Pharmaceutical dry powder compositions and methods for treating respiratory tract infections

Abstract

The present invention provides a pharmaceutical dry powder composition for use in the treatment, prevention, and / or amelioration of viral infections in the respiratory tract of a mammalian, e.g., human, subject, the composition comprising a micronized sodium salt as a primary therapeutic agent. The present invention further provides a pharmaceutical composition for use in the treatment and / or amelioration of cough. The subject treated with the pharmaceutical composition may be infected with SARS-CoV-2 and / or diagnosed with COVID-19 infection. The pharmaceutical composition is administered by dry powder inhalation.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for use in the treatment, prevention, and / or amelioration of a viral infection in the respiratory tract of a mammal, e.g., a human subject. The present invention further relates to a pharmaceutical composition for use in the treatment and / or amelioration of cough in a mammal, e.g., a human subject, e.g., a cough caused by a viral infection in the respiratory tract of the subject. The human subject treated with the pharmaceutical composition may be infected with SARS-CoV-2 and / or diagnosed with COVID-19 infection. The pharmaceutical composition of the present invention is a dry powder composition comprising a micronized sodium salt as a primary therapeutic agent. The pharmaceutical composition is administered by dry powder inhalation. [Background technology]

[0002] Respiratory tract infections are common infections of the upper respiratory tract (e.g., nose, ears, sinuses, and throat) and lower respiratory tract (e.g., trachea, bronchi, and lungs). Symptoms of upper respiratory tract infections include rhinorrhea or nasal congestion, irritability, restlessness, loss of appetite, decreased activity level, cough, and fever. Viral infections of the upper respiratory tract cause or are associated with, for example, sore throat, chills, croup, and influenza. Clinical signs of lower respiratory tract infections include shallow cough that produces phlegm in the lungs, fever, and difficulty breathing.

[0003] Respiratory viral infections cause a significant disease burden in infants, children, and adults. The clinical impact of common infections is even greater in individuals with underlying cardiopulmonary conditions. Current therapies for viral respiratory tract infections involve the administration of antiviral agents for treatment, prevention, or amelioration.

[0004] Some common classes of respiratory viruses associated with human disease include paramyxoviruses, orthomyxoviruses, adenoviruses, picornaviruses, parvoviruses, arenaviruses, herpesviruses, retroviruses, and coronaviruses.

[0005] Severe acute respiratory syndrome-associated coronavirus (SARSr-CoV or SARS-CoV) is a type of virus consisting of many known strains. The SARSr-CoV species is a member of the genus Betacoronavirus and the subgenus Saberbecovirus (SARSbetacoronavirus). The morphology of SARS-associated coronaviruses is characteristic of the entire coronavirus family. These viruses are enveloped, single-stranded, positive-sense RNA viruses. SARS-associated coronaviruses follow a replication strategy unique to all coronaviruses. Two viral strains, severe acute respiratory syndrome coronavirus 1 (SARS-CoV or SARS-CoV-1), which caused the 2002-2004 outbreak of severe acute respiratory syndrome (SARS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is causing the ongoing pandemic of COVID-19, have caused outbreaks of severe respiratory disease in humans.

[0006] Viral infections are often highly contagious, especially when spread by breathing. The recent pandemic caused by severe acute respiratory syndrome-associated coronavirus demonstrates how rapidly infectious diseases can spread around the world.

[0007] Potential ways the virus may spread include touching another person's skin or an object contaminated with infectious droplets and then touching one's own eyes, nose, or mouth. Additionally, airborne transmission is also a likely route of viral transmission. It is also possible that the virus can spread more widely through the air via aerosols, which are composed of mucus droplets originating from the lungs and / or nasal passages that are produced when people or animals cough or simply breathe. Because these aerosols may contain the virus, the disease is transmitted by inhalation by exposed subjects.

[0008] Coronaviruses can cause both upper and lower respiratory tract infections.

[0009] Although several means of treating and / or preventing viral infections are available, there is a great demand for new treatments to address growing medical problems such as the emergence of new pathogen strains and the high cost of traditional medicines.

[0010] The present invention encompasses methods for reducing viral growth, infectivity, load, shedding, and the development of antiviral resistance, and for enhancing the effectiveness of conventional antiviral therapies. Summary of the Invention

[0011] In one aspect, the present invention provides a dry powder composition comprising at least 50 wt%, such as at least 60 wt%, for example at least 70 wt%, such as at least 80 wt%, for example at least 90 wt% NaCl for use in the prevention and / or treatment of viral, such as coronavirus, such as SARS-CoV-2, respiratory tract infection, wherein the composition is to be administered to a mammalian subject by dry powder inhalation, for example by using a dry powder inhaler.

[0012] In one embodiment, the dry powder compositions of the invention reduce the time for resolution of one or more symptoms caused by a viral respiratory tract infection, such as coughing.

[0013] In one embodiment, the dry powder composition of the invention has a particle size distribution in which at least 80% of the particles are in the range of 1 to 10 μm.

[0014] In one embodiment, the dry powder composition of the present invention further comprises an anti-agglomerating agent, such as a carbohydrate or carbohydrate derivative, such as lactose, mannitol, and maltodextrin, preferably lactose.

[0015] In one embodiment, the dry powder composition of the present invention consists essentially of NaCl and lactose, hi one embodiment, the ratio of NaCl to lactose is 99:1 to 75:25 by wt%.

[0016] In one embodiment, the only therapeutic agent in the dry powder composition of the present invention is NaCl, or NaCl in combination with an anti-agglomerating agent.

[0017] In one embodiment, the dry powder composition of the present invention is administered in a daily dose of 0.5 to 200 mg, hi one embodiment, the daily dose is divided into 1 to 10 daily sessions of 4 to 10 mg per session.

[0018] In one embodiment, the dry powder compositions of the present invention are administered orally and / or intranasally. In one embodiment, the dry powder compositions of the present invention are administered to a human subject.

[0019] In one embodiment, the dry powder composition of the present invention reduces the time to cough resolution in a mammalian subject by at least 30% compared to a mammalian subject not treated with the dry powder composition.

[0020] In one embodiment, the NaCl of the dry powder compositions of the present invention is provided by a composition obtained from Voornom Salt Diapir, Denmark (N56 36.834 E009 42.070). [Brief explanation of the drawings]

[0021] [Figure 1] Mean % reduction of SARS-CoV-2 viral RNA (y-axis) in the supernatant of CCL-81 Vero cells treated with various concentrations of BREATHOX® powder (x-axis). Vero cells were pretreated with various concentrations of BREATHOX® powder for 1 hour prior to viral infection, followed by incubation with virus in the presence of BREATHOX® powder for 1 hour. [Figure 2] Luciferase assay that measures intracellular ATP concentration in cells 1 hour, 24 hours, and 72 hours after treatment with various concentrations of BREATHOX® powder by generating luminescence. [Figure 3]Indirect measurement of total cellular ATP concentration. Total ATP concentration of Vero cells treated with high concentrations of BREATHOX® powder for 1 hour was measured by luciferase assay. BREATHOX® powder was diluted in cell culture medium containing 0.6% NaCl (110 mM NaCl) with or without ouabain (5 μM). Data are plotted as the mean (*p<0.05) with standard error bars from 12 replicates. Sham = cells in the presence of high concentrations of BREATHOX® powder (white bars); Ouabain = cells in the presence of ouabain and high concentrations of BREATHOX® powder (gray bars). [Figure 4] Mitochondrial status of cultured Vero cells (40,000 cells / well) treated with various concentrations of BREATHOX® powder (0% wt / vol, 0.2% wt / vol, 0.4% wt / vol, 0.8% wt / vol, 0.9% wt / vol, 1.1% wt / vol, 1.2% wt / vol, and 1.4% wt / vol). Using Seahorse technology, (A) oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) were measured with a MitoStress Test Kit. Sequential injections of oligomycin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and rotenone plus antimycin A are shown in the cultured cells. A glycolysis stress test kit (Agilent Technologies) was applied to verify ECAR rates. Data are representative of three independent experiments and are shown as mean ± SEM; two-way ANOVA (*p ≤ 0.05). (C) The correlation between OCR and ECAR is plotted as an energy map. (D) Overview of mitochondrial respiration assessment under four different conditions: (i) basal respiration (corresponding to basal cellular oxygen consumption); (ii) proton leak (after addition of oligomycin (ATP synthase blocker)); (iii) uncoupled (after addition of the respiratory chain uncoupler FCCP, consumed oxygen reflects maximal respiration and is irreversibly uncoupled from ATP synthesis); and (iv) inhibition through total inhibition of complex I and complex III by rotenone and antimycin A, respectively. [Figure 5]Changes in intracellular Na+ concentration (Δ[Na+]i) in Vero cells. Vero cells cultured in medium containing 0.6% NaCl were incubated with sodium green (11 μM) and pluronic acid (0.07%) for 45 minutes. BREATHOX® powder was added to the cell culture at various concentrations. Changes in Na+ concentration were recorded using a Nikon Eclipse Ti inverted fluorescence microscope connected to an Andor CCD camera. (A) Cellular response lines representing cellular sodium influx detected by sodium green dye over time for three different concentrations of BREATHOX® powder (0.4% wt / vol, 0.8% wt / vol, and 1.1% wt / vol). Arrows indicate the time of BREATHOX® powder application. (B) Data from (A) plotted as the mean with standard error bars for 70 cells / 2 replicates. [Figure 6] Real-time PCR assay of Ato1B1 mRNA, encoding the Na+ / K+ ATPase channel protein, in Vero cells after 72 hours of incubation with BREATHOX® powder. [Figure 7] Study design to test the antiviral efficacy of BREATHOX® in patients with COVID-19. [Figure 8] Cough as a symptom of Covid-19 affecting the airways. Time to recovery from the onset of symptoms for patients treated with BREATHOX® 5 sessions / day for 10 days (Group 1), BREATHOX® 10 sessions / day for 10 days (Group 2), or standard care (Group 3). 1 session = 4 inhalations. The y-axis shows the percentage of patients experiencing cough at the time indicated on the x-axis (1.00 = 100%). The values ​​on the graphs below show the number of patients in each group experiencing cough at the time indicated on the x-axis of the graph. (A) Data plot for the first 10 days of treatment. (B) Same data as (A), but shown up to 20 days. [Figure 9]An inhaler suitable for administering a dry powder composition of the present invention: (A) horizontal cross-sectional view of the inhaler in the "CLOSED" and "OPEN" positions, (B) side perspective view of the inhaler in the "CLOSED" and "OPEN" positions, (C) exploded view of the inhaler. DETAILED DESCRIPTION OF THE INVENTION

[0022] Abbreviations, Terms and Definitions: As used herein, the term "respiratory tract" includes the upper respiratory tract (e.g., nasal cavity, nasal cavity, throat, pharynx), respiratory airways (e.g., pharynx, trachea, bronchi, bronchioles) and lungs (e.g., respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli).

[0023] The term "respiratory tract infection" is a technical term that refers to upper respiratory tract infections (e.g., infections of the nasal passages, pharynx, and throat) and lower respiratory tract infections (e.g., infections of the trachea, bronchus, and lungs) and combinations thereof. Typical symptoms associated with respiratory tract infections include nasal congestion, cough, rhinorrhea, sore throat, fever, facial pressure, nausea, chest pain, and shortness of breath.

[0024] As used herein, the term "dry powder" refers to a composition comprising finely dispersed, respirable dry particles that can be dispersed in an inhalation device and then inhaled by a subject. Such dry powders or dry particles are substantially free of water and other solvents or are anhydrous (i.e., water is absent) to avoid agglomeration. The dry powder of the present invention may be referred to as "NaCl powder," which should be understood herein as a dry powder composition comprising NaCl according to the present disclosure.

[0025] The term "micronized" as used herein in reference to a dry powder composition refers to a dry powder having particles of a size suitable for inhalation through the respiratory tract of a mammal, e.g., a human. Typically, such particle sizes are between 0.1 μm and 20 μm. The term "micronized" is not limited to the micronization process.

[0026] As used herein, the term "aerosol" refers to any preparation of fine cloud particles. In the present invention, specifically, non-liquid particles, i.e., dry powders, constitute an aerosol. Typically, the fine cloud particles in an aerosol have a volume median geometric diameter of about 0.1 to about 30 microns or a mass median aerodynamic diameter of about 0.5 to about 10 microns.

[0027] The term "excipient" as used herein means any suitable compound to support and / or enhance / improve the dryness and / or flowability of the sodium chloride powder.

[0028] The term "anti-agglomerating agent" as used herein means a compound, such as a sugar, particularly a micronized sugar, especially lactose, that prevents clumping of a dry powder composition, particularly sodium chloride.

[0029] As used herein, the term "viral infection" refers to any clinical sign or symptom caused by the invasion of the respiratory tract of a mammal, e.g., a human, by an inhaled virus. Viral particles may be deposited in the nasal or oral cavity, pharynx, trachea, primitive bronchi, secondary bronchi, terminal bronchi, and / or alveoli.

[0030] As used herein, the term "standard of care" refers to paracetamol.

[0031] As used herein, the terms "treatment" and "treating" refer to the management and care of a patient for the purpose of combating a condition, e.g., a disease or disorder. This term is intended to include any treatment for the specific condition from which a patient is suffering, such as the administration of a dry powder composition of the present invention to alleviate symptoms or complications, slow the progression of the disease, disorder or condition, alleviate or relieve symptoms and complications, and / or cure or eliminate the disease, disorder or condition, as well as to prevent a condition; "prevention" is to be understood as the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of a dry powder composition of the present invention to prevent the onset of symptoms or complications.

[0032] The terms "a," "an," and "the" and similar referents as used in the context of describing the present invention should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0033] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is intended to serve merely as a shorthand method of referring individually to each value falling within the range, and each individual value is incorporated herein as if it were individually set forth herein. Unless otherwise indicated, all exact values ​​provided herein represent the corresponding approximation (e.g., all exact exemplary values ​​given with respect to a particular factor or measurement can be considered to also provide the corresponding approximate measurement, modified by "about" as appropriate).

[0034] Detailed description of the invention: The present invention relates to a pharmaceutical composition for use in the treatment, prevention and / or amelioration of viral infections in the respiratory tract of a mammal, e.g., a human subject. In a preferred embodiment, the human subject treated with the pharmaceutical composition is infected with SARS-CoV-2 and / or diagnosed with COVID-19 infection.

[0035] More specifically, the present invention relates to an inhalable dry composition comprising sodium chloride (NaCl) for use in therapy to help prevent or reduce viral growth and inhibit viral replication in the respiratory tract of mammals, such as humans.

[0036] The present invention further relates to an inhalable dry composition comprising sodium chloride (NaCl) for use in reducing the duration of symptoms in viral infections. Typical symptoms are selected from one or more of fever, shortness of breath, nasal congestion, cough, sore throat, rhinorrhea, runny nose, fever, fatigue, body aches, and chest pain. In a further embodiment, the dry composition comprising sodium chloride promotes relief from post-COVID symptoms.

[0037] The compositions may be inhalable by the oral and / or nasal route - thus the dry powder composition comprises inhalable sodium chloride particles of a size suitable to reach the respiratory tract of a mammal. In a preferred embodiment, the sodium chloride dry powder is a micronized dry powder.

[0038] Inhalation therapy can provide a drug delivery system that is simple and safe to use in hospital or outpatient settings. Because nebulization can potentially expose others to contaminated aerosols, the present invention can preferably replace treatments using nebulized formulations, ultimately protecting medical personnel and other subjects in the immediate vicinity of the patient.

[0039] Various embodiments of the present invention are described below, and unless an embodiment is explicitly stated to relate to a particular aspect or aspects of the invention, it should be considered to refer to any one of the aspects described herein as well as any one of the embodiments described herein.

[0040] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0041] I. Pharmaceutical Dry Powder Formulations In one aspect, the present invention provides an inhalable dry powder formulation of a pharmaceutical agent comprising NaCl as its major component.

[0042] II. Composition of pharmaceutical dry powder formulations NaCl is a major component of pharmaceutical dry powder formulations.

[0043] Without wishing to be bound by theory, the inventors observed that sodium chloride reduces intracellular ATP concentrations; this is one mechanism to explain how the dry powder compositions of the present invention reduce SARS-CoV-2 replication in mammalian cells. When Vero cells were treated with BREATHOX® powder with or without ouabain (Na+ / K+ ATPase transporter inhibition), it was observed that the reduced levels of ATP were caused by increased activity of the cellular Na+ / K+ ATPase transporter (see Example 2).

[0044] In one embodiment, the amount of sodium chloride in the dry powder composition is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% by weight, preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147 , 74%, 75%, 76%, 77%, 78%, or 79% by weight, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% by weight, and most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight.

[0045] In one embodiment, the amount of sodium chloride in the dry powder composition is 70-99% by weight, 71-99% by weight, 72-99% by weight, 73-99% by weight, 74-99% by weight, 75-99% by weight, 76-99% by weight, 77-99% by weight, 78-99% by weight, 79-99% by weight, 80-99% by weight, 81-99% by weight, 82-99% by weight, 83-99% by weight, 84-99% by weight, 85-99% by weight, 86-99% by weight, 87-99% by weight, 88-99% by weight, 89-99% by weight, 90-99% by weight, 91-99% by weight, 92-99% by weight, 93-99% by weight, 94-99% by weight, 95-99% by weight, 96-99% by weight, 97-99% by weight, or 98-99% by weight. In one embodiment, the amount of sodium chloride in the dry powder composition is 89-99% by weight, 90-98% by weight, 91-97% by weight, 92-97% by weight, 93-97% by weight, 94-96% by weight, for example, preferably about 95% by weight.

[0046] A high wt. % of NaCl in the dry powder composition ensures localized delivery of high concentrations of NaCl to the desired location, e.g., within the respiratory tract, as disclosed in more detail herein. Targeted delivery of high concentrations of NaCl is advantageous because the higher the concentration of NaCl, the greater the penetration power of the compound. Example 1 also showed that increasing the concentration of BREATHOX® powder reduced SARS-CoV-2 replication in proportion to the concentration of BREATHOX®. The higher the concentration of BREATHOX® powder, the greater the reduction in viral replication.

[0047] In one embodiment, the sodium chloride, e.g., SANAL® P+ (available from Dansk Salt A / S), is of pharmaceutical quality. Typically, the sodium chloride is SANAL® (Permian raw salt produced in accordance with GMP-ICH Q7). Preferably, the sodium chloride is extracted from the Vornom Salt Diapir (N56 36.834 E009 42.070), Denmark; for example, sodium chloride is extracted from the Danish underground by Maricogen A / S and / or Nouryon. Salt compositions derived from the Vornom Salt Diapir are known to be of very high purity—this is relevant given the important role that salt in its purest form, free of additives, plays in the pharmaceutical industry. In a preferred embodiment, the NaCl in the dry powder composition of the present invention is provided by a composition obtained from the Vornom Salt Diapir (N56 36.834 E009 42.070), Denmark.

[0048] In one embodiment, sodium chloride can be prepared in accordance with the European Pharmacopoeia Monograph on Sodium Chloride, current version no. 193. Micronized sodium chloride is the main component of pharmaceutical dry powder formulations. Inhalable dry powder compositions may contain small amounts of additional powders, preferably micronized powders, of other salts and / or minerals and / or excipients.

[0049] Excipient carrier particles may be part of a pharmaceutical formulation and may be co-delivered with a therapeutic aerosol to aid in achieving efficient aerosolization, among other possible benefits. In one embodiment, the dry powder composition includes an excipient. In certain preferred embodiments, the excipient is an anti-agglomerating agent. In one embodiment, the excipient is an anti-agglomerating agent and is a carbohydrate. In one embodiment, the excipient is an anti-agglomerating agent and is a sugar or sugar derivative. In one embodiment, the excipient is an anti-agglomerating agent selected from a disaccharide, a sugar alcohol, and a polysaccharide. In one embodiment, the anti-agglomerating agent is selected from various amino acids. In a preferred embodiment, the anti-agglomerating agent is selected from lactose, mannitol, and maltodextrin.

[0050] To enhance the flowability of the dry powder and reduce aggregation of salt particles due to external factors, anti-agglomerating agents such as lactose, amino acids (e.g., leucine) and mannitol are added, which act in part as carriers.

[0051] In one embodiment, the amount of anti-agglomerating agent, e.g. lactose, in the dry powder composition is at most 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, or 31% by weight, preferably at most 30%, 29%, 28%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, or 31% by weight. %, 27%, 26%, 25%, 24%, 23%, 22%, or 21% by weight, more preferably at most 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, or 11% by weight, and even more preferably at most 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight. In one embodiment, the amount of anti-agglomerating agent, such as lactose, in the dry powder composition is 1-10%, 2-8%, 3-7%, 4-6%, or preferably about 5% by weight.

[0052] In a preferred embodiment, the anti-agglomerating agent is lactose, hi one embodiment, the lactose is pharmaceutical grade alpha-lactose monohydrate, e.g., CAS number 7647-14-5, conforming to the European Pharmacopoeia.

[0053] In one embodiment, the dry powder composition essentially consists of NaCl and lactose. In one embodiment, NaCl and lactose together account for at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or at least 89% by weight of the total dry powder composition, preferably at least 90%, 91%, 92%, 93%, 94%, or at least 95% by weight of the total dry powder composition, more preferably at least 95%, 96%, 97%, 98%, or at least 99% by weight of the total dry powder composition; for example, at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or even 100% by weight of the total dry powder composition. In one embodiment, the dry powder composition of the present invention consists of NaCl and lactose.

[0054] In one embodiment, the weight ratio between sodium chloride and the anti-agglomerating agent, such as lactose, is 99 / 1 to 50 / 50, 99 / 1 to 55 / 45, 99 / 1 to 60 / 40, 99 / 1 to 65 / 35, or 99 / 1 to 70 / 30, 99 / 1 to 75 / 25, 99 / 1 to 70 / 30, 99 / 1 to 85 / 25, 99 / 1 to 90 / 10, 98 / 2 to 90 / 10, 97 / 3 to 90 / 10, 96 / 4 to 90 / 10, 95 / 5 to 90 / 10, 99 / 1 to 95 / 5, 98 / 2 to 95 / 5, 97 / 3 to 95 / 5, or 96 / 4 to 95 / 5. Preferably, the weight ratio between sodium chloride and lactose is approximately 95 / 5.

[0055] In one embodiment, the dry powder composition comprises 50-99% by weight NaCl and 1-50% by weight lactose, 60-99% by weight NaCl and 1-40% by weight lactose, or 70-99% by weight NaCl and 1-30% by weight lactose, for example preferably 75-99% by weight NaCl and 1-25% by weight lactose, more preferably 80-99% by weight NaCl and 1-20% by weight lactose, more preferably 85-99% by weight NaCl and 1-25% by weight lactose, more preferably 90-99% by weight NaCl and 1-10% by weight lactose; most preferably the dry powder composition comprises approximately 95% by weight NaCl and 5% by weight lactose.

[0056] In one embodiment, the composition consists essentially of NaCl and lactose, in weight ratios as disclosed above.

[0057] In a preferred embodiment, the dry powder composition of the present invention consists essentially of NaCl and lactose in a ratio of approximately 95:5 by weight. "Consisting essentially of" and "consisting essentially of" means that only minor impurities are present in the composition, which is considered inactive with respect to having a therapeutic effect and is also considered inactive with respect to functioning as an anti-agglomerating agent.

[0058] As further disclosed herein and as evidenced by the examples provided herein, the NaCl in the dry powder composition acts as a therapeutic agent when administered to a mammalian subject. In one embodiment, NaCl is the only therapeutic agent in the pharmaceutical composition, and all other ingredients are inactive with respect to a therapeutic effect, such as treating and / or preventing and / or ameliorating a viral infection in the respiratory tract of a mammal. In one embodiment, the dry powder composition includes lactose, and the only therapeutic agents in the composition are NaCl and lactose. In another embodiment, additional therapeutic agents, e.g., other antiviral agents such as oseltamivir, zanamivir (a misspelling of zanamivir), amantadine, rimantadine, ribavirin, ganciclovir, valganciclovir, foscavir, Cytogam® (cytomegalovirus immune globulin), pleconaril, rupintrivir, palivizumab, motavizumab, cytarabine, docosanol, denotivir, cidofovir, acyclovir, or, for example, Paquilobid™ (nilmatrervir and ritonavir) or Lagebrio™ (molnupiravir), may be present in the composition.

[0059] I.ii Particle size Dry powder formulations are prepared with particle size and density suitable for local delivery to selected areas of the respiratory tract.For example, higher density and / or larger particles can be used in upper respiratory tract transport, while lower density and / or smaller particles can be used in lower respiratory tract transport.Similarly, a mixture of particles of different sizes can be administered to different areas of the lungs to target in one administration.

[0060] For the present invention, delivery to the respiratory tract and airways is preferred, and therefore the dry compositions of the present invention are preferably micronized to a respirable particle size range of 1-10 μm.

[0061] The dry powder composition is administered by dry inhalation as an aerosol, i.e., a cloud of fine dry particles. Preferably, dry powder aerosols for inhalation therapy are produced having a mean diameter within the range of 1 to 10 microns. Preferably, the volume median geometric diameter of the aerosol particles is less than about 10 microns.

[0062] A preferred volume median geometric diameter for the aerosol particles is about 5 microns. For example, the aerosol can include particles having a volume median geometric diameter of about 0.1 to about 30 microns, about 0.5 to about 20 microns, about 0.5 to about 10 microns, about 1.0 to about 5.0 microns, or about 2.0 to 5.0 microns.

[0063] All dry powder particles have a particle size (i.e., average diameter) of less than 20 microns, less than 19 microns, less than 18 microns, less than 17 microns, less than 16 microns, less than 15 microns, less than 14 microns, less than 13 microns, less than 12 microns, or less than 11 microns, preferably less than 10 microns, less than 9 microns, less than 8 microns, less than 7 microns, or less than 6 microns, more preferably all dry particles have a particle size of about 5 microns.

[0064] Malvern particle size analyzers, such as the Malvern Mastersizer 3000 instrument, are used to define and measure particle size distribution. The particle size distribution (PSD) of a powder defines the relative amount of particles present according to particle size.

[0065] In one embodiment, the dry powder composition has a particle size distribution in which at least 50% of the particles are in the 1-10 μm range. In a preferred embodiment, the dry powder composition has a particle size distribution in which at least 60% of the particles are in the 1-10 μm range. In a more preferred embodiment, the dry powder composition has a particle size distribution in which at least 70% of the particles are in the 1-10 μm range. In a most preferred embodiment, the dry powder composition has a particle size distribution in which at least 80% of the particles are in the 1-10 μm range. In an even more preferred embodiment, the dry powder composition has a particle size distribution in which at least 90% of the particles are in the 1-10 μm range, more preferably at least 95% of the particles are in the 1-10 μm range.

[0066] In one embodiment, the dry powder composition has a particle size distribution in which at least 50% of the particles are in the 2-5 μm range. In a preferred embodiment, the dry powder composition has a particle size distribution in which at least 60% of the particles are in the 2-5 μm range. In a more preferred embodiment, the dry powder composition has a particle size distribution in which at least 70% of the particles are in the 2-5 μm range. In a most preferred embodiment, the dry powder composition has a particle size distribution in which at least 80% of the particles are in the 2-5 μm range. In an even more preferred embodiment, the dry powder composition has a particle size distribution in which at least 90% of the particles are in the 2-5 μm range, more preferably at least 95% of the particles are in the 2-5 μm range.

[0067] I.iii Preparation of pharmaceutical dry powder formulations Generally, dry powder pharmaceutical formulations can be produced by any of spray drying, freeze drying, jet milling, single and double emulsion solvent evaporation, and supercritical fluids. Preferably, dry powder formulations are produced by jet milling. Jet milling is a particle size reduction method in which unmilled powder is fed into a milling chamber. Within the chamber, compressed air and / or nitrogen, usually in a vortex motion, promotes interparticle collisions. Typically, jet mills are designed to eject particles below a certain size while continuing to grind particles above that size, resulting in a narrow particle size distribution of the resulting product. Jet-milled powders containing the salts of the present invention, such as the sodium salts, can be easily prepared using conventional methods.

[0068] Dry powder formulations can also be prepared by blending individual components into a final pharmaceutical formulation. For example, a first dry powder containing a salt can be blended with an additional dry powder containing an excipient (e.g., lactose) to be included in the blend. This blend can contain the salt, excipient, and optionally other additional components in any desired relative amounts or ratios, in accordance with the teachings of the present invention.

[0069] II. Pharmaceutical Dry Powder Compositions for Use in the Treatment and / or Prevention of Viral Respiratory Tract Infections One aspect of the present invention provides a pharmaceutical composition as described herein for use as a medicament, wherein the pharmaceutical composition is administered by dry powder inhalation to a mammal in need thereof. The mammal may be a human, a primate, a mouse, a rat, a dog, a cat, a horse, or a livestock or animal raised for food, such as a cow, a sheep, a pig, a chicken, and a goat. In a preferred embodiment, the mammal is a human.

[0070] As shown herein, the pharmaceutical compositions of the present invention can inhibit viral infections, e.g., reduce the viral load of SARS-CoV-2 (Example 1). As further demonstrated herein, administration of the pharmaceutical compositions to the respiratory tract of a mammal suffering from a viral infection, e.g., COVID, promotes significant relief of cough symptoms in the patient (Example 3).

[0071] The present invention provides a pharmaceutical composition as described herein for use in the treatment and / or prevention of infectious diseases of the respiratory tract, preferably viral infections of the respiratory tract, wherein the composition is administered by dry powder inhalation.

[0072] The present invention further provides methods for the treatment (including prophylactic treatment) of infectious diseases of the respiratory tract, such as viral infections of the respiratory tract. In one embodiment, the present invention provides a method for treating (including prophylactically treating) an individual having, exhibiting symptoms of, or at risk of contracting a viral infection of the respiratory tract, comprising administering to the individual's respiratory tract an effective amount of a pharmaceutical formulation described herein.

[0073] In certain embodiments, the viral infection is caused by a virus selected from the group consisting of influenza virus (e.g., influenza virus A, influenza virus B), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, adenovirus, coxsackievirus, echovirus, herpes simplex virus, poxvirus (e.g., smallpox), enterovirus, and coronavirus. In a preferred embodiment, the viral infection is a coronavirus, such as SARS-coronavirus. In a more preferred embodiment, the viral infection is caused by a betacoronavirus, such as a subgenus Saberbekovirus (SARS betacoronavirus). In a most preferred embodiment, the viral infection is specifically SARS-CoV-2.

[0074] In a preferred embodiment, the present invention provides a pharmaceutical composition as described herein for use in the treatment and / or prevention of a viral respiratory tract infection caused by a coronavirus, wherein the composition is administered to a mammalian subject by dry powder inhalation.In a preferred embodiment, the present invention provides a pharmaceutical composition as described herein for use in the treatment and / or prevention of a viral respiratory tract infection caused by SARS-CoV-2, wherein the composition is administered to a mammalian subject by dry powder inhalation.

[0075] In one embodiment, the present invention provides a pharmaceutical composition described herein for use in the treatment and / or prevention of infectious diseases of the respiratory tract, preferably viral infections of the respiratory tract, wherein the composition is administered by dry powder inhalation and alleviates the symptoms of viral respiratory tract infections. In some embodiments, the symptoms may be selected from one or more of fever, shortness of breath, nasal congestion, cough (e.g., non-productive), sore throat, rhinorrhea, vomiting, nausea, diarrhea, sneezing, fatigue, body pain, muscle pain, taste disorders, anosmia, headache, and chest pain.

[0076] In a preferred embodiment, the pharmaceutical composition of the present invention reduces coughing, such as dry coughing, caused by a viral respiratory tract infection. In a preferred embodiment, the pharmaceutical composition of the present invention reduces the frequency of coughing caused by a viral respiratory tract infection compared to a person not treated with the composition. In one embodiment, a reduction in the frequency of coughing of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 95% is achieved 5 days after treatment compared to an untreated patient. In one embodiment, a reduction in the frequency of coughing of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 95% is achieved 10 days after treatment compared to an untreated patient. Cough frequency is typically measured by patient self-assessment, such as by the American Thoracic Society St. George's Respiratory Questionnaire (SGRQ), a disease-specific measure designed to measure the impact on overall health, daily activities, and perceived well-being in patients with obstructive airway disease (www.thoracic.org / members / assemblies / assemblies / srn / questionaires / sgrq.php).

[0077] In a preferred embodiment, the pharmaceutical composition of the present invention reduces the severity of cough, e.g., dry cough, caused by a viral respiratory tract infection, compared to a person not treated with the composition. In one embodiment, a reduction in cough severity of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 95% is achieved 5 days after treatment compared to an untreated patient. In one embodiment, a reduction in cough severity of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 95% is achieved 10 days after treatment compared to an untreated patient. Cough severity is typically measured by patient self-assessment, for example by the St. George's Respiratory Questionnaire.

[0078] In one embodiment, use of a composition according to the present invention promotes a significantly reduced recovery time in a patient suffering from a viral infection compared to the same patient not being treated with such a composition. In one embodiment, the dry powder composition of the present invention reduces the time for resolution of one or more symptoms caused by a viral respiratory tract infection. In one embodiment, the mean time to recovery from cough (e.g., as a symptom of Covid-19 affecting the respiratory tract) is significantly improved. The terms "time to recovery" and "time to resolution" are used interchangeably herein. In one embodiment, an average of 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or even 70% of patients suffering from a viral respiratory tract infection and treated with a dry powder composition of the invention recover from cough within 10 days of illness onset. In one embodiment, an average of 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or even 70% of coronavirus patients treated with a dry powder composition of the invention recover from cough within 10 days of illness onset. The term "recover from cough" covers complete cessation of coughing in the patient or reduction of coughing to the patient's normal cough level when not suffering from said viral respiratory tract infection.

[0079] In one embodiment, an average of 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or even up to 90% of patients suffering from a viral respiratory tract infection and treated with a dry powder composition of the invention will recover from cough within 18 days of illness onset. In one embodiment, an average of 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or even up to 90% of coronavirus patients treated with a dry powder composition of the invention recover from cough within 18 days of illness onset.

[0080] As shown herein (Example 2), on average, coronavirus patients treated with the dry powder composition of the present invention recover from COVID-19-induced cough 30-40% faster than untreated controls.

[0081] In one embodiment, the invention provides a dry powder composition disclosed herein for use in treating one or more symptoms of a viral respiratory tract infection, wherein the time to resolution of one or more symptoms in a patient is improved, on average, by at least 10%, 12%, 14%, 16%, 18%, or 20% compared to an untreated control group, such as by at least 22%, 24%, 26%, 28%, or 30% compared to an untreated control group, such as by at least 32%, 34%, 36%, 38%, or 40% compared to an untreated control group, such as by at least 42%, 44%, 46%, 48%, or 50% compared to an untreated control group.

[0082] In one embodiment, the invention provides a dry powder composition disclosed herein for use in treating cough, e.g., non-productive cough, caused by a viral respiratory tract infection, wherein the time to cough resolution in a patient suffering from said viral respiratory tract infection is improved by at least 10%, 12%, 14%, 16%, 18%, or 20% compared to an untreated control group, such as an average improvement of at least 22%, 24%, 26%, 28%, or 30% compared to an untreated control group, such as an improvement of at least 32%, 34%, 36%, 38%, or 40% compared to an untreated control group, such as an improvement of at least 42%, 44%, 46%, 48%, or 50% compared to an untreated control group. In a preferred embodiment, the cough in said patient is caused by a coronavirus.

[0083] In a further aspect, the present invention provides a pharmaceutical composition as described herein for use in the treatment and / or prevention of pulmonary disease (i.e., a type of disease that affects the lungs and other parts of the respiratory system), wherein the composition is administered by dry powder inhalation.

[0084] In one embodiment, the present invention provides a method for treating (including prophylactically treating) an individual with a pulmonary disease, such as an individual having, exhibiting symptoms of, or susceptible to a pulmonary disease, comprising administering to the individual's airways an effective amount of a pharmaceutical formulation according to the present invention by dry powder inhalation.

[0085] Preferably, the present invention provides a dry powder composition disclosed herein for use in the prevention and / or treatment of a viral respiratory tract infection caused by a coronavirus, such as SARS-CoV-2, which is administered to a mammalian subject by dry powder inhalation. Preferably, the dry powder composition contains at least 80%, 85%, or 90% by weight of sodium chloride, or more preferably about 95% by weight of sodium chloride. Pharmaceutical formulations used to treat (including prophylactically treat) respiratory tract infections preferably contain sodium chloride salt and lactose, preferably in a ratio of NaCl salt to lactose of about 95:5 (wt / wt).

[0086] More preferably, the present invention provides a dry powder composition disclosed herein for use in treating and / or alleviating cough caused by a viral respiratory tract infection, the composition being administered to a mammalian subject by dry powder inhalation. Preferably, the dry powder composition contains at least 80%, 85%, or 90% by weight of sodium chloride, or more preferably about 95% by weight of sodium chloride. Pharmaceutical formulations used to treat and / or alleviate cough preferably contain sodium chloride salt and lactose, preferably in a ratio of NaCl salt to lactose of about 95:5 (wt / wt).

[0087] III. Administration of Pharmaceutical Dry Powder Formulations It is an essential feature of the present invention that the dry powder composition is administered by dry powder inhalation, thus providing NaCl particle inhalation therapy with significant advantages over the use of standard nebulized solutions for both patients and healthcare professionals, due to the safety considerations of minimizing viral contamination of the aerosol.

[0088] III.i Dry powder inhalation When selecting an appropriate method for generating and delivering aerosols of pharmaceutical formulations to the respiratory tract, the geometry of the respiratory tract is an important consideration. The lungs are designed to trap inhaled foreign matter, such as dust particles. As disclosed herein, dry powder inhalation is selected for administration with an appropriate particle size to preferentially deliver to the desired region of the respiratory tract. Particles with a diameter of 0.6 to 5 microns generally reach the deep lungs, while particles with a diameter of about 3 microns or larger generally remain in the upper respiratory tract.

[0089] Widespread clinical application of dry powder inhalation delivery has been limited by the difficulty of producing dry powders with appropriate particle size, particle density, and dispersibility, keeping the dry powders dry when stored, and developing a convenient, handheld device that effectively disperses the inhaled respirable dry particles into the air.

[0090] The pharmaceutical preparations described herein are intended to be administered to the respiratory tract (for example, to the mucosal surface of the respiratory tract) in dry powder form.Preferably, the pharmaceutical preparations described herein are aerosolized for administration.Many suitable methods and devices known in the art, such as dry powder inhalers, can be used to aerosolize the formulation into dry powder.

[0091] III.ii Dry powder inhaler Inhalable dry powders are typically administered to the respiratory tract, including the lungs and nasal membranes, by a dry powder inhaler.

[0092] In one embodiment, the dry powder inhaler is suitable for nasal inhalation. In another embodiment, the dry powder inhaler is suitable for oral inhalation. In yet another embodiment, the dry powder composition can be inhaled orally and nasally. Several suitable dry inhaler devices exist. The composition is preferably administered in a single breath-actuated step using a breath-actuated dry powder inhaler (DPI).

[0093] In certain embodiments, the dry powder inhaler is as described in WO2015004227 (U.S. Pat. No. 10,583,261), which is incorporated by reference in its entirety.

[0094] Specifically, the dry powder inhaler shown in Figure 9 (a copy of Figures 1a, 1b, 2a, 2b, and 3 of WO2015004227) is a preferred embodiment. Accordingly, in a preferred embodiment of the present invention, a dry powder inhaler used to administer a dry powder composition of the present invention has, in an axial direction, a proximal end (P) for insertion into a user's mouth and a distal end (D) opposite the proximal end (P), the inhaler including an inlet, an outlet disposed at the proximal end (P), an air passageway extending from the inlet to the outlet, and a reservoir communicating with the air passageway through a discharge orifice, the reservoir containing a dispersible material, the inhaler having a proximal portion including the outlet and a distal portion attached to the proximal portion, and having an "OPEN" position in which the proximal portion is deployed from the distal portion and an "OPEN" position in which the proximal portion is directed toward the distal portion. The proximal portion is linearly slidable along an axial direction (A) relative to the distal portion between a retracted "CLOSED" position and a retracted "CLOSED" position, and the inhaler further includes an inlet valve member, an outlet valve member, and a reservoir valve member, the inlet, outlet, and reservoir valve members being arranged to simultaneously close the inlet, outlet, and discharge orifice when the proximal portion moves from the "OPEN" position to the "CLOSED" position, and to simultaneously open the inlet and open the outlet when the proximal portion moves from the "CLOSED" position to the "OPEN" position, delivering a quantity of the dispersible substance from the reservoir through the discharge orifice to the air passageway.

[0095] In a further preferred embodiment, the dry powder inhaler is an inhaler (1) having, in an axial direction, a proximal end (P) for insertion into a user's mouth and a distal end (D) opposite the proximal end (P), the inhaler (1) including an inlet (4), an outlet (5) disposed at the proximal end (P), air passages (10, 11, 12, 13) extending from the inlet (4) to the outlet (5), and reservoirs (8, 9) in communication with the air passages (10, 11, 12, 13) through discharge orifices (6, 7), the reservoirs (8, 9) contains a dispersible substance, the inhaler (1) has a proximal portion (2) including an outlet (5), and a distal portion (3) attached to the proximal portion (2), the proximal portion (2) being linearly slidable along an axial direction (A) relative to the distal portion (3) between an "OPEN" position in which the proximal portion (2) is extended from the distal portion (3) and a "CLOSED" position in which the proximal portion (2) is retracted toward the distal portion (3), the inhaler (1) further including an inlet valve member (14) and an outlet valve member (15); - reservoirs (8, 9) are arranged in the proximal part (2); - the inlet (4) comprises one or more openings in the housing wall (20) around the proximal portion (2), one or more openings facing radially outward away from the axial direction (A), and when the proximal portion (2) is in the "CLOSED" position, the inlet valve member (14) is formed by the housing wall (30) around the distal portion (3) covering the openings; the outlet (5) comprises an axial opening; The outlet valve member (15) is formed as a plug attached to the distal part (3), the plug blocking said outlet (5) when the proximal part (2) is in the "CLOSED" position; - the discharge orifices (6, 7) are oriented in the axial direction (A); - the inhaler (1) further comprises reservoir valve members (16, 17), which are formed as pegs that move in an axial direction (A), and which are fixed to the distal part (3) via an axially extending body, and which block the discharge orifices (6, 7) when the proximal part (2) is in the "CLOSED" position; Thereby, the inlet valve member (14), the outlet valve member (15) and the reservoir valve members (16, 17) - when the proximal part (2) is retracted axially towards the distal part from the "OPEN" position to the "CLOSED" position, it simultaneously closes the inlet (4), the outlet (5) and the discharge orifices (6, 7); and - when the proximal portion (2) is deployed axially from the distal portion from the "CLOSED" position to the "OPEN" position, simultaneously opening the inlet (4) and opening the outlet (5) to deliver a quantity of dispersible substance from the reservoir (8, 9) through the discharge orifices (6, 7) to the air passages (10, 11, 12, 13); are arranged as follows.

[0096] Further preferred embodiments are specifically described in WO 2015004227. In one preferred embodiment, the dry powder inhaler for administering the dry powder compositions of the invention is a BREATHOX®.

[0097] The BREATHOX® inhaler contains 1000 mg ± 150 mg of dry powder, referred to herein as BREATHOX® powder. The device delivers 90% of doses within 2.1 ± 0.6 mg per inhalation and 95% of doses within the specified critical value of 2.0 ± 1.0 mg. This equates to a minimum of 300 inhalations per device. The 300 lifetime doses are based on a worst-case scenario where 90% of doses are 2.7 mg (2.1 + 0.6 mg). 300 doses of 2.7 mg equate to 810 mg of salt, which is 40 mg below the minimum fill tolerance (850 mg). 40 mg remains in the device, significantly reducing the likelihood of a maximum lifetime dose.

[0098] III.iii Dosage regimen The pharmaceutical formulations of the present invention can be delivered to the upper respiratory tract (e.g., nostrils, nasal cavity, throat, pharynx), respiratory airways (e.g., pharynx, trachea, bronchi, bronchioles) and / or lungs (e.g., respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli).

[0099] In one aspect, the invention relates to a dry powder composition disclosed herein for use in inhibiting or reducing viral growth and inhibiting viral replication in the respiratory tract of a mammal, e.g., a human, wherein an effective amount of the dry powder composition is administered to the mammal by dry powder inhalation.

[0100] In a further aspect, the present invention relates to a method for preventing or reducing viral growth and inhibiting viral replication in the respiratory tract of a mammal, e.g., a human, wherein an effective amount of a dry powder composition of the present invention is administered by dry powder inhalation to a mammal in need of said treatment.

[0101] An effective amount of the pharmaceutical formulations described herein is administered to an individual in need thereof, such as an individual having a respiratory tract infection, an individual exhibiting symptoms of a respiratory tract infection, or an individual at risk of contracting a respiratory tract infection. An "effective amount" is an amount sufficient to achieve the desired therapeutic or prophylactic effect, e.g., an amount sufficient to alleviate the symptoms of infection.

[0102] Preferably, the inhalation device should be capable of delivering a therapeutically effective amount of the compositions described herein in a single inhalation, although in some cases multiple inhalations and / or frequent administration may be required to achieve the desired therapeutic result.

[0103] Generally, pharmaceutical formulations are administered once, twice, three times, four times, five times, six times, or more times per day as needed. The appropriate interval between doses to provide the desired therapeutic effect can be determined based on the severity of the condition (e.g., infection).

[0104] The composition of the present invention is non-toxic to human subjects and most mammals.Dosage can be based on the desired amount of salt delivered to the respiratory tract.The daily dose to be administered can be determined according to the concentration of virus particles in the respiratory tract and the frequency of inhalation of such virus particles.

[0105] During the COVID-19 trial (Example 3), patients in Group 1 received 5 sessions of BREATHOX® per day, equivalent to 22.5-30 mg of BREATHOX® powder (NaCl powder), while patients in Group 2 instead received 10 sessions of BREATHOX® per day, equivalent to 45-60 mg of BREATHOX® powder (NaCl powder).

[0106] In a preferred embodiment, the total daily dose administered is 4.5-60 mg, which corresponds to 1-10 sessions per day when using, for example, a BREATHOX® device.

[0107] In one embodiment, the total daily dose of the composition of the invention administered to a human subject is 0.5 to 200 mg, 1 to 180 mg, 2 to 150 mg, 3 to 120, 4 to 90 mg, or preferably 5 to 60 mg, such as 10 to 55 mg, 20 to 50 mg, 30 to 45 mg, for example about 40 mg; such as 5 to 55 mg, 5 to 50 mg, 5 to 45 mg, 5 to 40 mg, 5 to 35 mg, 5 to 30 mg, 5 to 25 mg, 5 to 20 mg, 5 to 15 mg, or 5 to 10 mg; such as 55 to 60 mg, 50 to 60 mg, 45 to 60 mg, 40 to 60 mg, 35 to 60 mg, 30 to 60 mg, 25 to 60 mg, 20 to 60 mg, 15 to 60 mg, or 10 to 60 mg. These daily doses are particularly preferred for the treatment and / or prevention of coronaviruses, such as the treatment and / or prevention of COVID-19.

[0108] In one embodiment, the total daily dose of NaCl administered to a human subject is 0.5 to 200 mg, 1 to 180 mg, 2 to 150 mg, 3 to 120 mg, 4 to 90 mg, or preferably 5 to 60 mg, such as 10 to 55 mg, 20 to 50 mg, 30 to 45 mg, for example about 40 mg; such as 5 to 55 mg, 5 to 50 mg, 5 to 45 mg, 5 to 40 mg, 5 to 35 mg, 5 to 30 mg, 5 to 25 mg, 5 to 20 mg, 5 to 15 mg, or 5 to 10 mg; such as 55 to 60 mg, 50 to 60 mg, 45 to 60 mg, 40 to 60 mg, 35 to 60 mg, 30 to 60 mg, 25 to 60 mg, 20 to 60 mg, 15 to 60 mg, or 10 to 60 mg. These daily doses are particularly preferred for the treatment and / or prevention of coronaviruses, such as the treatment and / or prevention of COVID-19.

[0109] In preferred embodiments, the total daily dose of the compositions of the invention administered to a human subject is 0.005-5 mg / kg body weight, 0.005-4 mg / kg body weight, 0.005-3 mg / kg body weight, 0.005-2 mg / kg body weight, 0.005-1 mg / kg body weight, 0.005-0.5 mg / kg body weight, 0.005-0.2 mg / kg body weight, 0.005-0.1 mg / kg body weight, or 0.005-0. 0.05 mg / kg body weight, for example, 4 to 5 mg / kg body weight, 3 to 5 mg / kg body weight, 2 to 5 mg / kg body weight, 1 to 5 mg / kg body weight, 0.5 to 5 mg / kg body weight, 0.2 to 5 mg / kg body weight, 0.1 to 5 mg / kg body weight, or 0.05 to 5 mg / kg body weight, for example, 0.05 to 4 mg / kg body weight, 0.1 to 3 mg / kg body weight, 0.2 to 2 mg / kg body weight, or 0.5 to 1 mg / kg body weight.

[0110] In preferred embodiments, the total daily dose of NaCl administered to a human subject is 0.005-5 mg / kg body weight, 0.005-4 mg / kg body weight, 0.005-3 mg / kg body weight, 0.005-2 mg / kg body weight, 0.005-1 mg / kg body weight, 0.005-0.5 mg / kg body weight, 0.005-0.2 mg / kg body weight, 0.005-0.1 mg / kg body weight, or 0.005-0.0 5 mg / kg body weight, for example, 4 to 5 mg / kg body weight, 3 to 5 mg / kg body weight, 2 to 5 mg / kg body weight, 1 to 5 mg / kg body weight, 0.5 to 5 mg / kg body weight, 0.2 to 5 mg / kg body weight, 0.1 to 5 mg / kg body weight, or 0.05 to 5 mg / kg body weight, for example, 0.05 to 4 mg / kg body weight, 0.1 to 3 mg / kg body weight, 0.2 to 2 mg / kg body weight, or 0.5 to 1 mg / kg body weight.

[0111] In one embodiment, the dry powder composition is administered to a patient once daily. In a preferred embodiment, the dry powder composition of the present invention is administered to a patient several times daily, for example, two, three, four, five, six, seven, eight, nine, ten, or more times daily, such that the total daily dose is administered in several unit doses. This may also be referred to as administration in several sessions throughout the day. In one embodiment, the dry powder composition is administered to a patient 1-10 times daily, 1-9 times daily, 1-8 times daily, 1-7 times daily, 1-6 times daily, 1-5 times daily, 1-4 times daily, 1-3 times daily, or 1-2 times daily. In one embodiment, the dry powder composition is administered to a patient 2-10 times daily, 3-10 times daily, 4-10 times daily, 5-10 times daily, 6-10 times daily, 7-10 times daily, 8-10 times daily, or 9-10 times daily. In one embodiment, the dry powder composition is administered to a patient, preferably 1 to 5 times daily, such as 2 to 5 times daily, for example 2 to 4 times daily, such as 3 times daily.

[0112] When a mammal, e.g., a human, inhales a single dose of the dry powder composition, such a dose is considered a unit dose. The unit dose is determined by the type of dry powder inhaler used. A single administration session may include multiple inhalations—i.e., multiple dose units, e.g., 2, 3, 4, 5, 6, or more dose units per session. Typically, dry powder inhalers are configured to deliver 0.25 to 25 mg, e.g., 0.25 to 4 mg, of dry powder per inhalation. In a most preferred embodiment, the inhaler delivers 1.5 to 2 mg of dry powder per inhalation.

[0113] As an illustrative example, and as a preferred embodiment, a BREATHOX® inhaler contains 1000 mg ± 150 mg of BREATHOX® powder (NaCl powder according to the present invention). This device delivers 90% of doses within 2.1 ± 0.6 mg per inhalation and 95% of doses within the specified critical value of 2.0 ± 1.0 mg. This equates to a minimum of 300 inhalations per device. The 300 lifetime doses are based on a worst-case scenario of 90% of a 2.7 mg (2.1 + 0.6 mg) dose. 300 doses at 2.7 mg equate to 810 mg of salt, which is 40 mg below the minimum fill tolerance (850 mg). 40 mg remains in the device, significantly reducing the likelihood of a maximum lifetime delivery.

[0114] For COVID-19 disease, the optimal recommended dose has been found to be 4 inhalations (2 nasally and 2 orally) 5 times daily until symptoms resolve (see Example 3). This is equivalent to approximately 40 mg of NaCl powder. Users can also inhale as needed (10 sessions per day is the maximum recommended use, with 1 session = 3 inhalations). BREATHOX® should be inhaled each time a dose is released.

[0115] In a preferred embodiment, the unit dose administered to a patient is 0.25 to 25 mg, for example 0.25 to 4 mg. In a most preferred embodiment, the unit dose administered to a patient is 1.5 to 2 mg of dry powder.

[0116] In one embodiment, the pharmaceutical dry powder formulation of the present invention is administered to a patient via a dry powder inhaler in an amount of 0.001-0.5 mg dry powder / kg body weight / dose, for example 0.002-0.2 mg dry powder / kg body weight / dose, preferably 0.01-0.04 mg dry powder / kg body weight / dose.

[0117] In a further embodiment, the dry powder composition is administered sequentially or simultaneously with one or more other drugs or medications.It is contemplated that the composition of the present invention can be used in combination with other antibacterial agents, to enhance the activity of other antibacterial agents, or to create synergy between multiple drugs so that the combined effect is more effective than the sum of the effects of any drug considered independently.Combination with other drugs can be useful because it allows the use of such other drugs at lower doses, thereby reducing toxicity concerns.This combination may inhibit microbial replication, alleviate symptoms, shorten the duration of infection, and / or reduce the microbial burden in patients.

[0118] In some embodiments, the dry powder compositions of the present invention are administered with different drugs to treat, ameliorate, and / or prevent the same disease, disorder, or condition. In other embodiments, the dry powder compositions are used in combination with different drugs to treat, ameliorate, and / or prevent co-morbidities.

[0119] In some embodiments, combination therapy requires the simultaneous administration of both agents / therapies. This may be achieved by administering a single composition or pharmacological formulation containing both agents, or by simultaneously administering two separate compositions or formulations, each composition containing one agent. Alternatively, treatment with the dry powder composition of the present invention may precede or follow the "other" treatment by intervals ranging from minutes to weeks.

[0120] In one embodiment, the present invention contemplates the use of one or more conventional antiviral therapies in combination with the dry powder compositions of the present invention. In another embodiment, the dry powder composition is administered together with, and / or before, and / or after, treatment with an antiviral agent. In a further embodiment, the dry powder composition is administered prior to, sequentially with, or simultaneously with a pulmonary medication, such as an inhaled corticosteroid.

[0121] III.iv Reducing transmission or spread The use of dry powder formulations administered by dry powder inhalation reduces the transmission and / or spread of viral infections, as compared to the use of, for example, a nebulizer.

[0122] In a further aspect, the present invention provides a method for reducing the transmission or spread of a viral respiratory tract infection, comprising administering an effective amount of a pharmaceutical formulation as described herein to the respiratory tract (e.g., lungs, nasal passages) of an individual infected with a virus that causes a respiratory tract infection, an individual showing symptoms of a respiratory tract infection, or an individual at risk of contracting a respiratory tract infection due to a virus.

[0123] Example The present invention is illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, separately or in any combination thereof, be material for realizing the invention in diverse forms thereof.

[0124] BREATHOX® is a dry powder inhaler. It is a non-sterile, breath-actuated dry particle inhaler based on a material consisting of five injection-molded acrylonitrile butadiene styrene (ABS) plastic polymer components assembled into a single unit. This device is disclosed in International Publication No. 2015004227.

[0125] The BREATHOX® device contains BREATHOX® powder, which contains 95 wt% NaCl and 5 wt% lactose. The NaCl is obtained from Vornom Salt Diapir, Denmark. The NaCl is mixed with alpha-lactose monohydrate and micronized. The powder has a NaCl:lactose ratio of 95%:5% (by weight) with a tolerance of ±2%. This is also referred to as "NaCl powder."

[0126] NaCl is pharmaceutical grade manufactured in accordance with the European Pharmacopoeia monograph Sodium Chloride, current version no. 193. Alpha-lactose monohydrate is pharmaceutical grade and confirmed by the European Pharmacopoeia.

[0127] BREATHOX® powder is micronized to a respirable particle size range of 1-10 μm and is considered a pharmaceutical product.

[0128] One inhalation (ie, one unit dose) delivers approximately 1.5-2 mg of BREATHOX® powder to a subject.

[0129] Example 1: Antiviral Efficacy of BREATHOX® - In Vitro Study Design A preclinical in vitro study, performed in an NB-3 laboratory (biosafety level 3) in accordance with all WHO biosafety regulations and in compliance with Good Laboratory Practices (GLP), investigated the antiviral efficacy of the dry powder composition (containing 95 wt% NaCl and 5 wt% lactose) contained in the BREATHOX® device - i.e., BREATHOX® powder - in reducing the viral load of SARS-CoV-2.

[0130] Methods: To analyze the antiviral activity of BREATHOX®, there were three steps: first, prepare cell, virus, and BREATHOX® dilutions; then, infect cells and add BREATHOX® powder at various times; finally, extract nucleic acids and perform quantitative real-time qPCR.

[0131] Vero cells (a cell line derived from kidney epithelial cells extracted from African green monkeys; ATCC CCL-81-VHG) and Calu-3 cells (a human non-small cell lung carcinoma cell line with epithelial morphology grown in adherent cell culture; ATCC Calu-3-HTB-55) were maintained in DMEM containing 0.6% NaCl at 37°C in a humidified atmosphere containing 5% CO. Viral titers were determined in terms of plaque-forming units per milliliter.

[0132] BREATHOX® powder was dissolved in free DMEM (Dulbecco's Modified Eagle's Medium). Seven concentrations of BREATHOX® powder in cell culture medium were prepared: 0.2%, 0.4%, 0.8%, 0.9%, 1.1%, 1.2%, and 1.4% (weight / volume basis). Free DMEM contained 110 mM NaCl, so the total NaCl concentrations for the seven samples were 135 mM, 160 mM, 185 mM, 210 mM, 235 mM, 260 mM, and 285 mM, respectively.

[0133] For antiviral activity, four different addition times were tested. Vero cells were seeded in clear-bottom 96-well plates (5 × 10 cells). 4Cells were incubated at 37°C for 24 hours at 37°C for cell attachment. Cells were then treated with high concentrations of BREATHOX® Powder at various stages of viral infection, as described below. Four different BREATHOX® Powder addition times were evaluated, including virus preincubation (VPI), adsorption (AD), postinfection (PI), and adsorption plus postinfection, designated total time (FT).

[0134] VPI: SARS-CoV-2 mutants were pre-incubated with high concentrations of BREATHOX® powder for 1 hour before infecting cells. After adsorption, the inoculum was removed and replaced with culture medium, which was maintained until the end of the experiment. AD: One hour before virus infection, various concentrations of BREATHOX® powder were added to the cell monolayer and allowed to stand for one hour for the virus attachment process. After that, the virus-BREATHOX® powder mixture was replaced with fresh DMEM until the end of the experiment. PI:virus was added to the cells and allowed to infect for 1 hour, after which the virus-containing supernatant was replaced with medium containing various concentrations of BREATHOX® powder until the end of the experiment. FT: Vero cells were pretreated with various concentrations of BREATHOX® powder 1 hour prior to virus infection and then incubated with virus for 1 hour in the presence of BREATHOX® powder.

[0135] The virus mixture was then removed, and cells were cultured in medium containing the same concentration of BREATHOX® powder until the end of the experiment. For all experimental groups, cells were infected with the virus at a multiplicity of infection (MOI) of 0.02, and cell supernatants were collected for real-time qPCR (RT-qPCR) at 72 hours post-infection (hpi).

[0136] Supernatants collected after 72 hours were subjected to real-time RT-PCR for detection and quantification of SARS-CoV-2 viral RNA. Total nucleic acid (RNA and DNA) extraction was performed using the semi-automated NucliSENS EASYMAG platform (bioMérieux, Lyon, France) according to the manufacturer's instructions. Viral RNA quantification was performed using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Weiterstadt, Germany) on an ABI 7500 SDS real-time PCR machine (Applied Biosystems) using the reference published sequences for the E gene primers and probes (Corman et al., 2020). RNA copies per milliliter were quantified using specific in vitro transcribed synthetic RNA quantification standards.

[0137] Findings: In Table 1 (VPI), Table 2 (AD), Table 3 (PI) and Table 4 (FT) the RT-qPCR results are shown in RNA copies / mL and the percentage of inhibition is expressed for each treatment.

[0138] [Table 1]

[0139] [Table 2]

[0140] [Table 3]

[0141] [Table 4]

[0142] Based on the observation of cytopathic effects and comparison of real-time RT-PCR results, it was found that no BREATHOX® Powder concentration in the VPI and AD treatments was able to reduce SARS-CoV-2 viral titers. However, PI and FT treatments demonstrated the ability to reduce viral load (see Figure 1 for FT treatment). The best results for PI were achieved with PI treatments at 0.8% and 0.9% BREATHOX® Powder concentrations, with average percent reductions of 66.17 and 81.76, respectively. However, there was a wide variability in the inhibition results for PI treatments. Inhibition was not consistent, with higher BREATHOX® Powder concentrations showing lower percent inhibition, as confirmed in Table 3. Meanwhile, for FT, the best results were achieved with BREATHOX® Powder concentrations of 0.9%, 1.1%, 1.2%, and 1.4%, with average percent reductions of 73.80, 99.85, 95.36, and 100.00, respectively. In this experiment, FT treatment showed the best results, with consistent viral reduction, inhibiting the visible cytopathic effect of a 1.1% BREATHOX® powder concentration by 1.4%, without causing irreversible damage to the cell monolayer.

[0143] Conclusion: The antiviral activity of BREATHOX® Powder was confirmed by in vitro laboratory findings, which showed that BREATHOX® Powder at concentrations of 0.9%, 1.1%, 1.2%, and 1.4% (weight / volume basis) had average viral inhibition of 73.80%, 99.85%, 95.36%, and 100.00%, respectively.

[0144] Example 2: Mechanism of Action of BREATHOX® We investigated the mechanism of action of BREATHOX® Powder in reducing SARS-CoV-2 viral load. To clarify this mechanism of action, we performed various assays.

[0145] First, membrane potential fluctuations were analyzed by microfluorimetry. Changes in membrane potential of Vero cells exposed to high concentrations of NaCl were measured by plate microfluorimetry recording using a FlexStation III microplate reader and a FLIPR membrane potential assay kit (Molecular Devices, Sunnyvale, CA) according to the manufacturer's instructions. 5 × 10 cells were cultured in black clear-bottom 96-well plates at rest (time point 0), 1 h, 24 h, and 72 h after NaCl challenge (a depolarizing agent). 4 Fluorescence intensity recordings were taken.

[0146] A luciferase assay was then performed to confirm available ATP. Changes in total ATP concentration in Vero cells exposed to high concentrations of NaCl were measured at 1 hour or 72 hours by plate microfluorimetry using a FlexStation III microplate reader and an ATP assay kit (Sigma-Aldrich) according to the manufacturer's instructions. This kit provides highly sensitive results, detecting ATP release from 10–100 mammalian cells per well based on the oxidation of D-luciferin catalyzed by firefly luciferase in the presence of ATP, where the amount of ATP is quantified by the amount of light produced (hν). 5 × 10 cells were cultured in a black, clear-bottom 96-well plate at rest (time point 0), 1 hour, 24 hours, and 72 hours after NaCl challenge (a depolarizing agent). 4 Records of individual luminescence intensities were obtained.

[0147] To assess the effect of BREATHOX® Powder on cellular ATP production, mitochondrial status was measured after each treatment condition (varying BREATHOX® Powder concentrations). For this purpose, Vero cells were tested using the Seahorse assay, which measures mitochondrial metabolism. Intact Vero cells were evaluated in a high-resolution respirometry assay. Two parameters were assessed: oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).

[0148] Mitochondrial respiration was assessed under four different conditions: (i) basal respiration, which corresponds to the basal oxygen consumption of cells without the addition of substrates or inhibitors; (ii) proton leak after the addition of oligomycin (an ATP synthase blocker), in which oxygen consumption occurs due to proton leak across the inner mitochondrial membrane; (iii) uncoupled (uncoupled to ATP synthesis), in which oxygen consumed reflects maximal respiration after the addition of the respiratory chain uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP); and (iv) inhibition with the addition of rotenone (a complex I blocker) and antimycin (a complex III blocker), in which oxygen consumption reflects non-mitochondrial activity. The ECAR rate was verified using a glycolysis stress test kit (Agilent Technologies).

[0149] Another objective was to evaluate the Na+ influx into cells upon treatment with BREATHOX® powder by fluorescence microscopy. Vero cells were stained with the intracellular sodium indicator sodium green and exposed to NaCl (3%) for method calibration. Once the method conditions were established (45 min incubation in the presence of 11 μM sodium green), cells were exposed to BREATHOX® powder (0.4% wt / vol, 0.8% wt / vol, and 1.1% wt / vol), and Na+ influx was measured for 100 s. Changes in Na+ concentration upon application of BREATHOX® powder were recorded using a Nikon fluorescence microscope coupled to a CCD camera and automatic shutter.

[0150] Based on the luciferase assay (scheme for measuring ATP concentration via luminescence generation - Figure 2), it is clear that concentrations of BREATHOX® powder above 0.9% (i.e., 1.1%, 1.2%, and 1.4%) result in a significant decrease in intracellular ATP concentration, which is one mechanism that may explain the reduced replication of SARS-CoV-2 in Vero cells at 1 hour and, more clearly, at 72 hours.

[0151] To test whether increased activity of the Na+ / K+ ATPase transporter causes a decrease in ATP levels, Vero cells were treated with 0.4%, 0.8%, and 1.1% BREATHOX® powder with or without 5 μM ouabain (a Na+ / K+ ATPase transporter inhibitor). Changes in total ATP concentration in Vero cells exposed to increasing concentrations of BREATHOX® powder with or without 5 μM ouabain for 1 hour were measured by plate microfluorimetry using a FlexStation III microplate reader and an ATP assay kit (Sigma-Aldrich) according to the manufacturer's instructions. The Sigma-Aldrich ATP assay kit provides highly sensitive results, detecting ATP release from 10–100 mammalian cells per well based on the oxidation of D-luciferin catalyzed by firefly luciferase in the presence of ATP, where the amount of ATP is quantified by the amount of light produced (hν). 5 x 10 cells in black clear-bottom 96-well plates at rest (time point 0) and 1 hour after BREATHOX® powder challenge (depolarizing agent) 4 Luminescence intensity recordings were obtained. Basal fluorescence intensity, which represents the luminescence emission rate before the addition of the ATP-releasing agent, was monitored for 10 seconds, followed by measurements over 120 seconds at 1.52-second intervals to obtain time kinetics. Responses were calculated as peak luminescence minus basal luminescence using SoftMax2Pro software (Molecular Devices). Figure 3 shows that the decrease in ATP levels was significantly reversed by ouabain treatment.

[0152] To assess the effect on ATP production, we also measured mitochondrial status after each treatment condition. To this end, we tested Vero cells using the Seahorse assay, which measures mitochondrial metabolism. Intact Vero cells were evaluated using a high-resolution respirometry assay. Two parameters were assessed: oxygen consumption rate (OCR; Figure 4A) and extracellular acidification rate (ECAR; Figure 4B). When mitochondrial metabolism is impaired, glycolytic enzymes enhance their activity to compensate for ATP production (Figure 4D). The end product of this pathway is lactate, whose synthesis and accumulation results in medium acidification. Figure 4D shows oxygen consumption in four states: (i) basal, (ii) proton leak, (iii) uncoupled, and (iv) inhibited. Basal indicates normal cell respiration; proton leak reflects the integrity of the mitochondrial inner membrane; uncoupled indicates respiratory chain activity of complexes I–IV (i.e., maximal respiratory capacity); and inhibited reflects oxygen-consuming non-mitochondrial processes. Seventy-two hours after the addition of BREATHOX® powder to cultured Vero cells, maximal respiration was upregulated at 0.2% BREATHOX® powder compared to control values. However, concentrations of BREATHOX® powder greater than 0.8% downregulated respiration (Figure 4A). Consistently, ECAR assessment (Figure 4B) showed a similar pattern. The correlation between OCR and ECAR was plotted on an energy map (Figure 4C), which indicates that treatment with 0.2% and 0.4% BREATHOX® powder resulted in enhanced energy status, while higher concentrations shifted metabolism toward anaerobic pathways. The OCR and ECAR results indicate that low concentrations of BREATHOX® powder positively affected cellular metabolism, while higher doses impaired mitochondrial function in Vero cells. Because mitochondrial dysfunction is commonly observed during new drug development, these results are important for any study of potential therapeutic compounds.

[0153] An additional objective was to evaluate Na+ influx upon BREATHOX® powder treatment by fluorescence microscopy. Vero cells were stained using the intracellular sodium indicator sodium green and exposed to NaCl (3%) for method calibration. Once the method conditions were established (45 minutes of incubation in the presence of 11 μM sodium green), Na+ influx was measured for 100 seconds after exposure of cells to BREATHOX® powder (0.4%, 0.8%, and 1.1%), as seen in Figures 5A, B, and B.

[0154] The Δ[Na+]i data show that application of BREATHOX® Powder induced Na+ influx. At a BREATHOX® Powder concentration of 0.8%, Na+ influx was significantly higher than at BREATHOX® Powder concentrations of 0.4% and 1.1%. This supports the mitochondrial status data that doses of BREATHOX® Powder above 0.8% were toxic to mitochondria.

[0155] It is important to note that BREATHOX® Powder was diluted in cell culture medium containing 0.6% NaCl (110 mM NaCl), so 0% BREATHOX® Powder corresponds to a NaCl concentration of 0.6% and 1.7% BREATHOX® Powder corresponds to 2.1% NaCl.

[0156] Sodium channel gene expression in cell culture was examined by measuring the relative abundance of Na+ / K+ ATPase mRNA (Atp1B1, NCBI reference sequence: NM_001677.4), and primers were standardized. As seen in Figure 6, Na+ / K+ ATPase expression increased at a BREATHOX® concentration of 0.4% as a positive feedback response to Na+ / K+ ATPase overstimulation. However, doses of BREATHOX® higher than 0.8% were toxic and reduced Na+ / K+ ATPase channel expression. ENaC expression in Vero cells was not observed.

[0157] Due to its ability to enhance Na+ / K+ ATPase transporter activity, BREATHOX® powder can induce a significant decrease in ATP levels. Because SARS-CoV-2 requires ATP for its replication, the experiments presented herein demonstrated that BREATHOX® powder has important properties in viral inhibition (replication inhibition) of SARS-CoV-2.

[0158] Example 3: Antiviral effect against COVID-19 An open-label, randomized, three-arm feasibility study was conducted to evaluate the effect of a BREATHOX® device containing NaCl dry powder according to the present invention (specifically BREATHOX® powder as defined herein) on reducing COVID-19-related symptoms and preventing referral to healthcare services in adult patients diagnosed with COVID-19. Data were collected from 100 patients with COVID-19 randomized into three treatment groups.

[0159] The objective of this study was to demonstrate the benefit of using BREATHOX® Powder to reduce the severity of COVID-19 symptoms and prevent further symptom exacerbation and development, based on the antiviral effects observed in preclinical in vitro studies.

[0160] This study provides evidence of the feasibility of a full-scale clinical trial of the effect of BREATHOX® Powder in COVID-19 patients. The primary objective of this study was to determine the effectiveness of using BREATHOX® containing the NaCl dry powder composition of the present invention (BREATHOX® Powder) as dry powder inhalation therapy compared to standard of care (symptom treatment / alleviation with paracetamol only) for clinical improvement in symptomatic COVID-19 patients. Secondary objectives were to evaluate the effectiveness of using BREATHOX® Powder to reduce health service utilization and to assess the safety of adverse events following discontinuation of treatment within 28 days.

[0161] Outcomes were measured through telemedicine monitoring of vital signs and symptoms, physical assessments, diary data, and participants' daily self-reporting. To evaluate the efficacy of BREATHOX® Powder and measure changes in the subjects' health status, the World Health Organization (WHO) recommended eight-point ordinal scale was used to measure health status in COVID-19 patients. The 100 participants enrolled were adult men or women (aged 18 years or older) with mild to moderate SARS-CoV-2 infection diagnosed by RT-PCR or swab antigen PCR testing. Symptoms at the time of enrollment were required to include at least one of the following: fever or fever lasting for more than 24 hours, headache, sore throat, cough (e.g., dry cough), fatigue, chest pain or choking sensation (without associated respiratory distress), myalgia, anosmia, loss of taste, or gastrointestinal symptoms within 10 days of illness onset. Participants were not hospitalized. Subjects were randomized 1:1:1 into three groups.

[0162] The two experimental treatment groups were Group 1 and Group 2. Group 1 received standard care treatment for 10 days, with 10 sessions of BREATHOX® per day during the day, i.e., one session every hour. Group 2 received standard care treatment for 10 days, with 5 sessions of BREATHOX® per day, i.e., one session every three hours. Each participant in these two groups received two BREATHOX® devices, one nasal and one oral. One BREATHOX® session consisted of four inhalations of BREATHOX® powder, one into each nostril using the BREATHOX® device and two oral inhalations. Group 3 was the control group, receiving standard care only. The following is a summary of the three randomized groups: Group 1: Standard care + 1 session of BREATHOX® every hour (4 inhalations / session) for 10 days for a total of 10 sessions per day - 33 subjects. Group 2: Standard care + 1 session of BREATHOX® every 3 hours (4 inhalations / session), for a total of 5 sessions per day for 10 days - 33 subjects. Group 3: Standard care only (no BREATHOX® sessions) - 34 subjects.

[0163] The total duration of the study was 28 days, including a 10-day treatment period and an 18-day treatment break. At the first visit, informed consent forms were collected, vital signs were measured, and a physical assessment was performed by a healthcare professional. Ten days after the start of the study, patients visited the clinic remotely via telephone or video conversation to assess use of the assigned treatment, adverse events, and the need for unscheduled medical care. During this call, symptom diaries were assessed. After the visit, treatment was suspended for the remaining 18 days of the study. At the end of the study (day 28), a final visit was conducted to assess the presence of adverse events and collect symptom diaries. This end-of-treatment visit was conducted virtually. Figure 7 illustrates the study design.

[0164] Outcomes were measured based on RT-PCR COVID-19 testing, telemedicine monitoring of vital signs and symptoms, diary data, and participants' daily self-reporting. The principal investigator was responsible for ensuring adherence to the correct intervals for visits and follow-up. An independent safety monitoring committee was established to continuously review and evaluate clinical efficacy and safety data collected during the study at biweekly intervals.

[0165] This feasibility study included 100 patients and the first interim analysis of efficacy and safety of the trial was performed. All participants were analyzed based on an intention-to-treat analysis. The results of this study were analyzed using analysis of variance (ANOVA) for continuous variables and the Kruskal-Wallis test for nonparametric variables. The chi-square test was used to calculate the rates of health resource use and hospitalization. Poisson regression was used to assess the frequency of health resource use.

[0166] After analyzing data from the clinical trial using Kaplan-Meier survival analysis, it was observed that the use of BREATHOX® Powder during treatment (both treatment groups) resulted in a significant improvement in symptom resolution compared to the control group. The duration of COVID-19-induced cough was significantly shortened in the treatment groups.

[0167] Figure 8 shows the number of days it takes to recover from coughing as a symptom of Covid-19 affecting the respiratory tract. This dataset includes only patients who experienced coughing at the start of the trial. The values ​​reported below the graph in Figure 8 represent the symptom prevalence - i.e., patients experiencing coughing in each group - while in the graph, the y-axis shows the proportion of patients experiencing coughing in each group at the time indicated on the x-axis (1.00 = 100%).

[0168] As illustrated in Figure 8, already 4 days after the first day of coughing, the following was observed: 50% of patients in groups 1 and 2 recovered from their cough. However, in group 3, only 25% of patients recovered from their cough.

[0169] Cough symptoms resolved twice as quickly in patients treated with BREATHOX® compared to patients receiving usual care.

[0170] Furthermore, as illustrated in Figure 8, over the 10 days from the first day of coughing, the following was found: 75% of patients in Group 1 recovered from their cough. 64% of patients in the second group recovered from their cough. However, in group 3, only 42% of patients recovered from their cough.

[0171] Furthermore, as illustrated in Figure 8, over the 20 days from the first day of coughing, the following was found: 88% of patients in Group 1 recovered from their cough. 72% of patients in the second group recovered from their cough. However, in group 3, only 58% of patients recovered from their cough.

[0172] Thus, BREATHOX® containing the NaCl dry powder of the present invention has been shown to reduce cough recovery time in COVID-19 patients compared to standard of care.

[0173] Overall, no adverse events were reported related to the use of BREATHOX® Powder, and it was well tolerated by patients.

[0174] Conclusion: This clinical study confirmed the in vitro findings, demonstrating that the use of BREATHOX® containing the NaCl dry powder of the present invention during treatment (both treatment groups) significantly improved symptom recovery compared to the control group (p=0.01). COVID-19-induced cough resolution was significantly shorter in the treatment groups. The "BREATHOX® Group 1" recovered from COVID-19-induced cough 40% faster than the control group, and the "BREATHOX® Group 2" recovered 30% faster than the control group (p=0.01).

[0175] Improvements in the resolution of other COVID-19-induced symptoms were also observed, but due to the small sample size, the results were not statistically significant. [Explanation of symbols]

[0176] 1 inhaler 2 Proximal 3. Distal part 4 entrance 5 exit 6 Discharge Orifice 7 Discharge Orifice 8 Reservoir 9 Reservoir 10 Air passage 11 Air passage 12 Air passage 13 Air passage 14 inlet valve member 15 Outlet valve member 16 Reservoir valve member 17 Reservoir valve member 20. Enclosure wall 30 Enclosure wall A axis direction D. Distal end P proximal end

Claims

1. 1. A dry powder composition comprising at least 50 wt% NaCl for use in the prevention and / or treatment of viral respiratory tract infections, the composition being administered to a mammalian subject by dry powder inhalation.

2. 2. The dry powder composition for use according to claim 1, wherein the viral respiratory tract infection is caused by a coronavirus.

3. 3. The dry powder composition for use according to claim 1 or 2, which reduces the time for resolution of one or more symptoms caused by said viral respiratory tract infection.

4. 4. The dry powder composition for use according to any one of claims 1 to 3, wherein the one or more symptoms caused by the viral respiratory tract infection is cough.

5. 5. A dry powder composition for use according to any one of claims 1 to 4, comprising at least 60% NaCl, preferably at least 70% NaCl, more preferably at least 80% NaCl, most preferably at least 90% NaCl.

6. 6. A dry powder composition for use according to any one of claims 1 to 5, wherein at least 80% of the particles have a particle size distribution in the range 1 to 10 μm.

7. 7. A dry powder composition for use according to any one of claims 1 to 6, further comprising an anti-agglomerating agent.

8. 8. The dry powder composition for use according to any one of claims 1 to 7, wherein the only therapeutic agent in the composition is the NaCl.

9. 8. The dry powder composition for use according to claim 7, wherein said only therapeutic agents are said NaCl and said anti-agglomerating agent.

10. 10. The dry powder composition for use according to any one of claims 7 to 9, wherein the anti-agglomerating agent is a carbohydrate or carbohydrate derivative.

11. 11. A dry powder composition for use according to any one of claims 7 to 10, wherein the anti-agglomerating agent is selected from lactose, mannitol and maltodextrin, preferably lactose.

12. 12. The dry powder composition for use according to any one of claims 1 to 11, to be administered in a daily dose of 0.5 to 200 mg.

13. 13. The dry powder composition for use according to claim 12, wherein the daily dose is divided into 1 to 10 daily sessions of 4 to 10 mg per session.

14. 14. A dry powder composition for use according to any one of claims 1 to 13, which is administered orally and / or nasally.

15. 15. A dry powder composition for use according to any one of claims 1 to 14, consisting essentially of NaCl and lactose.

16. 16. A dry powder composition for use according to any one of claims 1 to 15, comprising lactose, wherein the ratio of NaCl to lactose is from 99:1 to 75:25 by wt%.

17. 17. The dry powder composition for use according to claim 16, wherein the ratio of NaCl to lactose is approximately 95:5 by wt%.

18. 18. The dry powder composition for use according to any one of claims 2 to 17, wherein the coronavirus is SARS-CoV-2.

19. 19. The dry powder composition for use according to any one of claims 2 to 18, wherein the viral respiratory tract infection is COVID-19.

20. 20. The dry powder composition for use according to any one of claims 1 to 19, wherein the mammalian subject is a human.

21. 21. The dry powder composition for use according to any one of claims 4 to 20, which reduces the time for resolution of a cough in a mammalian subject by at least 30% compared to a mammalian subject not treated with said dry powder composition.

22. 22. A dry powder composition for use according to any one of claims 1 to 21, wherein the NaCl is provided by a composition obtained from Voornom Salt Diapir, Denmark (N56 36.834 E009 42.070).

23. 23. A dry powder composition for use according to any one of claims 1 to 22, which is administered using a dry powder inhaler.

24. The dry powder inhaler is an inhaler (1) having, in an axial direction, a proximal end (P) for insertion into a user's mouth and a distal end (D) opposite the proximal end (P), the inhaler (1) including an inlet (4), an outlet (5) disposed at the proximal end (P), air passages (10, 11, 12, 13) extending from the inlet (4) to the outlet (5), and reservoirs (8, 9) communicating with the air passages (10, 11, 12, 13) through discharge orifices (6, 7), the reservoirs (8, 9) containing a dispersible material the inhaler (1) has a proximal portion (2) including the outlet (5), and a distal portion (3) attached to the proximal portion (2), the proximal portion (2) being linearly slidable along the axial direction (A) relative to the distal portion (3) between an "OPEN" position in which the proximal portion (2) is extended from the distal portion (3) and a "CLOSED" position in which the proximal portion (2) is retracted toward the distal portion (3), the inhaler (1) further comprising an inlet valve member (14) and an outlet valve member (15); - said reservoirs (8, 9) are arranged in said proximal part (2); - the inlet (4) comprises one or more openings in a housing wall (20) around the proximal part (2), the one or more openings facing radially outward, away from the axial direction (A), and the inlet valve member (14) is formed by a housing wall (30) around the distal part (3) covering the openings when the proximal part (2) is in the "CLOSED" position; - said outlet (5) comprises an axial opening, the outlet valve member (15) is formed as a plug attached to the distal portion (3), the plug blocking the outlet (5) when the proximal portion (2) is in the "CLOSED" position; - said discharge orifices (6, 7) are oriented in said axial direction (A); the inhaler (1) further comprises a reservoir valve member (16, 17), the reservoir valve member (16, 17) being formed as a peg moving in the axial direction (A), the peg being fixed to the distal part (3) via an axially extending barrel, the peg blocking the discharge orifice (6, 7) when the proximal part (2) is in the "CLOSED" position; Thereby, the inlet valve member (14), the outlet valve member (15) and the reservoir valve members (16, 17) - when said proximal part (2) is retracted along said axial direction towards said distal part from said "OPEN" position to said "CLOSED" position, it simultaneously closes said inlet (4), said outlet (5) and said discharge orifices (6, 7); and - when the proximal portion (2) is deployed from the distal portion along the axial direction from the "CLOSED" position to the "OPEN" position, it simultaneously opens the inlet (4) and opens the outlet (5) to deliver a quantity of the dispersible substance from the reservoir (8, 9) through the release orifices (6, 7) to the air passages (10, 11, 12, 13). It is arranged as follows:

24. A dry powder composition for use according to claim 23.

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