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" as used in the claims of the present invention is not intended to be interpreted as equivalent to "comprise."

[0024] As used herein, the words "increase", "increase", "enhance", "enhance", "improvement" and "improve" (as well as grammatical variations thereof) refer to an increase of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more as compared to another measurable characteristic or amount (e.g., a control value).

[0025] As used herein, the words "reduction", "reduced", "reduce", "reduce" and "decrease" (as well as grammatical variations thereof) refer to a decrease of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%, for example, compared to another measurable characteristic or amount (e.g., a control value). In some embodiments, the decrease may result in no detectable activity or amount, or essentially no decrease (i.e., an insignificant amount, e.g., less than about 10%, or even less than 5%).

[0026] According to embodiments of the invention provided herein, there is provided a nitric oxide (NO)-releasing composition. The NO-releasing composition of the invention may be a nasal composition and / or may be configured for intranasal delivery. In some embodiments, the composition of the invention comprises a nitric oxide-releasing active pharmaceutical ingredient; and a buffer configured to maintain the pH of the composition in the range of from about 3, from about 3.5, from about 4 or from about 4.5, to about 5, to about 5.5, to about 6, to about 6.5, to about 7, to about 7.5, to about 8 or to about 8.5. The buffer can include a weak acid and a salt of the weak acid, or a weak base and a salt of the weak base. In some embodiments, the buffer of the invention comprises a weak acid having at least two pKa values ​​in the range of about 2.5 to about 6.5. In some embodiments, the weak acid has a pKa value of about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, and / or about 6.5. The buffer can have a pH of from about 3, about 3.5, or from about 4, to about 4.5, to about 5, to about 5.5, or to about 6, e.g., about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, or about 6.

[0027] Exemplary buffers include, but are not limited to, citrate, acetate, phosphate, and / or maleate buffers. Acetate buffers can include acetic acid and / or a salt form of acetic acid (e.g., sodium acetate). Phosphate buffers can include phosphoric acid and / or a salt form of phosphoric acid (e.g., monobasic dihydrogen phosphate and dibasic monohydrogen phosphate). Maleate buffers can include maleic acid and / or a salt form of maleic acid (e.g., maleic acid tromethamine salt). The buffering agent in the salt form of citric acid may include citric acid and / or a salt form of citric acid (e.g., sodium citrate tribasic and / or tri sodium citrate dihydrate). In some embodiments, the compositions of the present invention include a buffering agent in the salt form of citric acid. The buffering agent in the salt form of citric acid may include a citrate ion (e.g., a tricarboxylate trianion) and / or a mono-hydrogen citrate ion.

[0028] The buffer of the present invention may have an acid and / or a corresponding salt of the acid in an amount of at least 50 mM or at least 100 mM or more. In some embodiments, the buffer has an acid and / or a corresponding salt of the acid in an amount of about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 mM or more. In some embodiments, the buffer has a concentration of the acid and / or the corresponding salt of the acid in an amount of about 50 mM to about 150 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 200 mM to about 1000 mM, about 200 mM to about 500 mM, about 250 mM to about 600 mM, or about 500 mM to about 1000 mM. In some embodiments, the buffer contains a salt form of citric acid (e.g., citrate salt) and / or citric acid in an amount of about 200 mM or more. In some embodiments, the buffer has a concentration of the acid and / or the corresponding salt of the acid in an amount that would not be expected to be tolerated by a subject and / or in an amount suitable for intranasal administration. In some embodiments, the buffer has a concentration of an acid and / or a corresponding salt of the acid in an amount that exceeds the amount typically established as being tolerated by a subject and / or in an amount that exceeds the amount typically established as being suitable for intranasal administration.

[0029] Buffers of the present invention may contain at least one additional acid (e.g., HCl) and / or base (e.g., NaOH) in an amount sufficient to adjust the pH of the buffer, for example, to a pH of from about 3, from about 3.5, or from about 4, to about 4.5, to about 5, to about 5.5, or to about 6. In some embodiments, the buffer has a pH of about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, or about 6.

[0030] In some embodiments, the buffer is a citric acid salt buffer. The citric acid salt buffer may include a citric acid salt (e.g., citrate salt) and / or citric acid. Exemplary citric acid salts include, but are not limited to, trisodium citrate, potassium citrate, calcium citrate, and / or their hydrates and / or their anhydrous forms. In some embodiments, the citric acid salt buffer includes a citric acid salt and citric acid. In some embodiments, the citric acid salt is sodium citrate tribasic. In some embodiments, the citric acid salt is trisodium citrate dihydrate. In some embodiments, the buffering agent of the present invention in the form of a salt of citric acid is citric acid in an amount of about 1 w / w%, about 1.25 w / w%, about 1.5 w / w%, or about 1.75 w / w% to about 2 w / w%, about 2.25 w / w%, or about 2.5 w / w%; about 1.5 w / w%, about 1.75 w / w%, about 2 w / w%, about 2.25 w / w%, about 2.5 w / w%, about 2.75 w / w%, or about 3 w / w% to about 3.25 w / w%, about 3.5 w / w%, about 3.75 w / w%. salt forms of sodium citrate tribasic, trisodium citrate dihydrate, and / or anhydrous sodium citrate in an amount of about 4 w / w% or about 5 w / w%; optionally additional acid (e.g., an acid (e.g., HCl) and / or a base (e.g., NaOH) in an amount sufficient to adjust the pH of the buffer to from about 3, from about 3.5 to 5, or from about 4, to about 4.5, to about 5, to about 5.5, or to about 6 (e.g., about 4.5); and the balance water.In some embodiments, the buffer salt form of citric acid of the present invention comprises a salt form of sodium citrate tribasic, trisodium citrate dihydrate and / or anhydrous sodium citrate in an amount of about 1 w / w%, about 1.25 w / w%, about 1.5 w / w%, about 1.75 w / w%, about 2 w / w%, about 2.25 w / w% or about 2.5 w / w%, and about 1.5 w / w%, about 1.75 w / w%, about 2 w / w%, about 2.25 w / w%, about 2.5 w / w%, about 2.75 w / w%, about 3 w / w%, about 3.25 w / w%, about 3.5 w / w%, about 3.75 w / w% or about 4 w / w%, and has a pH of about 4.5.

[0031] The nitric oxide-releasing active pharmaceutical ingredient (API) may be suspended in the buffer and / or composition of the present invention. The NO-releasing API may be insoluble, particularly, for example, an insoluble, non-biodegradable particulate. In some embodiments, the NO-releasing API does not include and / or is not prepared from a biopolymer. In some embodiments, as nitric oxide is released from the NO-releasing API, the pH of the composition in which the NO-releasing API is present may increase.

[0032] In some embodiments, the compositions of the invention comprise a nitric oxide-releasing active pharmaceutical ingredient in an amount of about 0.1 mg / mL to about 30 mg / mL, and a buffer (e.g., a buffer in the salt form of citric acid) configured to maintain the pH of the composition in the range of about 3, about 3.5, about 4, or about 4.5 to about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, or about 8.5. In some embodiments, the buffer is configured to maintain the pH of the composition comprising the NO-releasing API at about 8.5 or less, e.g., a pH of about 8, about 7.5, about 7, about 6.5, about 6, about 5.5, about 5, or less. In some embodiments, the buffer is configured to maintain the pH of the composition comprising the NO-releasing API at a pH in the range of about 4.5 to about 5.5, or about 4.5 to about 7. In some embodiments, the NO-releasing API is present in the composition in an amount of about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 mg / mL. In some embodiments, the NO-releasing API is present in the composition in an amount of about 1 mg / mL to about 20 mg / mL. In some embodiments, the NO-releasing API is present in the composition in an amount of about 2 mg / mL, about 4 mg / mL, about 6 mg / mL, about 8 mg / mL, about 10 mg / mL, about 1 mg / mL, or about 14 mg / mL.

[0033] The compositions of the present invention may have a pH that changes over time, optionally due to the presence of the NO-releasing API in the composition and / or the release of nitric oxide from the NO-releasing API. In some embodiments, the pH of the composition is about 4.5 to about 6, depending on the initial formation of the composition (e.g., NO-releasing API together with the buffer). The buffer may be configured to maintain the pH of the composition comprising the NO-releasing API and the buffer in the range of from about 3, about 3.5, about 4 or about 4.5 to about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5; in this manner, the composition may have a pH of from about 3, about 3.5, about 4 or about 4.5 to about 8.5. In some embodiments, the composition has a pH of about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, or about 8.5. In some embodiments, the composition has a pH of about 7 or less. In some embodiments, the composition has a pH of about 5.5.

[0034] As used herein, "nitric oxide-releasing active pharmaceutical ingredient", "nitric oxide-releasing API" and "NO-releasing API" refer to a compound or other composition that provides nitric oxide to the skin (e.g., mucous membrane) and / or tissue of a subject, but is not gaseous nitric oxide. In some embodiments, the NO-releasing API is also not an acidified nitrite. In some embodiments, the NO-releasing API also includes a nitric oxide-releasing compound (hereinafter referred to as "NO-releasing compound"). The NO-releasing compound includes at least one NO donor, which is a functional group that can release nitric oxide under certain conditions.

[0035] In some embodiments, the NO-releasing compound comprises a small molecule compound comprising an NO-donor group. As used herein, a "small molecule compound" is defined as a compound having a molecular weight of less than 500 Daltons, and includes organic and / or inorganic small molecule compounds. In some embodiments, the NO-releasing compound comprises a macromolecule comprising an NO-donor group. As used herein, a "macromolecule" is defined as any compound having a molecular weight of 500 Daltons or greater. In some embodiments, the NO-releasing macromolecule comprises a crosslinked or non-crosslinked polymer, a dendrimer, a metal compound, an organometallic compound, an inorganic-based compound, and / or other macromolecular scaffold. In some embodiments, the macromolecule has a nominal diameter ranging from about 0.1 nm to about 100 μm, and may comprise an aggregate of two or more macromolecules, whereby the macromolecular structure is further modified with an NO-donor group.

[0036] In some embodiments, the NO-releasing compound comprises a diazeniumdiolate functional group as the NO donor. The diazeniumdiolate functional group can generate nitric oxide under certain conditions, for example, in response to exposure to water or protons. As another example, in some embodiments, the NO-releasing compound comprises a nitrosothiol functional group as the NO donor. The NO donor can generate nitric oxide under certain conditions, for example, in response to exposure to light. Examples of other NO donor groups include nitrosamines, hydroxylnitrosamines, hydroxylamines and hydroxyureas. In some embodiments, a combination of NO donors and / or NO-releasing compounds may be present in the composition of the present invention. In addition, the NO donor may be incorporated into and / or onto small molecules or macromolecules through covalent and / or non-covalent interactions.

[0037] The NO-releasing macromolecule may be in the form of an NO-releasing particle, for example, as described in U.S. Pat. No. 8,282,967, U.S. Pat. No. 8,962,029, or U.S. Pat. No. 8,956,658, the disclosures of which are incorporated herein by reference in their entireties. Other non-limiting examples of NO-releasing compounds include NO-releasing zeolites, such as those described in U.S. Pat. App. Pub. No. 2006 / 0269620 or U.S. Pat. App. Pub. No. 2010 / 0331968; NO-releasing metal organic frameworks (MOFs), such as those described in U.S. Pat. App. Pub. No. 2010 / 0239512 or U.S. Pat. App. Pub. No. 2011 / 0052650; "Tunable Nitric Oxide-Releasing Macromolecules Having Multiple Nitric Oxide Donors"; and ... No. PCT / US2012 / 052350, entitled "Tunable Nitric Oxide Releasing Macromolecules with Multiple Nitric Oxide Donor Structures"; NO-releasing dendrimer or metallostructures, such as those described in U.S. Patent Application Publication No. 2009 / 0214618; nitric oxide-releasing coatings, such as those described in U.S. Patent Application Publication No. 2011 / 0086234; and compounds described in U.S. Patent Application Publication No. 2010 / 0098733. The disclosures of each of the references in this paragraph are incorporated herein by reference in their entirety. Additionally, NO-releasing macromolecules can be prepared as described in International Application No. PCT / US2012 / 022048, entitled "Temperature Controlled Sol-Gel Co-Condensation," filed January 20, 2012, the disclosure of which is incorporated herein by reference in its entirety.

[0038] As an example, in some embodiments of the present invention, the nitric oxide releasing active pharmaceutical ingredient may include NO-loaded precipitated silica. The NO-loaded precipitated silica may be formed from nitric oxide donor modified silane monomers in a co-condensed siloxane network. In one embodiment of the present invention, the nitric oxide donor may be an N-diazeniumdiolate. In some embodiments of the present invention, the nitric oxide releasing active pharmaceutical ingredient may include, consist essentially of, or consist of a co-condensed siloxane network including a diazeniumdiolate (e.g., an N-diazeniumdiolate).

[0039] In some embodiments, the nitric oxide donor may be formed from aminoalkoxysilane by a pre-charging method, and the co-condensed siloxane network may be synthesized from the condensation of a silane mixture containing an alkoxysilane and an aminoalkoxysilane to form a co-condensed siloxane network modified with a nitric oxide donor. As used herein, the "pre-charging method" means that the aminoalkoxysilane is "pre-treated" or "pre-charged" with nitric oxide before co-condensation with the alkoxysilane. In some embodiments, pre-charging with nitric oxide may be achieved by chemical methods. In another embodiment, the "pre-charging" method may be used to create a co-condensed siloxane network and a material that is more densely functionalized with NO donors. In some embodiments of the present invention, the nitric oxide-releasing active pharmaceutical ingredient may comprise, consist essentially of, or consist of a co-condensed silica network synthesized from the condensation of a silane mixture comprising an alkoxysilane and at least one aminoalkoxysilane having an amine substituted with a diazeniumdiolate (e.g., an N-diazeniumdiolate).

[0040] The co-condensed siloxane network may be silica particles having a uniform size, a collection of silica particles having various sizes, amorphous silica, fumed silica, nanocrystalline silica, ceramic silica, colloidal silica, silica coatings, silica films, organo-modified silica, mesoporous silica, silica gels, bioactive glasses, and / or silica in any suitable form or state.

[0041] In some embodiments, the alkoxysilane is a tetraalkoxysilane having the formula Si(OR)4, where R is an alkyl group. The R groups may be the same or different. In some embodiments, the tetraalkoxysilane is selected as tetramethylorthosilicate (TMOS) or tetraethylorthosilicate (TEOS). In some embodiments, the aminoalkoxysilane has the formula R"-(NH-R')n-Si(OR)3, where R is alkyl, R' is alkylene, branched alkylene, or aralkylene, n is 1 or 2, and R" is selected from the group consisting of alkyl, cycloalkyl, aryl, and alkylamine.

[0042] In some embodiments, the aminoalkoxysilane can be selected from N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3); N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3); (3-trimethoxysilylpropyl)diethylenetriamine (DET3); (aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3); [3-(methylamino)propyl]trimethoxysilane (MAP3); N-butylaminopropyltrimethoxysilane (n-BAP3); t-butylaminopropyltrimethoxysilane (t-BAP3); N-ethylaminoisobutyltrimethoxysilane (EAiB3); N-phenylaminopropyltrimethoxysilane (PAP3); and N-cyclohexylaminopropyltrimethoxysilane (cHAP3).

[0043] In some embodiments, the aminoalkoxysilane has the formula NH[R'-Si(OR)3]2, where R is alkyl and R' is alkylene. In some embodiments, the aminoalkoxysilane can be selected from bis(3-triethoxysilylpropyl)amine, bis-[3-(trimethoxysilyl)propyl]amine, and bis-[(3-trimethoxysilyl)propyl]ethylenediamine.

[0044] In some embodiments, the aminoalkoxysilane is precharged for NO release, as described herein, and the amino group is substituted with a diazeniumdiolate. Thus, in some embodiments, the aminoalkoxysilane has the formula R″-N(NONO - X + )-R'-Si(OR)3, where R is alkyl, R' is alkylene or aralkylene, R" is alkyl or alkylamine, and X + is Na + , K + and Li + The cation is selected from the group consisting of:

[0045] The composition of the siloxane network (e.g., the amount or chemical composition of the aminoalkoxysilane) and the nitric oxide charging conditions (e.g., solvent and base) may be varied to optimize the amount and duration of nitric oxide release. Thus, in some embodiments, the composition of the silica particles may be altered to adjust the half-life of NO release from the silica particles.

[0046] In some embodiments, the amino group of the aminoalkoxysilane is substituted with a diazeniumdiolate, and the aminoalkoxysilane has the formula R″-N(NONO - X + )-R'-Si(OR)3, where R is alkyl, R' is alkylene or aralkylene, R" is alkyl or alkylamine, and X + is Na + and K + is a cation selected from the group consisting of:

[0047] In some embodiments, the NO-releasing API may comprise a co-condensed silica network comprising or formed from diazenium diolated aminoethylaminopropyltrimethoxysilane (AEAP3-NONOate) and tetramethyl orthosilicate (TMOS), and / or a co-condensed silica network comprising or formed from diazenium diolated aminoethylaminopropyltrimethoxysilane (AEAP3-NONOate) and tetraethyl orthosilicate (TEOS). In some embodiments, the NO-releasing API may comprise a co-condensed silica network comprising or formed from diazenium diolated methylaminopropyltrimethoxysilane (MAP3-NONOate) and tetramethyl orthosilicate (TMOS), and / or a co-condensed silica network comprising or formed from diazenium diolated methylaminopropyltrimethoxysilane (MAP3-NONOate) and tetraethyl orthosilicate (TEOS). In some embodiments, the NO-releasing API may comprise a co-condensed silica network comprising or formed from diazenium diolated methylaminopropyltrimethoxysilane (MAP3-NONOate), ethylaminoisobutylsiloxane (EAIB3), and tetraethylorthosilicate (TEOS). In some embodiments, the NO-releasing API may comprise a co-condensed silica network comprising or formed from diazenium diolated ethylaminoisobutylsiloxane (EAIB3-NONOate), tetraethylorthosilicate (TEOS) and / or tetramethylorthosilicate (TMOS). In some embodiments, the NO-releasing API may be ethylaminoisobutylsiloxane / methylaminopropylsiloxane-co-polysiloxane (EAIB3:MAP3-NONOate / TEOS). In some embodiments, the NO-releasing API may comprise an amorphous polymer.

[0048] In some embodiments, the particle size (e.g., diameter) of the NO-releasing API can be in the range of about 20 nm to about 20 μm or any range therein, such as, but not limited to, about 100 nm to about 20 μm or about 1 μm to about 20 μm or about 30 μm. The particle size can be adjusted to minimize or prevent toxicity and / or penetration through the epidermis (or compromised dermis) into the blood vessels. In some embodiments, the particle size is about an average particle size (e.g., average diameter) of less than 20 μm or any range therein, and the particle size can allow the particles to enter follicles. In some embodiments, the NO-releasing API may have a particle size distribution of about 20 μm, about 19 μm, about 18 μm, about 17 μm, about 16 μm, about 15 μm, about 14 μm, about 13 μm, about 12 μm, about 11 μm, about 10 μm, about 9 μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, or about 1 μm. In some embodiments, the NO-releasing API may have a particle size distribution of about less than 10 μm or any range therein, such as about 2 μm to about 10 μm or about 4 μm to about 8 μm. In some embodiments, the particle size may be more than 20 μm or any range therein, and the particle size may prevent the particles from entering into follicles. In some embodiments, a mixture of particles having an average particle size distribution of two or more average particle sizes may be provided. The NO-releasing API may be micronized (e.g., ball milled and / or jet milled). Methods for providing the desired particle size and / or micronization include, but are not limited to, those described in U.S. Patent Application Publication No. 2013 / 0310533, which is incorporated herein by reference in its entirety. In some embodiments, the nitric oxide-releasing active pharmaceutical ingredient has an average particle size of about 20 nm to about 30 μm. In some embodiments, the nitric oxide-releasing active pharmaceutical ingredient has an average particle size of about 2 μm to about 20 μm.

[0049] In some embodiments, the NO-releasing API can have a low charge. In some embodiments, the charge on the NO-releasing API can be controlled and / or regulated.

[0050] The compositions of the present invention may include a NO-releasing API and may store and / or release nitric oxide in an amount of about 0.001% to about 10% by weight of the composition, such as, but not limited to, about 0.001% to about 0.05%, about 0.01% to about 0.1%, about 0.15% to about 2%, about 0.15% to about 1%, about 0.3% to about 1.2%, about 0.15% to about 6%, about 1% to about 10%, about 3% to about 6%, or about 1% to about 5% by weight of the composition. In some embodiments, the compositions of the present invention may comprise a nitric oxide releasing active pharmaceutical agent and may be present in an amount of about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.15%, 0.3%, 0.6%, 0.9%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75% by weight of the composition. The composition may store and / or release nitric oxide in an amount of 0.01 wt%, 4 wt%, 4.25 wt%, 4.5 wt%, 4.75 wt%, 5 wt%, 5.25 wt%, 5.5 wt%, 5.75 wt%, 6 wt%, 6.25 wt%, 6.5 wt%, 6.75 wt%, 7 wt%, 7.25 wt%, 7.5 wt%, 7.75 wt%, 8 wt%, 8.25 wt%, 8.5 wt%, 8.75 wt%, 9 wt%, 9.25 wt%, 9.5 wt%, 9.75 wt%, or 10 wt%. The amount of nitric oxide released may be determined using a real-time in vitro release test. In some embodiments, nitric oxide release may be determined using a chemiluminescent nitric oxide analyzer.

[0051] The compositions of the present invention may include one or more excipients. In some embodiments, the compositions include a flavoring agent. In some embodiments, the buffer and / or composition does not include a diluent (e.g., additional diluent), a cosolvent, a preservative, an antioxidant, a suspending agent, a permeation enhancer, a surfactant, a viscosity enhancer, a humectant, a stabilizer, and / or a wetting agent.

[0052] Also provided according to an embodiment of the present invention is a kit. The kit of the present invention may include a first composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and a second composition comprising a buffer of the present invention, wherein the first composition and the second composition are stored separately in the kit. In some embodiments, the buffer is configured to maintain the pH of a composition comprising the first composition and the second composition (e.g., a combined combination) in the range of about 3, about 3.5, about 4 or about 4.5 to about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5. In some embodiments, the buffer is a citrate salt form buffer, for example, a citrate salt form buffer containing at least 100 mM of citrate salt form and / or citrate.

[0053] The first composition comprising the nitric oxide-releasing active pharmaceutical ingredient may be a solid, and / or the nitric oxide-releasing active pharmaceutical ingredient may be in particulate form. The second composition may be a solution. In some embodiments, the kit is configured to combine the first composition with the second composition to provide a combined composition. The combined composition can be a composition described herein. In some embodiments, the kit is configured to provide a combined composition comprising the nitric oxide-releasing active pharmaceutical ingredient in an amount of about 0.1 mg / mL to about 30 mg / mL, and the buffer (e.g., a buffer in the form of a salt of citric acid) is configured to maintain the pH of the combined composition in the range of about 3, about 3.5, about 4, or about 4.5 to about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, or about 8.5.

[0054] In some embodiments, the kit is configured to administer and / or deliver the composition. In some embodiments, the kit of the present invention includes a device configured to administer the composition. The kit and / or device may administer and / or deliver volumes of about 15 μL, about 25 μL, about 50 μL, about 75 μL, or about 100 μL to about 150 μL, about 200 μL, about 300 μL, about 400 μL, or about 500 μL. In some embodiments, the kit and / or device is configured to administer and / or deliver volumes of about 15 μL, about 25 μL, about 50 μL, about 75 μL, about 100 μL, about 150 μL, about 200 μL, about 250 μL, about 300 μL, about 350 μL, about 400 μL, about 450 μL, or about 500 μL. In some embodiments, the kit and / or device is configured to aerosolize and / or nebulize the composition of the present invention (e.g., a composition comprising the first composition and the second composition). In some embodiments, the aerosolized and / or nebulized composition of the present invention has an average droplet size (e.g., droplet diameter) of about 10 μm, about 20 μm, about 30 μm, or about 40 μm to about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the aerosolized and / or nebulized composition of the present invention has an average droplet size (e.g., droplet diameter) of about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the aerosolized and / or nebulized compositions of the present invention have an average droplet size that limits or avoids inhalation of the droplets / aerosol into the lungs.

[0055] The compositions of the present invention may be configured for intranasal administration. In some embodiments, the compositions of the present invention are configured to be sprayed and / or are sprayable compositions. The compositions may be sprayed onto the nasal and / or oral mucosa. In some embodiments, the compositions may contact oropharyngeal tissue. In some embodiments, the compositions of the present invention may be configured to be aerosolized and / or atomized and / or may be in the form of an aerosol and / or may be in the form of droplets in the gas phase. In some embodiments, the compositions of the present invention are configured to be administered to the nasal cavity of a subject and / or the oral mucosa of a subject. The NO-releasing API may be delivered locally to a subject (e.g., to the nasal and / or oral mucosa of the subject). In some embodiments, the local delivery of the NO-releasing API has a local effect and / or a systemic effect.

[0056] The compositions of the invention may be antiviral, antimicrobial and / or antibacterial. The buffers of the invention may not be antiviral and / or not viricidal. In some embodiments, the compositions of the invention administer and / or deliver nitric oxide in an amount sufficient to induce apoptosis in cells infected with a pathogen (e.g., a virus and / or a bacterium). In some embodiments, the compositions of the invention administer and / or deliver nitric oxide in an amount sufficient to induce apoptosis in virally infected cells. In some embodiments, the compositions of the invention administer and / or deliver nitric oxide in an amount sufficient to reduce or eliminate viral replication, optionally with less than about 50% host cell cytotoxicity.

[0057] According to some embodiments, there is provided a method of treating and / or preventing an infection (e.g., a viral infection and / or a bacterial infection) in a subject, the method comprising administering to the subject a composition of the invention.

[0058] In some embodiments, the method includes combining a nitric oxide-releasing active pharmaceutical ingredient with a buffer of the present invention to provide the composition before administering the composition to the subject. Combining a nitric oxide-releasing active pharmaceutical ingredient with a buffer can include adding the NO-releasing API to the buffer or adding the buffer to the NO-releasing API. In some embodiments, the nitric oxide-releasing active pharmaceutical ingredient and the buffer are present in a device (e.g., a kit) configured to combine the nitric oxide-releasing active pharmaceutical ingredient and the buffer. In some embodiments, the NO-releasing API and the buffer are combined and then provided in a device for administration. In some embodiments, the kit of the present invention can be a device configured for administration of the composition. The device of the present invention can be for single use or multiple use. In some embodiments, the device is refillable. In some embodiments, the device is a nasal spray device and / or a nebulizing device. In some embodiments, the device comprises a syringe and a plug at the tip of the syringe configured to atomize the composition as it travels from the syringe through the plug.

[0059] In some embodiments, administering the composition comprises administering to the subject from about 15 μL, about 25 μL, about 50 μL, about 75 μL, or about 100 μL to about 150 μL, about 200 μL, about 300 μL, about 400 μL, or about 500 μL of the composition per administration. From about 15 μL, about 25 μL, about 50 μL, about 75 μL, or about 100 μL to about 150 μL, about 200 μL, about 300 μL, about 400 μL, or about 500 μL of the composition per administration may be administered to each nostril of the subject.

[0060] The methods of the invention can include administering a composition of the invention to a subject at a time when the composition has a pH of about 3, about 3.5, about 4, or about 4.5 to about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, or about 8.5. In some embodiments, the composition is administered to the subject at a time when the composition has a pH of about 5.5.

[0061] The method of the present invention may include administering and / or delivering exogenous, gaseous nitric oxide to the upper airway, lower airway and / or lungs of a subject. Exogenous, gaseous NO may be delivered to the upper airway, lower airway and / or lungs in response to administering a composition of the present invention to the nasal and / or oral mucosa of the subject. In some embodiments, the NO-releasing API and / or composition (e.g., an aerosolized composition) is not delivered to the lower airway and / or lungs of the subject. In some embodiments, the NO-releasing API and / or composition is delivered locally to the subject.

[0062] The methods of the invention may optionally reduce or prevent transmission of a pathogen to a non-infected subject, compared to the amount of transmission in the absence of the methods of the invention. In some embodiments, the methods optionally reduce or prevent transmission of a virus to a non-infected subject (i.e., viral transmission), compared to the amount of viral transmission in the absence of the methods of the invention.

[0063] In some embodiments, the method of the present invention reduces the amount of pathogens (e.g., viruses, bacteria, etc.) present in a subject (e.g., in the nasal passages and / or lungs of a subject) by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the initial amount of viruses present in the subject. In some embodiments, the method of the present invention reduces the amount of pathogens (e.g., viruses, bacteria, etc.) present in one or more lungs of the subject, optionally compared to the amount of pathogens present in one or more lungs of the subject in the absence of the method of the present invention. In some embodiments, the method of the present invention reduces or prevents the progression of a pathogen and / or infection (e.g., a viral infection) to one or more lungs of the subject, optionally compared to the progression of the pathogen and / or infection to the lungs of the subject in the absence of the method of the present invention. The method of the present invention may optionally reduce the severity of an infection (e.g., a viral infection) compared to the severity in the absence of the method of the present invention.

[0064] The compositions and / or methods of the invention may reduce or prevent the growth and / or replication of pathogens (e.g., viruses). In some embodiments, the compositions and / or methods reduce or prevent viral replication. In some embodiments, the compositions and / or methods reduce or prevent bacterial growth. The methods may reduce or inhibit the replication of pathogens (e.g., viruses) by disrupting protein function. In some embodiments, the compositions and / or methods of the invention may reduce or prevent viral shedding, optionally compared to methods in the absence of the methods of the invention. In some embodiments, the methods of the invention increase blood oxygenation in the subject, optionally compared to blood oxygen levels before administration. In some embodiments, the methods may provide a localized and / or transient increase in oxygen in the blood of the subject.

[0065] In some embodiments, the pathogen is a virus or a bacterium. The method of the present invention may treat and / or prevent an infection caused by a pathogen. The infection may be a nosocomial infection. In some embodiments, the method of the present invention may treat and / or prevent an infection caused by a pathogen selected from a coronavirus family virus, Staphylococcus aureus, influenza virus, and / or respiratory syncytial virus (RSV). In some embodiments, the pathogen is a coronavirus family virus, such as, but not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory coronavirus 2 (SARS-CoV-2), a common cold virus (e.g., hCoV-229E and / or hCoV-OC43) and / or variants thereof. Exemplary variants of SARS-CoV-2 include, but are not limited to, SARS-CoV-2 alpha variants, SARS-CoV-2 beta variants, SARS-CoV-2 gamma variants, SARS-CoV-2 delta variants, SARS-CoV-2 omicron variants, and / or variants thereof (e.g., new mutants). In some embodiments, the methods of the invention treat and / or prevent infection with a coronavirus family virus, e.g., SARS-CoV-2, and / or treat and / or prevent coronavirus disease (e.g., COVID-19). In some embodiments, the pathogen is an influenza virus, e.g., influenza A / California / 7 / 2009 (H1N1) and / or variants thereof. In some embodiments, the methods of the invention treat and / or prevent infection with an influenza virus, e.g., H1N1, and / or treat and / or prevent influenza disease.In some embodiments, the pathogen is a respiratory syncytial virus, e.g., respiratory syncytial virus strain A2 (RSV-A2) and / or variants thereof. In some embodiments, the methods of the invention treat and / or prevent infection with a respiratory syncytial virus, e.g., RSV-A2, and / or treat and / or prevent respiratory syncytial virus disease.

[0066] The methods of the invention can include administering a composition of the invention one or more times per day (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 times), optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. In some embodiments, the composition is administered once, twice, or three times per day for about 7 to about 14 days. The methods can include administering the composition to one or both nostrils of a subject, optionally once, twice, or three times per day, for a period of time (e.g., for 1 to 14 days or more).

[0067] In some embodiments, the compositions and / or methods of the invention provide a localized, topical treatment that can reduce pathogen load (e.g., viral load) in the nasal and / or oral epithelium and / or one or more lungs of an infected subject, and / or can reduce pathogen shedding (e.g., viral shedding) and / or transmission to another subject (e.g., an uninfected subject). The compositions and / or methods of the invention can treat and / or reduce one or more symptoms of an infection and / or inhibit the progression of a disease (e.g., an infection) before it spreads to the lower respiratory tract of a subject.

[0068] The compositions and / or methods of the invention may treat and / or prevent an infection (e.g., SARS-CoV-2 infection) in a subject by disrupting the interaction and / or activity of ACE2 and / or S protein. In some embodiments, the compositions and / or methods of the invention may inhibit Bruton tyrosine kinase (BTK) and / or NF-κB, and / or inhibit the formation of the NLRP3 inflammasome. In some embodiments, the compositions and / or methods of the invention may disrupt a protease function (e.g., a viral protease function) required for pathogen replication (e.g., viral replication).

[0069] As used herein, "treat", "treating" or "treatment of" (and grammatical variations thereof) refers to any type of treatment that confers a benefit to a subject, and may mean that the severity of the subject's condition is alleviated, at least partially improved or ameliorated, and / or some alleviation, reduction or reduction in at least one clinical symptom associated with the condition (e.g., viral infection) is achieved, and / or there is a delay in the progression of the condition. In some embodiments, for example, the severity of a condition, e.g., a viral infection (e.g., a viral infection caused by SARS-CoV-2), may be reduced in a subject compared to the severity of the condition in the absence of the method of the invention. In some embodiments, the method of the invention may treat a viral infection by eliminating at least one clinical symptom associated with the viral infection for a predetermined period of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, or more, etc.).

[0070] In some embodiments, the compositions of the present invention are administered in a therapeutically effective amount. "Treatment effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount sufficient to treat a subject (as defined herein). One skilled in the art will appreciate that the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject. In some embodiments, a therapeutically effective amount of the compositions of the present invention may be administered and may include administering a therapeutically effective amount of a nitric oxide-releasing active pharmaceutical ingredient. In some embodiments, a therapeutically effective amount of nitric oxide may be administered and / or applied in the methods of the present invention. In some embodiments, the methods of the present invention are carried out in a manner such that administration of a composition comprising a nitric oxide (NO)-releasing active pharmaceutical ingredient (API) does not result in systemic effects (e.g., adverse systemic effects) due to administration of nitric oxide, e.g., when the composition, NO-releasing API and / or NO are administered in a therapeutically effective amount.

[0071] The terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to the avoidance, alleviation and / or delay of the onset of a condition (e.g., a viral infection) and / or clinical symptoms associated therewith in a subject, and / or a reduction in the severity of the onset of the condition and / or clinical symptoms relative to that which would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the complete absence of the condition and / or clinical symptoms. The prevention can also be partial, such that the onset and / or severity of the onset of the condition and / or clinical symptoms in a subject is less than that which would occur in the absence of the methods of the invention. In some embodiments, the methods of the invention prevent a viral infection in a subject, e.g., a viral infection caused by SARS-CoV-2.

[0072] In some embodiments, the compositions of the present invention are administered in a prophylactically effective amount. As used herein, a "prophylactically effective" amount is an amount sufficient to prevent (as defined herein) a condition (e.g., viral infection) and / or clinical symptoms in a subject. One of skill in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject. In some embodiments, a prophylactically effective amount of the compositions of the present invention may be administered, and may include administering a prophylactically effective amount of a nitric oxide-releasing active pharmaceutical ingredient. In some embodiments, a prophylactically effective amount of nitric oxide may be administered and / or applied in the methods of the present invention. In some embodiments, the methods of the present invention are carried out in a manner such that administration of a composition comprising a NO-releasing API does not result in systemic effects (e.g., adverse systemic effects) due to administration of nitric oxide, e.g., when the composition, NO-releasing API and / or NO are administered in a prophylactically effective amount.

[0073] The present invention finds use in both veterinary and medical applications. Suitable subjects of the present invention include, but are not limited to, birds and mammals. As used herein, the term "birds" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, pheasants, parrots, parakeets, macaws, cockatiels, canaries, and finches. As used herein, the term "mammals" includes, but is not limited to, primates (e.g., apes and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), bovines, ovines, caprines, ungulates, porcines, equines, felines, canines, lagomorphs, pinnipeds, rodents (e.g., rats, hamsters, and mice), and the like. In some embodiments, the subject is a mammal, and in some embodiments, the subject is a human. Human subjects include both males and females, and subjects of any age, including fetal, neonatal, infant, juvenile, adolescent, adult, and geriatric subjects.

[0074] The methods of the invention may also be practiced in animal subjects, particularly mammalian subjects, such as mice, rats, dogs, cats, livestock and horses for veterinary purposes and / or for drug screening and development purposes.

[0075] In some embodiments, the subject is "in need of" or "in need of" the methods of the invention, e.g., the subject is in an at-risk population (e.g., the subject may be at risk of or susceptible to viral infection), the subject has a finding typically associated with a viral infection, and / or the subject is suspected of being exposed to or has been exposed to a virus. In some embodiments, the subject in need thereof has a viral infection and / or clinical signs or symptoms associated therewith that can be treated with the methods of the invention. The invention may be particularly suitable for pediatric, adolescent, adult and / or geriatric subjects.

[0076] In some embodiments, the methods of the invention may administer nitric oxide to the basal layer of the epithelium of a subject. The methods of the invention may administer a therapeutically effective amount and / or a prophylactically effective amount of nitric oxide to the basal layer of the epithelium of a subject. In some embodiments, nitric oxide may be administered to the basement membrane of the epithelium of a subject.

[0077] In some embodiments, the methods of the present invention may administer nitric oxide in an amount sufficient to induce apoptosis or other cell damage in cells infected with a virus. In some embodiments, the methods of the present invention may administer nitric oxide in an amount sufficient to inhibit and / or prevent viral replication in cells infected with a virus. The methods of the present invention may reduce viral replication by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100%, compared to the replication rate before the methods of the present invention are performed.

[0078] In some embodiments, the method of the present invention can treat and / or prevent viral infection in a subject without or with reduced cytotoxicity to host cells. The method can treat and / or prevent viral infection in a subject with reduced cytotoxicity to host cells compared to a different method for treating viral infection, for example, a method that does not administer nitric oxide to the skin and / or tissue of a subject or a method that uses acidified nitrite. In some embodiments, the method of the present invention can reduce cytotoxicity to host cells by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% compared to a different method for treating viral infection. The method of the present invention may reduce and / or eliminate viral replication with no or minimal host cell cytotoxicity. For example, the method may provide about 50% or less (e.g., about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less) host cell cytotoxicity. Cytotoxicity may be determined using methods known to those skilled in the art, such as qualitative reading of hematoxylin & eosin (H&E) slides, lactate dehydrogenase (LDH) assay, and / or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetratetrazolium bromide (MTT) assay. In some embodiments, the method of the present invention may not cause apoptosis. For example, the method may not cause apoptosis in the keratinocyte layer of skin and / or tissue.

[0079] In some embodiments, the method of the present invention can reduce the amount of viral DNA in the virally infected cells of a subject and / or in the nasal cavity of a subject.For example, the method of the present invention can reduce the amount of viral DNA (e.g., in virally infected cells) by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99% or about 100% compared to the amount of viral DNA present before performing the method of the present invention.

[0080] The invention is described in more detail in the following non-limiting examples.

[0081] Working Example

[0082] Example 1

[0083] The antiviral activity of verdazimer sodium (a polymeric scaffold containing polysiloxane; tetraethoxysilane (TEOS), N-methylaminopropyltrimethoxysilane (MAP3), and N,N-methylaminopropyldiazeniumdiolatetrimethoxysilane (MAP3-NONOate)) and MAP3-NONOate was evaluated against SARS-CoV-2 (USA-WA1 / 2020 strain) in a highly differentiated three-dimensional (3-D) in vitro model of normal human-derived tracheal / bronchial epithelia (TBE) cells. Compounds were tested at 4-fold concentrations in single or double infusions of a 3D tissue model of the human airway (Matek Life Sciences) as shown in Table 1. Antiviral activity was measured by a virus yield reduction assay 5 days after infection.

[0084] Materials and Methods

[0085] Compounds: Test compounds were provided as solids and stored at -20°C upon arrival. One aliquot of compound verdazimer sodium was dissolved in 100% DMSO at concentrations of 200, 160, 80 and 20 mg / mL immediately prior to the start of the experiment and then further diluted to the test dilution in MatTek medium (AIR-100-MM). One aliquot of compound MAP3-NONOate was dissolved in 100% methanol at concentrations of 200, 160, 80 and 20 mg / mL on the morning of the experiment and then further diluted to the test dilution in MatTek medium. Remdesivir (MedChemExpress, cat# HY-104077) was tested as a positive control in singlet wells at 1, 0.1, 0.01 and 0.001 μg / mL.

[0086] Cell culture: EpiAirway 商標 The model consists of normal human-derived tracheal / bronchial epithelial (TBE) cells that are cultured to form a multi-layered, highly differentiated model closely resembling respiratory epithelial tissue. The cell cultures were made to order by MatTek Life Sciences (https: / / www.mattek.com) (Ashland, MA) and delivered in kits of either 12-well or 24-well inserts. TBE cells were cultured on 6 mm mesh disks in transwell inserts. During shipping, the tissue was stabilized on an agarose sheet, which was removed upon receipt. One insert contains approximately 1.2 × 10 6 The cell insert kit (EpiAirway 商標The TBE cells (AIR-100, AIR-112) were derived from a single donor #9831, a 23-year-old healthy non-smoking Caucasian male. The cells have the unique property of forming a layer, the apical side of which is exposed only to air, which forms a mucin layer. Upon arrival, the cell transwell inserts were immediately transferred into individual wells of a 6-well plate according to the manufacturer's instructions, and 1 mL of MatTek's proprietary medium (AIR-100-MM) was added to the basal cell side, while the apical side was exposed to a humidified 5% CO2 environment. TBE cells were cultured at 37°C for 1 day before the start of the experiment. After a 24-hour equilibration period, the mucin layer secreted from the apical side of the cells was removed by washing three times with 400 μL of pre-warmed 30 mM HEPES-buffered saline. The medium was replenished after the washing steps.

[0087] Virus: SARS-CoV-2 strain USA-WA1 / 2020 was passaged three times in Vero76 cells to generate virus stocks. Virus was diluted in AIR-100-MM medium prior to infection to approximately 0.01 CCID per cell. 50 The cells were incubated at a multiplicity of infection (MOI) of 100 μg / ml.

[0088] Experimental design: Each 2X compound treatment (120 μL) and virus (120 μL) was applied to the apical side, and only 1X compound treatment was applied to the basolateral side (1 mL) and incubated for 2 hours. As virus controls, 3 wells of the cell wells were treated with verdazimer sodium medium (cell medium containing 0.5% DMSO), 3 wells were treated with MAP3-NONOate medium (cell medium containing 0.3% methanol), and 3 wells were treated with cell culture medium alone as remdesivir virus control. After 2 hours of infection, the apical medium was removed and the basolateral side was replaced with freshly prepared compound or medium. The cells were maintained at an air-liquid interface. On days 1, 2, 3, and 4 after infection, test compounds were freshly prepared and drugs were removed from the replicate wells and treated with fresh drugs. The basolateral side of the singlet wells was replaced with fresh growth medium containing only DMSO or methanol without test compound. On day 5, the medium was removed and discarded from the basolateral side. Virus released into the apical compartment of the tissue was harvested by adding 400 μL of medium pre-warmed at 37°C. The contents were incubated for 30 min, mixed thoroughly, collected, vortexed thoroughly, and plated onto Vero 76 cells for VYR titration. Triplicate wells and singlet wells were used for virus and cell controls, respectively.

[0089] Measurement of viral titers from each treated cell culture: Vero76 cells were seeded in 96-well plates and grown overnight (37°C) to 90% confluence. Virus-containing samples were diluted 10-fold in infection medium, and 200 μL of each dilution was transferred to each well of a 96-well microtiter plate. Four microwells were used for each dilution to determine the 50% viral endpoint. After 7 days of incubation, each well was considered positive for virus if a cytopathic effect (CPE) was observed compared to uninfected controls, and the endpoint count was confirmed on day 10. The amount of virus capable of infecting 50% of the cell culture (CCID per 0.1 mL) was determined. 50 ) was calculated by the Reed-Muench method (Reed, LJ, Muench, H., 1938. A simple method of estimating fifty percent endpoints. The American Journal of Hygiene 27, 493-497). Values ​​at day 7 are reported. Untreated uninfected cells were used as cell controls.

[0090] result

[0091] Viral yield results and EC 90 The values ​​are summarized in Table 1 below. Repeated once-daily treatments of each compound (vertazimer sodium and MAP3-NONOate) demonstrated an antiviral response, whereas single treatment regimens of either compound did not produce an antiviral response under the conditions tested. No toxicity was observed at any of the test compound concentrations.

[0092] [Table 1]

[0093] Each well was scored as virus positive if any CPE was observed compared to uninfected controls. Vero76 cells were scored on day 7 and confirmed on day 10.

[0094] a Titer results from virus yield reduction assays.

[0095] b EC 90 = 90% effective concentration (1 log viral yield) as determined by regression analysis 10 (concentration to reduce

[0096] Example 2

[0097] The objective of this study was to evaluate the efficacy of verdazimer sodium in treating SARS-CoV-2 infection in wild-type golden Syrian hamsters. In addition, the efficacy of verdazimer sodium in reducing direct transmission of SARS-CoV-2 from infected animals to naive littermates was also evaluated.

[0098] The primary endpoints were the effects of verdazimer sodium treatment on body weight loss, lung viral titers, nasal tissue titers, lung weights, and oropharyngeal swap titers in hamsters exposed to SARS-CoV-2.

[0099] Materials and Methods

[0100] Animals: For this study, 5-week-old female Golden Syrian hamsters were obtained from Charles River Laboratories (Wilmington, Mass.) The hamsters were quarantined for 3 days prior to use and maintained on Teklad Rodent Diet (Harlan Teklad) and tap water at the Laboratory Animal Research Center at Utah State University.

[0101] Virus: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) USA_WA1 / 2020 strain was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). The virus was passaged twice in Vero76 cells to generate a working stock for infecting hamsters.

[0102] The compound, verdazimer sodium, was provided in a form that could be administered intranasally to animals. EIDD-2801 was purchased by USU from MedChem Express. EIDD-2801 (Molnupravir) is a ribonucleoside analogue that has demonstrated activity across a broad range of RNA viruses, including coronaviruses (2).

[0103] Experimental Design: Efficacy Study (PHA-254): A total of 55 hamsters were divided into 5 groups of 10 animals per treatment, with 5 animals used as normal controls for weight gain (Table 2 below). For virus challenge, hamsters were anesthetized by IP (intraperitoneal) injection of ketamine / xylazine (50 mg / kg / 5 mg / kg) and then administered 1x10 virions in an inoculum volume of 100 μL. 4.3 50% cell culture infectious doses (CCID 50The animals were challenged intranasally with a dose of 0.1 mL of verdazimer sodium. This total volume of 100 μL was delivered to both nostrils simultaneously. Treatment with verdazimer sodium was given intranasally in a volume of 0.1 mL once daily for 7 days starting 24 hours prior to infection. All intranasal treatments were administered in a volume of 100 μL after the animals were anesthetized as for infection. Verdazimer sodium was prepared for administration by preparing stocks of 400, 200, and 100 mg / mL in 100% dimethyl sulfoxide (DMSO) which were then diluted 200-fold into a buffer consisting of a 1:1 ratio of Hyclone MEM from USU and the citrate salt form at 50 mM and 4.5 mM. DMSO stocks of verdazimer sodium prepared 20-30 minutes prior to administration were added to the MEM / citrate salt form buffer 5 minutes prior to administration to the animals. EIDD-2801 was administered orally (PO) twice daily starting 4 hours post-infection. EIDD-2801 was solubilized in 10% DMSO and 90% corn oil. Five animals per group were euthanized on study days 3 and 6 to assess lung virus titers, nasal virus titers, and lung weights. Oropharyngeal swabs were collected from five animals per group on study days 1-6.

[0104] Experimental Design - Transmission Study (PHA-254B): A total of 20 5-week-old female Golden Syrian hamsters were randomly assigned into two groups of 2 animals each and four groups of 4 animals each (Table 3 below). Animals in groups 1 and 4 served as infected donor animals. Animals in groups 2, 3, 5 and 6 were uninfected (naive) animals and were housed with animals from groups 1 or 4 for 4 hours daily on test days 1, 2 and 3. Animals in groups 1, 2 and 5 were treated with DMSO in MEM / citric acid salt form buffer as placebo. Animals in groups 3, 4 and 6 were treated with verdazimer sodium at 2 mg / mL (2 mg / kg / day) once a day, 2 hours before cohabitation with infected animals. Treatment with verdazimer sodium was started on the day of infection, 24 hours before the first cohabitation session. Hamsters were weighed before infection and daily thereafter to assess infection-associated weight loss. All animals were euthanized on study day 4 and assessed for lung virus titers, nasal tissue virus titers, lung weights, and virus transmission from infected to naive animals. Oropharyngeal swabs were taken daily in all animals.

[0105] Titration of tissue and oropharyngeal swab samples: Tissue homogenates and oropharyngeal swab samples were titrated by endpoint dilution. Serial log dilutions of tissue homogenates or oropharyngeal swab samples were titrated. 10The dilutions were plated in quadruplicate wells of a 96-well microplate containing a confluent monolayer of Vero76 cells. The plates were incubated in a 5% CO2, 37°C incubator for 6 days. The plates were then visually inspected under a light microscope for the presence or absence of cytopathic effect (CPE). The viral titer of each sample was calculated by linear regression using the Reed-Muench method. The viral titer of each sample was calculated by linear regression using the Reed-Muench method.

[0106] Statistics and Figures: Individual hamster body weights were converted to a percentage of initial body weight on the day of infection. Percentage of initial body weight curves were compared using one-way analysis of variance (ANOVA) comparing each treatment group with a placebo-treated group. Tissue virus titers and oropharyngeal swab samples were compared using two-way ANOVA comparing treated and placebo-treated animals. Lung weights were compared using one-way ANOVA because there was only one evaluation time point.

[0107] Ethics statement for laboratory animals: This study was performed in accordance with approval by the Institutional Animal Care and Use Committee at Utah State University, dated March 31, 2020 (expiration date March 30, 2023). It was conducted at the AAALAC-accredited Laboratory Animal Research Center at Utah State University. US government (National Institutes of Health) approval was updated on March 9, 2018 (PHS Assurance No. D16-00468 [A3801-01]) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Revision; 2011).

[0108] [Table 2]

[0109] [Table 3]

[0110] Results and Discussion

[0111] This study evaluated verdazimer sodium treatment on lung viral titers, nasal tissue titers, oropharyngeal swab titers, and lung weight reduction in SARS-CoV-2-infected hamsters. Additionally, the efficacy of intranasal verdazimer sodium treatment on SARS-CoV-2 transmission in golden Syrian hamsters was evaluated.

[0112] In efficacy studies, significant respiratory infection was observed as evidenced by high virus titers in lung tissue, nasal tissue and oropharyngeal swab titers. Lung virus titers 3 days post-infection were approximately 3 logs higher than the inoculum, indicating robust infection in the respiratory tissues of hamsters. Additionally, weight loss was observed in placebo-treated animals.

[0113] The percent of initial body weight of 5-week-old Golden Syrian hamsters after challenge with SARS-CoV-2 and treatment with verdazimer sodium is shown in Figure 1. Treatment with verdazimer sodium did not prevent weight loss after infection. Treatment with EIDD-2801 at a dose of 200 mg / kg / day prevented weight loss in hamsters infected with SARS-CoV-2. The percent of weight loss was assessed using one-way ANOVA comparing the mean weight loss across the experiment for each treatment group.

[0114] Figure 2 shows lung viral titers in 5-week-old golden Syrian hamsters after challenge with SARS-CoV-2 and treatment with verdazimer sodium. Treatment with verdazimer sodium did not significantly reduce lung viral titers in animals infected with SARS-CoV-2. Treatment with EIDD-2801 did not reduce lung viral titers in SARS-CoV-2-infected animals on day 6 post-infection. One animal in the group treated with verdazimer sodium 2 mg / kg / day did not recover from anesthesia post-infection and was therefore not included in the data set. Table 4 below shows lung and nasal viral titers on days 3 and 6 of the study.

[0115] [Table 4]

[0116] Figure 3 shows viral titers in nasal tissues of 5-week-old golden Syrian hamsters after challenge with SARS-CoV-2 and treatment with verdazimer sodium. Treatment with verdazimer sodium did not significantly reduce viral titers in nasal tissues in animals infected with SARS-CoV-2. Treatment with EIDD-2801 did not significantly reduce viral titers in the nasal cavity after infection.

[0117] Viral titers in oropharyngeal swabs from 5-week-old golden Syrian hamsters challenged with SARS-CoV-2 and treated with verdazimer sodium are shown in Figure 4. Treatment with verdazimer sodium did not reduce oropharyngeal swab viral titers in SARS-CoV-2-infected animals. EIDD-2801 did not reduce oropharyngeal swab viral titers on any day post-infection. Table 5 below shows oropharyngeal swab viral titers on days 1-6 of the study.

[0118] [Table 5] JPEG2024547022000007.jpg43170

[0119] Figure 5 shows lung weights of 5-week-old golden Syrian hamsters challenged with SARS-CoV-2 and treated with verdazimer sodium. Lung weights were not significantly affected by treatment with verdazimer sodium or EIDD-2801.

[0120] In the case of transmission studies, cohabitation of donor animals with naive animals resulted in robust infection in all of the placebo-treated naive animals as indicated by post-cohabitation lung virus titers.

[0121] Figure 6 shows the percent initial body weight of 5-week-old Golden Syrian hamsters after being treated with verdazimer sodium and cohabiting with SARS-CoV-2-infected hamsters. Animals with the same colored symbols were cohabited on study days 1-3. Placebo-treated animals were protected from weight loss when donor animals were treated with verdazimer sodium. Treatment with verdazimer sodium prevented weight loss when donor animals were also treated with verdazimer sodium after exposure to SARS-CoV-2-infected hamsters. Percent weight loss was assessed using one-way ANOVA, and weight loss for each treatment group was compared across studies.

[0122] The lung viral titers on day 4 of hamsters treated with verdazimer sodium and exposed to SARS-CoV-2 infected hamsters are shown in Figure 7. Animals housed on days 1-3 of the study are shown in brackets. Treatment with verdazimer sodium significantly reduced lung viral titers in treated naive animals housed with infected animals also treated with verdazimer sodium. Virus was detected in only one of four animals treated with verdazimer sodium and housed with donor animals also treated with verdazimer sodium. The viral titers detected in that one animal were reduced by more than 2 logs compared to placebo-treated naive animals.

[0123] Figure 8 shows viral titers in nasal tissues on day 4 of hamsters treated with verdazimer sodium and exposed to SARS-CoV-2-infected hamsters. Animals housed on days 1-3 of the study are shown in brackets. Treatment with verdazimer sodium did not significantly reduce viral titers in nasal tissues of animals housed with SARS-CoV-2-infected hamsters. Table 6 below shows viral titers in lung and nasal tissues of golden Syrian hamsters after treatment with verdazimer sodium prior to cohabitation with SARS-CoV-2-infected animals.

[0124] [Table 6]

[0125] Figure 9 shows the viral titers of oropharyngeal swabs of 5-week-old golden Syrian hamsters after being treated with verdazimer sodium and cohabiting with SARS-CoV-2 infected hamsters. Treatment with verdazimer sodium at a dose of 2 mg / kg / day did not significantly reduce the oropharyngeal swab titers. Table 7 below shows the viral titers of oropharyngeal swabs of golden Syrian hamsters on study days 1 to 4 after being treated with verdazimer sodium before cohabiting with SARS-CoV-2 infected animals.

[0126] [Table 7] JPEG2024547022000010.jpg155170

[0127] Lung weights of 5-week-old golden Syrian hamsters treated with vertazimer sodium and cohabiting with SARS-CoV-2-infected animals are shown in Figure 10. Lung weights were not statistically different between groups when compared by one-way ANOVA.

[0128] conclusion

[0129] The study evaluated the effect of verdazimer sodium treatment on weight loss, lung viral titers, nasal tissue titers, oropharyngeal swab titers, and lung weights. The ability of verdazimer sodium treatment to prevent transmission of SARS-CoV-2 to naive animals was also evaluated.

[0130] In efficacy studies, treatment with verdazimer sodium at any dose did not prevent weight loss or reduce viral titers in lungs, nasal tissues, or oropharyngeal swabs.

[0131] However, treatment with 2 mg / kg / day verdazimer sodium significantly reduced SARS-CoV-2 lung viral load in naive animals when donor animals were also treated with verdazimer sodium at a dose of 2 mg / mL. No virus was detected in the lungs of three of the four naive animals exposed to infected animals, and the remaining one animal had a 2-log reduction. Virus was detected in all four nasal tissue samples, indicating that the animals were infected, but treatment with verdazimer sodium prevented the virus from infecting the lungs. This is also supported by the data on initial body weight percentages, which show that the same animals were protected from weight loss after exposure to SARS-CoV-2-infected animals. Oral pharyngeal swab titers and lung weights were not affected by verdazimer sodium treatment in the transmission study.

[0132] Interestingly, verdazimer sodium treatment was able to reduce the transmission of SARS-CoV-2 from infected animals, despite the lack of efficacy in the efficacy study. No adverse events were observed in hamsters treated with verdazimer sodium at 2 mg / kg / day.

[0133] Example 3

[0134] The objective of this study was to determine the efficacy of vertazimer sodium in reducing direct transmission of SARS-CoV-2 from infected wild-type golden Syrian hamsters to naive littermates.

[0135] The primary endpoints were the effects of verdazimer sodium treatment on body weight loss, lung viral titers, nasal tissue titers, and lung weights in hamsters exposed to SARS-CoV-2.

[0136] Materials and Methods

[0137] Animals: For this study, 16-week-old female Golden Syrian hamsters were obtained from the colony at the State University of Utah. The hamsters were quarantined for 3 days prior to use and maintained at the Laboratory Animal Research Center at the University of Utah on Teklad Rodent Diet (Harlan Teklad) and tap water.

[0138] Virus: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) USA_WA1 / 2020 strain was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). The virus was passaged twice in Vero76 cells to generate a working stock for infecting hamsters.

[0139] Compounds: Verdazimer sodium were provided as solids and were dissolved in DMSO prior to treatment. DMSO stocks were added to a 1:1 mixture of minimum essential media (MEM) and 50 mM citrate salt form buffer at pH 4.5.

[0140] Experimental Design - Transmission Study: A total of 43 16-week-old female Golden Syrian hamsters were randomly assigned into 4 groups of 2 animals and 8 groups of 4 animals, with 3 animals kept as normal controls (Table 8 below). Animals in Groups 1, 4, 7 and 10 served as infected donor animals. Animals in Groups 2, 3, 5, 6, 8, 9, 11 and 12 were uninfected (naive) animals and were housed with animals from Groups 1, 4, 7 or 10 for 4 hours daily on days 1, 2 and 3 of the study. Animals in Groups 1, 2, 5, 8 and 11 were treated with DMSO in MEM / citric acid salt form buffer as placebo. Animals in groups 3, 4, 6, 7, 9, 10, and 12 were treated once daily with 1, 2, or 8 mg / mL (1, 2, or 8 mg / kg / day) verdazimer sodium 2 hours before cohabitation with infected animals. For infected donor animals, treatment with verdazimer sodium was initiated on the day of infection, 24 hours before the first cohabitation session. To assess the weight loss associated with infection, hamsters were weighed before infection and daily thereafter. All animals were euthanized on study day 4 and the lung virus titers, nasal tissue virus titers, lung weights, and transmission of virus from infected to naive animals were evaluated.

[0141] [Table 8] JPEG2024547022000012.jpg87170

[0142] Titration of tissue samples: Tissue homogenates were titrated by endpoint dilution. Serial log dilutions of tissue homogenates were 10The dilutions were plated into quadruplicate wells of a 96-well microplate containing a confluent monolayer of Vero76 cells. The plates were incubated in a 5% CO2, 37°C incubator for 6 days. The plates were then visually inspected under a light microscope for the presence or absence of cytopathic effect (CPE). The viral titer of each sample was calculated by linear regression using the Reed-Muench method.

[0143] Statistics and Figures: Individual hamster body weights were converted to a percentage of initial body weight on the day of infection. Percentage of initial body weight curves were compared using one-way analysis of variance (ANOVA) comparing each treatment group with placebo-treated hamsters. Tissue virus titers and lung weights were compared using one-way ANOVA comparing treated animals with placebo-treated animals.

[0144] Ethics statement for laboratory animals: This study was performed in accordance with the approval of the Institutional Animal Care and Use Committee of Utah State University, dated March 31, 2020 (expiration date March 30, 2023). It was conducted at the AAALAC-accredited Laboratory Animal Research Center at Utah State University. The approval of the U.S. government (National Institutes of Health) was updated on March 9, 2018 (PHS Assurance No. D16-00468 [A3801-01]) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Revision; 2011).

[0145] Results and Discussion

[0146] This study evaluated the efficacy of intranasal treatment with veratidine sodium against SARS-CoV-2 transmission in golden Syrian hamsters.

[0147] In the case of transmission studies, cohabitation of donor animals with naive animals resulted in robust infection in all of the placebo-treated naive animals as indicated by post-cohabitation lung virus titers.

[0148] Figure 11 shows the percent of initial body weight of 16-week-old Golden Syrian hamsters treated with verdazimer sodium (8 mg / mL) and housed with SARS-CoV-2-infected hamsters. Animals with the same colored symbols were housed on study days 1-3. No significant differences in body weight were observed between hamsters treated with verdazimer sodium (8 mg / mL) and housed with SARS-CoV-2-infected hamsters.

[0149] Figure 12 shows the percent of initial body weight of 16-week-old Golden Syrian hamsters after being treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and cohabiting with SARS-CoV-2-infected hamsters. Animals with the same colored symbols were cohabited on study days 1-3. No significant differences in body weight were observed in hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and cohabiting with SARS-CoV-2-infected hamsters.

[0150] The lung viral titers on day 4 of hamsters treated with verdazimer sodium (8 mg / mL) and exposed to SARS-CoV-2 infected hamsters are shown in FIG. 13. Animals housed on days 1-3 of the study are shown in brackets. Treatment with verdazimer sodium (8 mg / mL) significantly reduced the lung viral titers of treated naive animals housed with infected animals also treated with verdazimer sodium (8 mg / mL). Virus was only detected in two of four animals treated with verdazimer sodium (8 mg / mL) and housed with donor animals also treated with verdazimer sodium (8 mg / mL). The viral titers detected in the two animals were reduced by more than 5 logs compared to placebo-treated naive animals.

[0151] The lung viral titers on day 4 of hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and exposed to SARS-CoV-2 infected hamsters are shown in FIG. 14. Animals housed on days 1-3 of the study are shown in brackets. Treatment with verdazimer sodium (2 mg / mL) significantly reduced the lung viral titers of treated naive animals housed with infected animals also treated with verdazimer sodium (2 mg / mL). Virus was only detected in two of four animals treated with verdazimer sodium (2 mg / mL) and housed with donor animals also treated with verdazimer sodium (2 mg / mL). The viral titers detected in the two animals were reduced by more than 5 (log) compared to placebo-treated naive animals. Treatment with verdazimer sodium (1 mg / mL) did not significantly reduce lung viral titers in treated naive animals housed with infected animals also treated with verdazimer sodium (1 mg / mL). Table 9 below shows lung viral titers in golden Syrian hamsters after treatment with verdazimer sodium prior to cohabitation with SARS-CoV-2 infected animals.

[0152] [Table 9] JPEG2024547022000014.jpg255162

[0153] Figure 15 shows viral titers in nasal tissues of hamsters treated with verdazimer sodium (8 mg / mL) and exposed to SARS-CoV-2-infected hamsters on day 4. Animals housed on days 1-3 of the study are shown in brackets. Treatment with verdazimer sodium (8 mg / mL) did not significantly reduce viral titers in nasal tissues of animals housed with SARS-CoV-2-infected hamsters.

[0154] FIG. 16 shows viral titers in nasal tissues on day 4 of hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and exposed to SARS-CoV-2-infected hamsters. Animals housed on days 1-3 of the study are shown in brackets. Treatment with verdazimer sodium (2 mg / mL or 1 mg / mL) did not significantly reduce viral titers in nasal tissues in animals housed with SARS-CoV-2-infected hamsters. Table 10 below shows viral titers in nasal tissues of golden Syrian hamsters after treatment with verdazimer sodium before being housed with SARS-CoV-2-infected animals.

[0155] [Table 10]

[0156] Lung weights of 16-week-old golden Syrian hamsters treated with vertazimer sodium (8 mg / mL) and cohabited with SARS-CoV-2-infected animals are shown in Figure 17. Lung weights were not statistically different between groups when compared by one-way ANOVA.

[0157] Lung weights of 16-week-old golden Syrian hamsters treated with verdazimer sodium (2 mg / mL or 1 mg / mL) and cohabited with SARS-CoV-2-infected animals are shown in Figure 18. Lung weights were not statistically different between groups when compared by one-way ANOVA.

[0158] conclusion

[0159] This study evaluated the ability of verdazimer sodium treatment to prevent transmission of SARS-CoV-2 to naive animals.

[0160] Treatment with verdazimer sodium at a dose of 8 mg / mL or 2 mg / mL significantly reduced SARS-CoV-2 lung viral load in naive animals when donor animals were also treated with verdazimer sodium at a dose of 8 mg / mL or 2 mg / mL. In each dose group, virus was not detected in the lungs of two of the four naive animals exposed to infected animals, and the remaining animals were reduced by 5 logs. Virus was detected in all four nasal tissue samples per dose group, indicating that the animals were infected, but that treatment with verdazimer sodium prevented the virus from infecting the lungs. The lack of a positive effect from treatment at 1 mg / mL indicates a dose-response of verdazimer sodium in this study.

[0161] These lung viral titer findings confirm the results observed for 2 mg / mL in Example 2. However, improvements in weight loss were not observed in this study as opposed to Example 2. This may be due to the advanced age of the hamsters used in this study.

[0162] Example 4

[0163] The objective of this study was to evaluate the efficacy of verdazimer sodium for the treatment of SARS-CoV-2 infection in wild-type golden Syrian hamsters. Verdazimer sodium was evaluated in once-daily or twice-daily dosing regimens at a treatment dose of 2 mg / mL and once-daily dosing regimens at treatment doses of 4 mg / mL and 8 mg / mL.

[0164] The primary endpoints were the effects of verdazimer sodium treatment on body weight loss, lung viral titers, nasal tissue titers, and lung weights in hamsters exposed to SARS-CoV-2.

[0165] Materials and Methods

[0166] Animals: For this study, 5-week-old female Golden Syrian hamsters were obtained from Charles River Laboratories (Wilmington, Mass.) The hamsters were quarantined for 3 days prior to use and maintained on Teklad Rodent Diet (Harlan Teklad) and tap water at the Laboratory Animal Research Center at Utah State University.

[0167] Virus: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) USA_WA1 / 2020 strain was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). The virus was passaged twice in Vero76 cells to generate a working stock for infecting hamsters.

[0168] The compound: verdazimer sodium was provided in a form that could be administered intranasally to animals. EIDD-2801 was purchased by USU from MedChem Express. EIDD-2801 (Molnupravir) is a ribonucleoside analogue that has shown activity across a broad range of RNA viruses, including coronaviruses.

[0169] Experimental Design: Efficacy Study (PHA-262): A total of 85 hamsters were divided into 5 groups of 10 hamsters for once-daily treatment dosing and 2 groups of 15 hamsters for twice-daily treatment dosing (Table 11 below). For virus challenge, hamsters were anesthetized with IP (intraperitoneal) injection of ketamine / xylazine (50 mg / kg / 5 mg / kg) and then administered 1x10 hamsters in an inoculum volume of 100 μL. 4.3 50% cell culture infectious doses (CCID 50) via the nasal route. This total volume of 100 μL was delivered to both nostrils simultaneously. Treatment with verdazimer sodium was given by intranasal administration in a volume of 0.1 mL once or twice daily for 7 days, starting 24 hours before infection. The twice daily doses were given 12 hours apart. All intranasal treatments were administered in a volume of 100 μL after anesthetizing the animals as for infection. Verdazimer sodium was prepared for administration by first preparing stocks. Briefly, stocks were prepared at 1600, 800, and 400 mg / mL in 100% dimethyl sulfoxide (DMSO), which were then diluted 200-fold into a buffer consisting of a 1:1 ratio of Hyclone MEM from USU and the salt form of citric acid at 50 mM and pH 4.5. DMSO stocks of verdazimer sodium were prepared 20-30 min prior to dosing and added to the MEM / citric acid salt form of the buffer 5 min prior to dosing the animals. EIDD-2801 was administered orally (PO) twice daily starting 4 h post-infection. EIDD-2801 was solubilized in 10% DMSO and 90% corn oil. Five animals per group were euthanized on study days 3 and 6 to assess lung virus titers, nasal virus titers, and lung weights. Ten animals from each group were euthanized on day 6 because five additional animals were included in these groups in case of failure to recover from anesthesia in the first and second groups.

[0170] [Table 11]

[0171] Titration of tissue samples: Lung and nasal tissue homogenates were titrated by endpoint dilution. Serial log dilutions were performed. 10The dilutions were plated into quadruplicate wells of a 96-well microplate containing a confluent monolayer of Vero76 cells. The plates were incubated in a 5% CO2, 37°C incubator for 6 days. The plates were then visually inspected under a light microscope for the presence or absence of cytopathic effect (CPE). The viral titer of each sample was calculated by linear regression using the Reed-Muench method.

[0172] Statistics and Figures: Individual hamster body weights were converted to a percentage of initial body weight on the day of infection. Percentage of initial body weight curves were compared using one-way analysis of variance (ANOVA) comparing each treatment group with placebo-treated hamsters. Tissue viral titers were compared using two-way analysis of variance (ANOVA) comparing treated animals with placebo-treated animals. Lung weights were compared using two-way ANOVA.

[0173] Ethics statement for laboratory animals: This study was performed in accordance with approval by the Institutional Animal Care and Use Committee at Utah State University, dated March 31, 2020 (expiration date March 30, 2023). It was conducted at the AAALAC-accredited Laboratory Animal Research Center at Utah State University. US government (National Institutes of Health) approval was updated on March 9, 2018 (PHS Assurance No. D16-00468 [A3801-01]) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Revision; 2011).

[0174] Results and Discussion

[0175] The study evaluated treatment with verdazimer sodium on lung viral titers, nasal tissue titers, and lung weight reduction in hamsters infected with SARS-CoV-2. Additionally, twice-daily treatment with 2 mg / mL verdazimer sodium was compared with once-daily treatment with 2 mg / mL verdazimer sodium.

[0176] In this study, significant respiratory infection was observed as evidenced by high virus titers in lung and nasal tissues. The lung virus titers were approximately 3 logs higher than the inoculum dose 3 days after infection, indicating robust infection in the respiratory tissues of hamsters. In addition, weight loss was observed in placebo-treated animals.

[0177] Figure 19 shows the percent of initial body weight in hamsters treated once daily with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was initiated 24 hours prior to infection. No statistically significant protection from weight loss was observed in hamsters treated with 2, 4 or 8 mg / mL verdazimer sodium or 500 mg / kg / day EIDD-2801. Although not statistically significant, hamsters treated with 8 mg / mL verdazimer sodium lost less weight compared to placebo-treated animals. A similar trend was observed in hamsters treated with EIDD-2801.

[0178] FIG. 20 shows lung viral titers on days 3 and 6 in hamsters treated once daily with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was started 24 hours prior to infection. Treatment with verdazimer sodium at a dose of 4 mg / mL significantly reduced lung viral titers on day 3 post-infection. Treatment with EIDD-2801 at a dose of 500 mg / kg / day significantly reduced lung viral titers on day 3 post-infection. This log decrease in viral titers was 1 log decrease in viral titers. 10The reduction is comparable to other studies using EIDD-2801. In animals treated with 2 mg / mL or 8 mg / mL verdazimer sodium once daily, lung viral titers on day 3 were reduced, but the reduction was not statistically significant. Lung viral titers on day 6 were below the limit of quantification in all but one sample across all groups, suggesting that infection was cleared by day 6 in this model.

[0179] Figure 21 shows viral titers in nasal tissues on days 3 and 6 of hamsters treated with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was started 24 hours before infection. Treatment with verdazimer sodium at a dose of 2, 4 or 8 mg / mL once a day did not significantly reduce viral titers in nasal tissues. Treatment with EIDD-2801 at a dose of 500 mg / kg / day did not significantly reduce viral titers in nasal tissues.

[0180] Figure 22 shows lung weights on days 3 and 6 in hamsters treated with verdazimer sodium (2, 4 or 8 mg / mL) and infected with SARS-CoV-2. Treatment with verdazimer sodium was started 24 hours prior to infection. Treatment with verdazimer sodium at doses of 2, 4, or 8 mg / mL once daily did not significantly reduce lung weights. Treatment with EIDD-2801 at a dose of 500 mg / kg / day did not significantly reduce lung weights.

[0181] The percent of initial body weight of hamsters treated with verdazimer sodium (2 mg / mL) twice daily and infected with SARS-CoV-2 is shown in Figure 23. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Twice daily administration of verdazimer sodium at a dose of 2 mg / mL did not prevent weight loss in hamsters infected with SARS-CoV-2. Although not statistically significant, hamsters treated with EIDD-2801 at a dose of 500 mg / kg / day lost less weight than the placebo-treated group.

[0182] Lung viral titers on days 3 and 6 of hamsters treated twice daily with verdazimer sodium (2 mg / mL) and infected with SARS-CoV-2 are shown in Figure 24. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Twice daily treatment with verdazimer sodium at a dose of 2 mg / mL reduced lung viral titers by approximately 1 / 2 log on day 3 post-infection, but the difference was not statistically significant. EIDD-2801 treatment at 500 mg / kg / day significantly reduced lung viral titers on day 3 post-infection.

[0183] Viral titers in nasal tissues on days 3 and 6 of hamsters treated with verdazimer sodium (2 mg / mL) twice daily and infected with SARS-CoV-2 are shown in Figure 25. Treatment with verdazimer sodium was started 24 hours prior to infection. Treatment with verdazimer sodium twice daily at a dose of 2 mg / mL significantly reduced viral titers in nasal tissues on day 3 post-infection. Treatment with EIDD-2801 at 500 mg / kg / day significantly reduced viral titers in nasal tissues on day 3 post-infection. There was greater variability in the nasal tissue samples, with some animals having high viral titers and some animals having no detectable viral titers in the nasal tissue. The greater variability may be due to the small amount of homogenized tissue in these samples. Table 12 below shows the viral titers in lung and nasal tissues on study days 3 and 6 in golden Syrian hamsters following treatment with verdazimer sodium and infection with SARS-CoV-2.

[0184] [Table 12] JPEG2024547022000018.jpg255132

[0185] Lung weights on days 3 and 6 of hamsters treated twice daily with verdazimer sodium (2 mg / mL) and infected with SARS-CoV-2 are shown in FIG. 26. Treatment with verdazimer sodium was initiated 24 hours prior to infection. Lung weights were significantly increased on day 6 post-infection by twice daily administration of verdazimer sodium. This is not believed to be due to intranasal treatment, as a similar increase in lung weight was observed in hamsters treated with EIDD-2801.

[0186] conclusion

[0187] This study evaluated the effect of once-daily treatment with 2, 4, and 8 mg / mL verdazimer sodium on weight loss, lung viral titers, nasal tissue titers, and lung weights. The effect of twice-daily treatment with 2 mg / mL verdazimer sodium was also evaluated.

[0188] Although not statistically significant, hamsters treated with 8 mg / mL verdazimer sodium lost less weight than the placebo-treated group. However, two of the 10 animals treated with 8 mg / mL verdazimer sodium died on day 3 after infection. Wild-type hamsters infected with SARS-CoV-2 usually do not succumb to infection, but the intranasal treatment may have exacerbated the infection. However, a previous study was conducted with the same regimen and dose level (8 mg / mL) and no premature deaths were observed.

[0189] Administration of 4 mg / mL verdazimer sodium significantly reduced lung virus titers. Although there was some variability in lung virus titers, the general trend appeared to be dose-responsive with verdazimer sodium treatment. However, no dose of verdazimer sodium was able to reduce nasal tissue titers when administered once daily. A significant reduction in nasal tissue titers was observed when hamsters were treated twice daily with 2 mg / mL verdazimer sodium. For two of five animals infected with SARS-CoV-2 and treated twice daily with 2 mg / mL verdazimer sodium, virus was not detectable in nasal tissue.

[0190] Example 5

[0191] The purpose of this study was to determine the potential toxicity of a composition (also referred to as the test article) containing verdazimer sodium (API) and a buffering agent administered intranasally to dogs five times a day for 14 days, and to evaluate the potential reversibility of the results found. The buffering agent in the composition was a 200 mM citrate salt form buffer at pH 4.5. The experimental design is provided in Table 13 below. Approximate daily dose levels were based on average body weights of 8.5 kg for males and 6.5 kg for females, and therefore daily dose levels (mg / kg / day) were 0 mg / kg / day for Group 1, 0.47 mg / kg / day for Group 2, 0.94 mg / kg / day for Group 3, and 1.65 mg / kg / day for Group 4 for males, and 0 mg / kg / day for Group 1, 0.61 mg / kg / day for Group 2, 1.23 mg / kg / day for Group 3, and 2.15 mg / kg / day for Group 4 for females.

[0192] [Table 13]

[0193] The following parameters and primary endpoints were evaluated in this study: mortality, clinical observations, body weight, body weight gain, food consumption, ophthalmological examinations, electrocardiography, clinical pathology parameters (hematology, coagulation, clinical chemistry, methemoglobin and urinalysis), pharmacokinetic parameters, organ weights, and macroscopic and microscopic examinations.

[0194] The test article did not alter mortality, clinical observations, body weight, body weight gain, food consumption, ophthalmologic examinations, clinical pathology (hematology, coagulation, clinical chemistry, and urinalysis), organ weights, or macroscopic or microscopic examinations, and there were no adverse findings.

[0195] Electrocardiograms (ECGs) showed a statistically significant increase in QRS complex duration in males in Group 4 (14 mg / day) 1-2 hours after dosing on Day 11. This change was considered test substance related because of its magnitude, but was not considered to be adverse since it resolved during the recovery period.

[0196] On day 14, all MetHgb values ​​for the 14 mg / day male and female dogs decreased compared to baseline values ​​at each collection interval after dosing, except for a slight increase in the female dog at the 2 hour collection interval immediately prior to dosing. In addition, all control male and female dogs showed a decrease in MetHgb compared to baseline values ​​at each collection interval after dosing on day 14.

[0197] At 14 mg / day, there were test article-related microscopic changes in the nasal turbinates that were not considered adverse, including mild to moderate mixed inflammation and minimal to mild luminal exudate in male and female dogs. These findings showed complete resolution in dogs, but not at the end of the 7-day recovery period in female dogs, although the exudate appeared to clear during the recovery period.

[0198] In conclusion, administration of the test article by intranasal administration five times daily for 14 days was well tolerated in dogs at a level of 14 mg API / day. Target organ effects were observed at the 14 mg / day level, and there were test article-related microscopic changes in male and female dogs, including mild to moderate mixed inflammation and minimal to mild luminal exudate in male and female dogs, which were not considered adverse. These findings showed complete resolution in male dogs but only partial resolution in female dogs at the end of the 7-day recovery period. In addition, there was a statistically significant increase in QRS complex duration in male dogs at 1-2 hours after dosing on day 11. This change was considered test article-related due to the magnitude of the change, but resolved during the recovery period. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 14 mg / day (i.e., 1.65 mg / kg / day in males and 2.15 mg / kg / day in females). At the NOAEL, the mean C 最大 and AUC (0~24時間) Values ​​were 11,800 pg / mL and 179,000 h·pg / mL, respectively, in males and 10,700 pg / mL and 173,000 h·pg / mL, respectively, in females.

[0199] Example 6

[0200] In terms of the alkaline API (verdazimer sodium), buffer concentration, pH and osmolality were evaluated. For this study, test formulations 11b-17b were prepared at multiple verdazimer sodium concentrations from 2 mg / mL to 18 mg / mL using only buffer as the vehicle for the test formulation. All samples were prepared w / v by weighing the API into a glass vial and diluting with 10 mL of the respective buffer to target the required verdazimer sodium concentration. The samples were then evaluated for pH and osmolality of the test formulation. Vehicle samples were also evaluated for pH and osmolality. In the case of test formulations 6b and 7b, the samples were prepared at concentrations of 2 mg / mL and 24 mg / mL. All samples were prepared w / v by weighing the API into a glass vial and diluting with 5 mL of the respective buffer to target the required verdazimer sodium concentration. The samples were then evaluated for pH and osmolality of the test formulation. Vehicle samples were also evaluated for pH and osmolality. The test formulations evaluated can be seen in Table 14 below.

[0201] 2mg / mL sample Upon formulation, no foaming was observed and all samples produced finely dispersed suspensions. With regard to pH, the pH of all samples fell below the target pH of pH 5.50, with all samples returning to around pH 4.60-4.80. With regard to osmolality, the osmolality values ​​of the formulations increased with increasing buffer concentration. However, all osmolality values ​​returned to within the acceptable range for intranasal administration.

[0202] 8mg / mL sample Upon compounding, minimal foaming of a thin consistency was observed in all samples. Regarding API wettability and dispersibility, the API was easily wetted and formed a finely dispersed suspension. Regarding pH, all formulations except 11b returned near the target pH of pH 5.50. Osmolality trends were similar to the 2 mg / mL sample, with values ​​increasing with increasing buffer concentration. All osmolality values ​​returned within the acceptable range for intranasal administration.

[0203] 12mg / mL sample Upon compounding, test formulations 15b and 16b produced a large amount of thick foam, while test formulations 17b and 11b produced a small amount of foam. Regarding API wettability and dispersibility, the API was easily wetted and formed a finely dispersed suspension. Regarding pH, all test formulations except 15b returned near the target pH of pH 5.50. The pH value of test formulation 15b was pH 7.07, suggesting that the buffer concentration of 100 mM was not strong enough to buffer the API concentration for 12 mg / mL. A similar trend was observed for osmolality, with all values ​​returning within the acceptable range for intranasal administration.

[0204] 14mg / mL sample Upon compounding, test formulations 15b-17b produced a large amount of thick foam, whereas test formulation 11b produced a small amount of foam. Regarding API wettability and dispersibility, the API was easily wetted and formed a finely dispersed suspension. Regarding pH, all test formulations except 15b returned to near the target pH of 5.50. A similar trend was observed for osmolality, with all values ​​returning to within the acceptable range for intranasal administration.

[0205] 16mg / mL sample Upon compounding, test formulations 15b and 11b produced a large amount of thick foam, while test formulations 16b and 17b produced a small amount of foam. Regarding API wetting and suspension, a large amount of dry API remained at the bottom of each sample and could not be mixed and suspended due to a thick layer of foam on the top of each vial. Regarding pH, all test formulations except 15b returned near the target pH of pH 5.50. A similar trend was observed for osmolality, with all values ​​returning within the acceptable range for intranasal administration.

[0206] 18mg / mL sample Upon compounding, all samples generated a large amount of thick foam. Regarding API wetting and suspension, a large amount of dry API remained at the bottom of each sample and could not be mixed and suspended due to a thick layer of foam on the top of each vial. Regarding pH, all test formulations returned near the target pH of pH 5.50. A similar trend was observed for osmolality, with all values ​​returning within the acceptable range for intranasal administration.

[0207] All data from this study can be seen in Tables 15-22 below.

[0208] [Table 14]

[0209] [Table 15]

[0210] [Table 16]

[0211] [Table 17]

[0212] [Table 18]

[0213] [Table 19]

[0214] [Table 20]

[0215] [Table 21]

[0216] [Table 22]

[0217] Summary and conclusions:

[0218] The results of this study indicate that the API upper limit for excessive effervescence is between 8mg / mL and 12mg / mL for the buffers tested. Based on the pH data, the 200mM citrate salt buffer appears to effectively buffer the API in the concentration range of 2mg / mL to 16mg / mL and maintain the formulation pH near the target pH of 5.50. With regard to API wettability and dispersibility, API concentrations above 14mg / mL did not provide sufficient API suspension, resulting in a large amount of dry API remaining at the bottom of the vial. At an API concentration of 14mg / mL, complete API wettability and suspension can be achieved while maintaining the target pH of the tested formulation.

[0219] Example 7

[0220] The antiviral activity of verdazimer sodium against respiratory syncytial virus (RSV-A2) and influenza A / California / 7 / 2009 (H1N1) was evaluated in highly differentiated three-dimensional (3D) in vitro cultures of normal human-derived tracheal / bronchial epithelial (TBE) cells. The compound was tested at various concentrations using triplicate inserts of the human EpiAirway (MatTek Life Sciences) 3D tissue model. Antiviral activity was measured by viral yield reduction assays 3 (H1N1) and 6 (RSV-A2) days after infection.

[0221] Materials and Methods

[0222] Compounds: Compounds (Verdazimer Sodium) were received as solids and stored at -20°C until preparation. Each day, fresh Verdazimer Sodium drug was dissolved in 100% dimethylsulfoxide (DMSO) and then prepared as test dilutions in MatTek medium (AIR-100-MM).

[0223] Cell culture: EpiAirway 商標 The model consists of normal human-derived tracheal / bronchial epithelial (TBE) cells that are cultured to form a multi-layered, highly differentiated model closely resembling respiratory epithelial tissue. The cell cultures were made to order by MatTek Life Sciences (https: / / www.mattek.com) (Ashland, MA) and delivered in kits of either 12- or 24-well inserts. TBE cells were cultured on 6 mm mesh disks in transwell inserts. During shipping, the tissue was stabilized on a sheet of agarose, which was removed 24 hours after shipping. Each insert contains approximately 1.2 × 10 6The cell insert kit (EpiAirway 商標 AIR-100, AIR-112) were derived from a single healthy non-smoker donor #9831.

[0224] Upon arrival, the cell transwell inserts were immediately transferred into individual wells of a 6-well plate according to the manufacturer's instructions. Then, 1 mL of MatTek's proprietary medium (AIR-100-MM) was added to the basolateral side, while the apical side was exposed to a humidified 5% CO2 environment. TBE cells were cultured at 37°C for 2 days before the start of the experiment. After a 48-h equilibration period, the secreted mucin layer from the apical side of the cells was removed by washing three times with 400 μL of pre-warmed 30 mM HEPES-buffered saline. Culture medium was replenished on the basolateral side after the washing steps. The tissue was then rested for a minimum of 1 hour at 37°C and 5% CO2 environment before the assay.

[0225] Virus: The virus RSV-A2 (ATCC VR-1540) was passaged twice on MA-105 cells to generate a virus stock. The virus dose (CCID per 0.2 mL) capable of infecting 50% of the cell culture was 50 The viral stock was then diluted in AIR-100-MM and incubated at an MOI of 0.1 CCID per cell. 50 Influenza A (H1N1) was passaged twice in MDCK cells to generate virus stocks. The virus dose capable of infecting 50% of the cell culture (CCID per 0.2 mL) was 50 The viral stock was then diluted in AIR-100-MM and incubated at an MOI of 0.001 CCID per cell. 50 was infected.

[0226] Experimental design: Each compound treatment (140 μL) and virus (140 μL) were applied to the apical side, and only compound treatment was applied to the basolateral side (1 mL), with a 2-hour infection period. As a virus control, 3 wells of each virus cell well were treated with placebo (cell medium only). After the 2-hour infection period, the apical medium was removed and the basolateral side was replaced with freshly prepared compound or medium. The cells were maintained at an air-liquid interface. At approximately the same time each day (within 1-2 hours), the basolateral side was removed and replaced with freshly prepared compound or fresh medium (virus control). [Note that the data shown in Table 23 below are the results after the basolateral side was not replaced with fresh compound on days 3 and 4]. On day 3 (H1N1) or day 6 (RSV), the medium was removed and discarded from the basolateral side. Virus released into the apical compartment of the tissue was harvested by adding 400 μL of medium pre-warmed to 37° C. The contents were incubated for 30 min, mixed thoroughly, harvested, thoroughly vortexed, frozen at −80° C., and finally plated on MDCK cells (H1N1) or MA-105 cells (RSV) on the same day as harvest for virus yield reduction (VYR) titrations. Ribavirin was included in this study as a positive control for antiviral effect.

[0227] Determination of virus titer from each treated cell culture: Cells were seeded in 96-well plates and grown overnight (37°C) to 90% confluence. Virus-containing samples were diluted 10-fold in infection medium, and 200 μL of each dilution was transferred into each well of a 96-well microtiter plate. Four microwells were used for each dilution to determine the 50% viral endpoint. After 3 days (H1N1) or 7 days (RSV) of incubation, each well was considered virus positive if a cytopathic effect (CPE) was observed compared to uninfected controls. The 90% effective concentration (EC90 ), i.e., 1 log 10 The amount of compound required to reduce was determined by regression analysis.

[0228] Cytotoxicity Assay: The CCK-8 colorimetric assay is based on the reduction of WST-8, a highly water-soluble tetrazolium salt, which produces an orange water-soluble formazan dye in the presence of an electron mediator. The amount of formazan is directly proportional to the number of viable cells, and the resulting colored solution is quantified by measuring the absorbance at 450 nanometers using a multiwell spectrophotometer. Drug concentrations of 2 mg / mL and 1 mg / mL were tested in duplicate wells, and drug concentrations of 0.5 mg / mL were tested in single wells. On day 3 (H1N1) or day 6 (RSV), the apical side was washed once with 400 μL of pre-warmed PBS. CCK-8 solution was added (200 μL) to the apical side and incubated for 2 hours at 37°C. Formazan (100 μL) was placed in a 96-well plate, and the absorbance at 450 nm was recorded. Percent toxicity values ​​were calculated as a percentage of cell control test wells (no drug treatment) and the 50% cytotoxic concentration (CC 50 The tissues were also examined via microscope to assess cytotoxicity.

[0229] VYR titer data, EC 90 and C.C. 50 The values ​​are summarized in Tables 23 to 25 below.

[0230] conclusion

[0231] As shown in Table 23, EC from Verdazimer sodium treatment on days 1, 2, and 5 of RSV-A2 infection 90 The EC values ​​ranged from 0.79 to 1.07 mg / mL, whereas the EC values ​​from veratidine sodium treatment on days 1 to 5 of RSV-A2 infection, as shown in Table 24, were 0.79 to 1.07 mg / mL. 90The values ​​ranged from 0.96 to 1.44 mg / mL. Without wishing to be bound by a particular theory, no difference in efficacy was observed for the two RSV-A2 treatment regimens under the conditions tested. As shown in Table 25, the EC 90 The values ​​were 1.11-1.14 mg / mL. Tissues observed under a microscope showed no obvious cytotoxicity.

[0232] [Table 23]

[0233] Each well was scored as virus positive if any CPE was observed compared to the uninfected control.

[0234] a Titer results from viral yield reduction (VYR) assays.

[0235] b EC 90 = VYR 90% effective concentration (1 log viral yield) as determined by regression analysis 10 (concentration to reduce

[0236] c CC 50 = 50% cytotoxic concentration of compound (without virus).

[0237] d SI=CC 50 / EC 90 the selectivity index is a measure of the window between cytotoxicity and antiviral activity: the higher the SI ratio, the more effective and safe the drug is during in vivo treatment against a given viral infection.

[0238] [Table 24]

[0239] Each well was scored as virus positive if any CPE was observed compared to the uninfected control.

[0240] a Titer results from viral yield reduction (VYR) assays.

[0241] b EC 90 = VYR 90% effective concentration (1 log viral yield) as determined by regression analysis 10 (concentration to reduce

[0242] c CC 50 = 50% cytotoxic concentration of compound (without virus).

[0243] d SI=CC 50 / EC 90 the selectivity index is a measure of the window between cytotoxicity and antiviral activity: the higher the SI ratio, the more effective and safe the drug is during in vivo treatment against a given viral infection.

[0244] [Table 25]

[0245] Each well was scored as virus positive if any CPE was observed compared to the uninfected control.

[0246] a Titer results from viral yield reduction (VYR) assays.

[0247] b EC 90 = VYR 90% effective concentration (1 log viral yield) as determined by regression analysis 10 (concentration to reduce

[0248] c CC 50 = 50% cytotoxic concentration of compound (without virus).

[0249] d SI=CC 50 / EC 90 the selectivity index is a measure of the window between cytotoxicity and antiviral activity: the higher the SI ratio, the more effective and safe the drug is during in vivo treatment against a given viral infection.

[0250] The above is illustrative of the present invention and should not be construed as limiting the present invention thereto. The present invention is defined by the appended claims, including equivalents thereto. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entirety for teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

1. 1. A composition comprising: a nitric oxide-releasing active pharmaceutical ingredient; and a buffer configured to maintain the pH of the composition in the range of from about 3, from about 3.5, from about 4, or from about 4.5, to about 5, to about 5.5, to about 6, to about 6.5, to about 7, to about 7.5, to about 8, or to about 8.5 The composition comprising:

2. 10. The composition of claim 1, wherein the buffer has a pH of about 4 to about 6 and / or the buffer comprises a weak acid having at least two pKa values ​​in the range of about 2.5 to about 6.

5.

3. A composition comprising: a nitric oxide-releasing active pharmaceutical ingredient; and buffer wherein the buffer comprises a salt form of citric acid and / or citric acid in an amount of at least 100 mM. The composition.

4. The composition of claim 3, wherein the buffer has a pH of about 4 to about 6.

5. 5. The composition of claim 3 or 4, wherein the buffering agent comprises the salt form of citric acid and / or citric acid in an amount of at least about 100 mM to about 300 mM.

6. 5. The composition according to claim 3 or 4, wherein the salt forms of citric acid and / or citric acid are selected from citric acid, trisodium citrate, potassium citrate, calcium citrate, and / or hydrates thereof, or anhydrous forms thereof.

7. 5. The composition of claim 3, wherein the buffering agent comprises citric acid in an amount of from about 1%, to about 1.25%, to about 1.5%, or from about 1.75%, to about 2%, to about 2.25%, or to about 2.5% w / w; sodium citrate tribasic and / or trisodium citrate dihydrate in an amount of from about 1.5%, to about 1.75%, to about 2%, to about 2.25%, to about 2.5%, to about 2.75%, or to about 3%, to about 3.25%, to about 3.5%, to about 3.75%, or to about 4% w / w.

8. 5. The composition of claim 3 or 4, wherein the buffering agent consists of water, citric acid, a salt form of citric acid, an additional acid, and a base.

9. 4. The composition of claim 1 or 3, wherein the nitric oxide-releasing active pharmaceutical ingredient is suspended in the buffer.

10. 4. The composition of claim 1, wherein the nitric oxide-releasing active pharmaceutical ingredient is present in the composition in an amount of from about 0.1 mg / mL to about 30 mg / mL.

11. 4. The composition of claim 1, wherein the nitric oxide-releasing active pharmaceutical ingredient has a particle size of about 20 nm to about 30 μm.

12. 4. The composition of claim 1, wherein the composition has an initial pH of about 4.5 to about 6.

13. The composition of claim 1 or 3, wherein the composition has a pH of about 4.5 to about 8.

5.

14. The composition of claim 1 or 3, wherein the composition is adapted to be aerosolized and / or atomized.

15. The composition of claim 1 or 3, wherein the composition is antiviral.

16. 4. The composition of claim 1 or 3, wherein the composition consists of the nitric oxide-releasing active pharmaceutical ingredient and the buffering agent.

17. 4. The composition of claim 1 or 3, wherein the buffer and / or composition does not comprise a diluent, co-solvent, preservative, antioxidant, suspending agent, permeation enhancer, surfactant, viscosity increasing agent, humectant, stabilizer, and / or wetting agent.

18. 4. The composition of claim 1 or 3, wherein the composition releases nitric oxide in an amount of about 0.001% to about 10% by weight of the composition as measured by real-time in vitro release testing, and / or the nitric oxide-releasing active pharmaceutical ingredient stores and / or releases nitric oxide in an amount of about 0.001% to about 10% by weight of the nitric oxide-releasing active pharmaceutical ingredient and / or composition as measured by real-time in vitro release testing.

19. 4. The composition of claim 1 or 3, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a co-condensed silica network functionalized with a diazeniumdiolate.

20. 4. The composition of claim 1 or 3, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a co-condensed silica network containing diazeniumdiolated methylaminopropyltrimethoxysilane (MAP3-NONOate) and tetraethylorthosilicate (TEOS).

21. 4. The composition of claim 1 or 3, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a co-condensed silica network containing diazeniumdiolated methylaminopropyltrimethoxysilane (MAP3-NONOate), ethylaminoisobutylsiloxane (EAIB3), and tetraethylorthosilicate (TEOS).

22. 10. The composition of claim 1 or 3, wherein the composition provides a sufficient amount of nitric oxide to induce apoptosis in virally infected cells.

23. 10. The composition of claim 1 or 3, wherein the composition provides a sufficient amount of nitric oxide to reduce or eliminate viral replication.

24. A kit comprising: a first composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and a second composition comprising a buffer configured to maintain the pH of the first composition in the range of from about 3, from about 3.5, from about 4, or from about 4.5, to about 5, to about 5.5, to about 6, to about 6.5, to about 7, to about 7.5, to about 8, or to about 8.

5. It is equipped with wherein the first composition and the second composition are stored separately within the kit. The kit.

25. A kit comprising: a first composition comprising a nitric oxide-releasing active pharmaceutical ingredient; and A second composition comprising a salt form of citric acid and / or a buffer containing citric acid in an amount of at least 100 mM. Including, wherein the first composition and the second composition are stored separately within the kit. The kit.

26. 26. The kit of claim 24 or 25, wherein the first composition is a solid or particulate.

27. 26. The kit of claim 24 or 25, wherein the second composition is a solution.

28. 26. The kit of claim 24 or 25, wherein the buffer has a pH of about 4 to about 6.

29. 26. The kit of claim 24 or 25, wherein the kit is configured to administer and / or deliver a volume of from about 15 μL to about 150 μL, to about 200 μL, to about 300 μL, to about 400 μL, or to about 500 μL; and / or the kit comprises a device configured to administer and / or deliver a volume of from about 15 μL to about 150 μL, to about 200 μL, to about 300 μL, to about 400 μL, or to about 500 μL.

30. 26. The kit of claim 24 or 25, wherein the kit is configured to aerosolize and / or atomize a composition comprising the first composition and the second composition, and / or the kit comprises a device configured to aerosolize and / or atomize a composition comprising the first composition and the second composition.

31. 26. The kit of claim 24 or 25, wherein the buffer has a pH of about 4 to about 6.

32. 26. The kit of claim 25, wherein the buffering agent comprises a salt form of citric acid and / or citric acid in an amount of at least about 100 mM to about 300 mM.

33. 26. The kit of claim 25, wherein the salt forms of citric acid and / or citric acid are selected from citric acid, trisodium citrate, potassium citrate, calcium citrate, and / or hydrates thereof, and / or anhydrous forms thereof.

34. 26. The kit of claim 25, wherein the buffering agent comprises citric acid in an amount of from about 1%, to about 1.25%, to about 1.5% w / w, or from about 1.75% w / w, to about 2%, to about 2.25% w / w, or to about 2.5% w / w; sodium citrate tribasic and / or trisodium citrate dihydrate in an amount of from about 1.5%, to about 1.75%, to about 2%, to about 2.25%, to about 2.5%, to about 2.75% w / w, or from about 3%, to about 3.25%, to about 3.5%, to about 3.75%, or to about 4% w / w.

35. 26. The kit of claim 25, wherein the buffer consists of water, citric acid, a salt form of citric acid, an additional acid, and a base.

36. 25. The kit of claim 24, wherein the nitric oxide-releasing active pharmaceutical ingredient has a particle size of about 20 nm to about 30 μm.

37. 25. The kit of claim 24, wherein the first composition and / or the second composition does not comprise a diluent, a cosolvent, a preservative, an antioxidant, a suspending agent, a permeation enhancer, a surfactant, a viscosity increasing agent, a humectant, a stabilizer, and / or a wetting agent.

38. 25. The kit of claim 24, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a co-condensed silica network functionalized with a diazeniumdiolate.

39. 25. The kit of claim 24, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a co-condensed silica network containing diazeniumdiolated methylaminopropyltrimethoxysilane (MAP3-NONOate) and tetraethylorthosilicate (TEOS).

40. 25. The kit of claim 24, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a co-condensed silica network containing diazeniumdiolated methylaminopropyltrimethoxysilane (MAP3-NONOate), ethylaminoisobutylsiloxane (EAIB3), and tetraethylorthosilicate (TEOS).

41. 1. A method of treating and / or preventing an infection caused by a pathogen in a subject, comprising: Administering the composition of claim 1 or 3 to the subject. The method comprising:

42. 42. The method of claim 41, wherein said administering comprises intranasally administering said composition to said subject.

43. 42. The method of claim 41, further comprising combining the nitric oxide-releasing active pharmaceutical ingredient and the buffering agent to provide the composition prior to administering.

44. 42. The method of claim 41, wherein the composition has a pH of about 4.5 to about 8.5 when administered to the subject.

45. 42. The method of claim 41, wherein the method comprises delivering exogenous nitric oxide to the upper respiratory tract of the subject.

46. 42. The method of claim 41, wherein the method reduces or prevents transmission of the pathogen to an uninfected subject.

47. 42. The method of claim 41, wherein the method reduces the amount of the pathogen present in the subject by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the initial amount of the pathogen present in the subject.

48. 42. The method of claim 41, wherein the method reduces the amount of pathogen present in one or more of the lungs of the subject and / or reduces or prevents progression of the pathogen into one or more of the lungs of the subject.

49. 42. The method of claim 41, wherein the method reduces the severity of the infection in the subject.

50. 42. The method of claim 41, wherein the pathogen is selected from a coronavirus, Staphylococcus aureus, influenza, or respiratory syncytial virus (RSV).

51. 51. The method of claim 50, wherein the pathogen is severe acute respiratory coronavirus 2 (SARS-CoV-2) or a variant thereof.

52. 51. The method of claim 50, wherein the pathogen is influenza A / California / 7 / 2009 (H1N1).

53. 51. The method of claim 50, wherein the pathogen is respiratory syncytial virus strain A2 (RSV-A2).

54. 42. The method of claim 41, wherein the infection is a hospital-acquired infection.

55. 42. The method of claim 41, wherein said method reduces or prevents shedding of said pathogen.

56. 42. The method of claim 41, wherein said method reduces or inhibits the growth and / or replication of a pathogen.

57. 42. The method of claim 41, wherein said administering comprises delivering gaseous nitric oxide to one or more of the lungs of said subject.

58. 42. The method of claim 41, wherein the method increases oxygenation in the blood of the subject.

59. 42. The method of claim 41, wherein the composition is administered one or more times daily.

60. 42. The method of claim 41, wherein the composition is administered once, twice, or three times daily for about 7 to about 14 days.

61. 26. A method of using a composition according to claim 1 or 3 or a kit according to claim 24 or 25 for treating and / or preventing an infectious disease in a subject in need thereof.

Images