Methods of clearing mucus from the lungs

Abstract

Provided herein are compositions and methods for clearing mucus (e.g., excess mucus) from the lungs. In one aspect, provided herein are methods of clearing mucus from the lungs of a subject in need thereof comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an isotonic osmolyte. The liquid pharmaceutical composition (e.g., aqueous, isotonic sodium gluconate solution) may further comprise one or more additional agents selected from epithelial sodium channel (ENaC) inhibitors, osmolytes mucolytic agents, surfactants, DNases, anti-infectives, and anti-inflammatory agents. Also provided herein are liquid pharmaceutical compositions (i.e., "instillation solutions," "lavage solutions") useful in the methods described herein.

Description

[0001] METHODS OF CLEARING MUCUS FROM THE LUNGS

[0002] RELATED APPLICATIONS

[0003]

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application, U.S.S.N. 63 / 662,972, filed June 21, 2024, the entire contents of which is incorporated herein by reference.

[0004] BACKGROUND

[0005]

[0002] Muco-obstructive lung diseases (MOLDs), e.g., COPD, NCFB, CF, and PCD, are diseases that impair the respiratory health and lifespan of many Americans (see, e.g., Boucher RC. Muco- obstructive lung diseases. N Engl J Med. 2019 May 16; 380(20):1941-1953). The pathogenesis of MOLDs is understood and reflects mucus hyperconcentration (see, e.g., Boucher RC. Muco- obstructive lung diseases. N Engl J Med. 2019 May 16; 380(20):1941-1953; Hill D B, et al. Physiology and Pathophysiology of Human Airway Mucus. Physiol Rev. 2022; 102:1757-1836; Henderson et al. Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure. J Clin Invest. 2014; 124(7):3047-3060).

[0006]

[0003] Ironically, despite the nomenclature of “muco-obstructive” lung diseases, data establishing the quantitative contributions of pulmonary mucus accumulation to airflow obstruction, impaired pulmonary health, and mortality have been heretofore limited to sparse histologic studies (see, e.g., Hogg et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004; 350(26):2645-2653; Hogg et al. The contribution of small airway obstruction to the pathogenesis of chronic obstructive pulmonary disease. Physiol Rev. 2017; 97(2):529-552). However, in the past five years there has been a revolution in the utilization of thoracic CT scanning to quantitate mucus obstruction and related indices of mucus obstruction to clinical outcomes in subjects with respiratory disease often reported as mucus plugs. A striking example was reported from the NHLBI Spiromics COPD cohort (see, e.g., Dunican et al. Mucus plugs and emphysema in the pathophysiology of airflow obstruction and hypoxemia in smokers. Am J Respir Crit Care Med. 2021 Apr; 203(8):957- 968).

[0007]

[0004] Studies have shown that for COPD subjects without emphysema there is a remarkable contribution of mucus plugging to airflow reduction, and in some cases mucus plugging is associated with a loss of ~ 50% of airflow (see, e.g., Dunican et al. Mucus plugs and emphysema in the pathophysiology of airflow obstruction and hypoxemia in smokers. Am J Respir Crit Care Med. 2021 Apr; 203(8):957- 968). Companion COPD studies reported mucus plugging may persist for several years and is associated with excess mortality (see, e.g., Diaz et al. Airway-occluding mucus plugs and mortality in patients with chronic obstructive pulmonary disease. JAMA. 2023; 329(21): 1832- 1839). These data suggest that a major fraction of airflow obstruction in COPD subjects may be reversible if hyperconcentrated mucus plugs were cleared. If so, the definition of “chronic obstructive pulmonary disease” might pleasantly have to be revised, and both the quality of life and lifespan of the > 16 million subjects with COPD in the US would be improved dramatically. These data, coupled with similar data from COPD Gene and the Severe Asthma Research Program, highlight the urgent need to develop mucus clearance strategies for MOLD and asthmatic patients (see, e.g., Dunican et al. Mucus plugs in patients with asthma linked to eosinophilia and airflow obstruction. J Clin Invest. 2018; 128(3):997- 1009; Kim et al. Mucus plugging on computed tomography and chronic bronchitis in chronic obstructive pulmonary disease. Respir Res. 2021; 22(1): 110; Ramos et al. Clinical issues of mucus accumulation in COPD. Int J Chron Obstruct Pulmon Dis. 2014; 9:139-150).

[0008]

[0005] Quantitation of mucus plugs likely underestimates the mucus burden in a diseased lung due to the contribution of plugging in bronchioles that cannot be visualized by CT scans. Current CT scans estimates of mucus plugging suggest a mucus volume of approximately 1 mL is involved in mucus plugging (see e.g., Fahy et al. Persistent mucus plugs in proximal airways are consequential for airflow limitation in asthma. JCI Insight 2024; 8;9(3):el74124). However, recent studies have estimated that the total mucus volume of mucus in the lungs of patients with non-CF bronchiectasis or primary ciliary dyskinesia is about 50 mL (see, e.g., Hogg et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004; 350(26):2645-2653; Asakura et al. Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis. Am J Respir Crit Care Med. 2024;209(4):374-89; Saito et al. Morphological Analysis of Bronchiectasis in Primary Ciliary Dyskinesia. Am J Respir Crit Care Med. 2024;209:A4432).

[0009]

[0006] In most instances, hyperconcentrated mucus cannot be transported by cilia or cough and must be diluted e.g., with an isotonic fluid) to restore transportability. Mucus obstructed lungs can in many instances have 1-100 mL of retained mucus, and therefore need a large volume of diluting fluid to clear the hyperconcentrated mucus, e.g., 12% solids content [defined as 12% dry weight / volume] requires dilution with an isotonic fluid by a factor of 6, i.e., approximately 300 mL, to swell the 50 mL mucus to hydrate / dilute the retained intrapulmonary mucus to an approximately 2% solids content consistent with mucus clearability. This volume requirement is orders of magnitude above that achievable by aerosol. Furthermore, polymer physics first principles suggest that: 1) hyperconcentrated mucin gels will swell slowly (hours), reflecting slow polymer reptation kinetics; and 2) the rate of swelling is a function of the initial mucus polymer concentration (see e.g., Rubinstein & Colby. Polymer Physics. Oxford: Oxford University Press; 2003). Therefore, the slow swelling kinetics require that the fluid stay in the lungs for an extended period of time - typically longer than is afforded by normal saline (see, e.g., Martens et al. Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi. Am. J. Physiol. Lung Cell Mol. Physiol. 2011, 301: L236 -L246).

[0010]

[0007] Isotonic saline (0.9% NaCl in water) has been used to lavage the lung, but it is rapidly absorbed from the lung surface via an active not fluid transport mechanisms that are rate limited by the epithelial transport channel (ENaC), before mucus can be effectively cleared, (see Berthiaume Y, Matthay MA. Alveolar edema fluid clearance and acute lung injury. Respir Physiol Neurobiol. 2007;159(3):350-9; Bastarache JA, Ong T, Matthay MA, Ware LB. Alveolar fluid clearance is faster in women with acute lung injury compared to men. J Crit Care.

[0011] 2011;26(3):249-56; Kylstra JA, et al Volume-controlled lung lavage in the treatment of asthma, bronchiectasis, and mucoviscidosis. Am Rev Respir Dis . 1971;103(5):651-65).

[0012]

[0008] Hyperconcentrated mucus can take a long time to swell and dissolve due its polymeric nature, and therefore lavage solutions with longer retention times in the lung are important to effective mucus clearance. Alternative compositions and methods for clearing mucus from the lungs are needed in order to address muco-obstructive lung diseases.

[0013] SUMMARY

[0014]

[0009] Provided herein are improved compositions and methods for clearing mucus from the lungs and treating diseases such as muco-obstructive lung diseases. In one aspect, provided herein are methods of clearing mucus from the lungs of a subject in need thereof comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an osmolyte, an ENaC inhibitor, and / or a mucolytic agent. In preferred embodiments, the osmolyte is not sodium chloride. The liquid pharmaceutical composition e.g., an isotonic sodium gluconate solution) may further comprise one or more additional agents selected from epithelial sodium channel (ENaC) inhibitors, additional osmolytes, mucolytic agents, surfactants, DNases, anti-infectives, and anti-inflammatory agents. Also provided herein are liquid pharmaceutical compositions useful in the methods described herein.

[0015]

[0010] In one aspect, provided herein are methods of clearing mucus from the lungs of a subject in need thereof, the methods comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an osmolyte, wherein the osmolyte is not sodium chloride. In certain embodiments, the osmolyte has a longer retention time in the lungs than sodium chloride. In certain embodiments, the osmolyte is a sodium salt. In certain embodiments, the osmolyte is a gluconate salt. In certain embodiments, the osmolyte is sodium gluconate.

[0016]

[0011] In another aspect, provided herein are methods of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising sodium gluconate.

[0017]

[0012] In certain embodiments, the concentration of the osmolyte in the composition is from about 2.5% to about 5% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is about 3.25% (w / v). As described, the osmolyte can be sodium gluconate. In certain embodiments, the concentration of sodium gluconate in the composition is from about 2.5% to about 5% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is about 3.25% (w / v).

[0018]

[0013] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution. In certain embodiments, the liquid pharmaceutical composition is isotonic. In certain embodiments, the liquid pharmaceutical composition is an isotonic sodium gluconate solution.

[0019]

[0014] In certain embodiments, the liquid pharmaceutical composition further comprises an epithelial sodium channel (ENaC) inhibitor. Examples of ENaC inhibitors are provided herein. In certain embodiments, the ENaC inhibitor is:

[0020] (Compound 1), or a pharmaceutically acceptable salt thereof (e.g., HC1 salt).

[0021]

[0015] In certain embodiments, the ENaC inhibitor is:

[0022] (Compound 2), or a pharmaceutically acceptable salt thereof (e.g., HC1 salt).

[0023]

[0016] In one aspect, provided herein are methods of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an ENaC inhibitor. The composition may further comprise one or more components described herein, such as an osmolyte, e.g., NaCl or sodium gluconate.

[0024]

[0017] In certain embodiments, the concentration of the ENaC inhibitor in a composition described herein is from about 0.3 g / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in a composition described herein is from about 10 pM to about 300 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pM to about 30 pM (e.g., about 20 pM). In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 150 pM to about 250 pM (e.g., about 200 pM).

[0025]

[0018] In certain embodiments, the liquid pharmaceutical composition further comprises a mucolytic agent. Examples of mucolytic agents are provided herein. In certain embodiments, the mucolytic agent is N-acetyl cysteine (NAC).

[0026]

[0019] In another aspect, provided herein are methods of clearing mucus from the lungs of a subject in need thereof, the methods comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising a mucolytic agent. Examples of mucolytic agents are provided herein.

[0027]

[0020] In certain embodiments, the mucolytic agent is: or a pharmaceutically acceptable salt thereof (e.g., HC1 salt). In certain embodiments, the mucolytic agent is NAC.

[0028]

[0021] In certain embodiments, the concentration of the mucolytic agent in the composition is from about 10 mM to about 200 mM. In certain embodiments, the concentration of the mucolytic agent is about 100 mM.

[0029]

[0022] In certain embodiments of the methods described herein, the liquid pharmaceutical composition further comprises a chloride salt. In certain embodiments, the chloride salt is sodium chloride. In certain embodiments, the concentration of the chloride salt in the composition is from about 10 mM to about 30 mM (e.g., about 20 mM).

[0030]

[0023] In certain embodiments, the liquid pharmaceutical composition further comprises a surfactant. Examples of surfactants are provided herein.

[0031]

[0024] In certain embodiments, the liquid pharmaceutical composition further comprises an anti- infective (e.g., antibiotic). In certain embodiments, the liquid pharmaceutical composition further comprises an anti-inflammatory agent.

[0032]

[0025] In certain embodiments, at least a portion of the liquid pharmaceutical composition is instilled into the intrapulmonary airways of the lungs. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via bronchoscope. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via endotracheal tube. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via a double lumen endotracheal tube. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via a tracheostomy tube.

[0033]

[0026] In some embodiments, the liquid pharmaceutical composition has a pH of from about 6.4 to about 8.4. In certain embodiments, the liquid pharmaceutical composition has a pH of from about 7.2 to about 7.6. In some embodiments, the liquid pharmaceutical composition has a pH of at or around physiological pH (z.e., a pH of about 7.4).

[0034]

[0027] In certain embodiments, a volume of about 1 mL to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject. In certain embodiments, a volume of about 10 mL to about 10 L of the liquid pharmaceutical composition is instilled into the lungs of the subject. In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject. In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into a single lobe of the lungs of the subject.

[0035]

[0028] In certain embodiments, a volume of about 1 mL to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject as a single bolus dose. In certain embodiments, a volume of about 10 mL to about 10 L of the liquid pharmaceutical composition is instilled into one lobe of the lungs of the subject as a single bolus dose. In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject as a single bolus dose. In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into a single lobe of the lungs of the subject as a single bolus dose.

[0036]

[0029] In certain embodiments of the methods provided herein, the mucus is a mucus plug. In certain embodiments, the subject has a mucus obstructive lung condition. In certain embodiments, the subject has asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CL), primary ciliary dyskinesia (PCD), non-CL bronchiectasis, or Job syndrome. In certain embodiments, the subject is intubated post-surgery.

[0037]

[0030] In certain embodiments of any of the methods provided herein, the subject is a human.

[0031] Also provided herein as aspects of the disclosure are any liquid pharmaceutical compositions (z.e., “lavage solutions”) described herein. In other aspects, provided herein are liquid pharmaceutical compositions described herein for use in clearing mucus from the lungs of a subject. In other aspects, provided herein are liquid pharmaceutical compositions provided herein for use as medicaments and / or in the preparation of medicaments.

[0038]

[0032] The details of certain embodiments of the disclosure are set forth in the Detailed Description, as described below. Other embodiments of the disclosure will be apparent from the Definitions, Examples, Abstract, Drawings, and Claims.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040]

[0033] FIG. 1A shows a depiction of a method using a flexible bronchoscope. As shown in FIG. IB, PVC tubing can be connected to the cannula on the bronchoscope for instillation of a liquid pharmaceutical composition (z.e., “lavage solution” into the lung at a controlled rate, either by syringe or sterile PVC tubing equipped with a drip chamber and roller clamp. The lavage solution can be sequentially instilled into the desired regions of the lung, as shown in FIG. 1C.

[0034] FIG. 2 shows retention of fluid on the apical surface of HBE (human bronchial epithelial) cell cultures over 24h after addition of NaCl or isotonic Na Gluconate solutions without and with 10 pM Compound 1 (“Cmpd 1”). KBR = Krebs bicarbonate Ringer. The data shown here are means + / - standard deviations (n=4-22). ***p<0.0001; **p<0.005; and *p<0.01 vs. NaCl KBR at 24h.

[0041]

[0035] FIGs. 3A-3C show fluid retention with an ENaC blocker. FIG. 3A: Dorsal view of mouse lungs removed after left lung infusion of PBS+ 1% Evans blue (4pl / g). FIG. 3B: Percent water content measured gravimetrically in left lobe of ~8 wk-old pENaC-Tg mice with selective left lobe instillation of PBS, Cmpd 2 in PBS, or 3.25% Na Glue + / - Cmpd 2, immediately or 4h after instillation. ***p<0.001; ****p<0.0001 vs PBS t=4h by ANOVA; $ p<0.05 vs PBS t=4h by exploratory 2-tailed t-test analysis. FIG. 3C: Percent water content measured gravimetrically in left lobe of ~8 wk-old WT mice with selective left lobe instillation of PBS or 200 pM Cmpd 1 in 3.25% Na Glue, immediately or 4h after instillation. * p<0.05 vs PBS t=Oh; # p<0.05 vs PBS t=4h.

[0042]

[0036] FIGs. 4A-4B show that direct instillation of a lavage solution comprising sodium gluconate and Compound 1 (“Cmpd 1”) into the left lobe eliminates mucus burden in the P- ENaC mouse model. A single dose of 100 pL PBS, 3.25% Na Gluconate, or Cmpd 1 (20 or 200 pM) in the presence of 3.25% Na Gluconate, was directly instilled into the left lobe of the P- ENaC mouse model. Lungs were processed and stained with AB-PAS for mucus quantification. The mucus burden in the treated animals was compared with a naive control (no Rx). The combined mucus measurements are portrayed in FIG. 4A (**p<0.01 by ANOVA vs. No Rx) and representative AB-PAS images for no Rx and 200 pM Cmpd 1 in Na Gluconate are shown in FIG. 4B (dark grey = mucus), n = 6 for all dosing groups. Abbreviations: AB-PAS: Alcian Blue Periodic Acid Schiff; ENaC: epithelial sodium channel; No Rx: no treatment; PBS: phosphate buffered saline.

[0043]

[0037] FIGs. 5A-5B show direct instillation of a lavage solution comprising sodium gluconate and Compound 2 (“Cmpd 2”) into the left lobe eliminates mucus burden in the P-ENaC mouse model. A single dose of 100 pL PBS, 3.25% Na Gluconate, or 200 pM Cmpd 2 in the presence of 3.25% Na Gluconate, was directly instilled into the left lobe of the P-ENaC mouse model. Lungs were processed and stained with AB-PAS for mucus quantification. The mucus burden in the treated animals was compared with a naive control (no Rx). The representative AB-PAS images for no Rx and 200 pM Cmpd 2 in 3.25% Na Gluconate are shown in FIG. 5A (dark grey = mucus) and the combined mucus measurements are portrayed in FIG. 5B (*p<0.01 by ANOVA vs.. No Rx). n = 6 for all dosing groups. Abbreviations: AB-PAS: Alcian Blue Periodic Acid Schiff; ENaC: epithelial sodium channel; No Rx: no treatment; PBS: phosphate buffered saline.

[0044]

[0038] FIGs. 6A-6D show HDM / p-ENaC-Tg Mice Model of Asthma-COPD overlap. Representative images of airway mucus obstruction by FIG. 6A: Muc5b rich plugs in pENaC- Tg mice, and FIG. 6B: Muc5ac-rich plugs in pENaC-Tg mice chronically sensitized to house dust mites (HDM). Note the prominent mucus cell metaplasia and shift towards more acidic mucopolysaccharides in AB-PAS stain following HDM administration. FIG. 6C: shows western blot analysis of mucin composition in airway mucus plugs collected from P-ENaC-Tg (P) mice and HDM / p-ENaC-Tg (HDM / p) mice. HDM induced increased secretion of an asthma associated mucin, Muc5ac. FIG. 6D: shows direct instillation of sodium gluconate and Compound 1 (“Cmpd 1”) and Compound 1 / sodium gluconate combined with NAC into the left lobe eliminates mucus burden in the P-ENaC mouse model. A single dose of 100 pL PBS, Cmpd 1 (200 pM) in the presence of 3.25% Na Gluconate, and Cmpd 1 (200 pM), and NAC (50 mM) in the presence of 2.1% Na Gluconate (was decreased upon 50 mM NAC addition to match the tonicity of the 3.25% Na Gluconate), was directly instilled into the left lobe of the HDM challenged P-ENaC mouse model. Lungs were processed and stained with AB-PAS for mucus quantification. The mucus burden in the treated animals was compared with a naive control (no Rx). The combined proximal, mid and distal lung sections mucus measurements are portrayed above (**p<0.05 by ANOVA vs. No Rx). n=3 for no Rx and n=6 for the other three dosing groups. Abbreviations: AB-PAS: Alcian Blue Periodic Acid Schiff; ENaC: epithelial sodium channel; No Rx: no treatment; PBS: phosphate buffered saline. The addition of NAC to the combination of 200 pM Cmpd 1 plus Na gluconate was numerically superior to 200 p M Cmpd 1 plus Na gluconate in reducing mucus burden in the lung

[0045]

[0039] FIG. 7 shows direct instillation of 100 mM Compound 3 (“Cmpd 3”) solution into the left lobe reduces mucus burden in the proximal lung of a P-ENaC mouse model. A naive control (No Rx), single dose of 100 pL PBS, or a single dose of 100 mM aqueous Cmpd 3 (100 pL), was directly instilled into the left lobe of the P-ENaC mouse model. Lungs were processed and stained with AB-PAS for mucus quantification. The mucus burden in the treated animals was compared with a naive control (no Rx). (*p<0.05 by ANOVA vs. No Rx). n = 7 for all dosing groups. Abbreviations: AB-PAS: Alcian Blue Periodic Acid Schiff; No Rx: no treatment; PBS: phosphate buffered saline.

[0046] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0047]

[0040] Provided herein are compositions and methods for clearing mucus from the lungs and treating diseases such as muco-obstructive lung diseases. In one aspect, provided herein are methods of clearing mucus from the lungs of a subject comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an osmolyte. In preferred embodiments, the osmolyte is not sodium chloride. In another aspect, provided herein are methods of clearing mucus from the lungs of a subject comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising a mucolytic agent. The liquid pharmaceutical composition (e.g., isotonic sodium gluconate solution) may further comprise one or more additional agents selected from epithelial sodium channel (ENaC) inhibitors, osmolytes, mucolytic agents, surfactants, anti-infectives, and anti-inflammatory agents. Also provided herein are liquid pharmaceutical compositions useful in the methods described herein.

[0048] Direct Instillation

[0049]

[0041] Methods described herein comprise instilling into the lungs of a subject a liquid pharmaceutical composition. “Instilling,” “instillation,” and the like, as used herein, are art- understood terms that refer to introducing a substance directly into the lungs of a subject (e.g., via bronchoscope) in a manner that avoids the upper respiratory tract. Installation is used as an alternative to inhalation for introducing substances into the lungs. A key attribute of instillation is that a large volume of instillate is not diluted as an aerosol may be when deposited on the airway surface. Installation into the lungs is also referred to as “intratracheal instillation,” “oropharyngeal instillation,” and the like. The term “lungs” and “lung” are used interchangeably and refer to either a single lobe, multiple lobes, one lung, or both lungs.

[0050]

[0042] As described herein, a liquid pharmaceutical composition can be instilled into the lungs of a subject via a device such as a bronchoscope, endotracheal tube, or tracheostomy tube. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via bronchoscope. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via endotracheal tube. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via tracheostomy tube. In certain embodiments, the liquid pharmaceutical composition is instilled into the lungs of the subject via double lumen endotracheal tube.

[0051]

[0043] In certain embodiments, at least a portion of the liquid pharmaceutical composition is instilled into the small and / or large airways of the lungs (e.g., the small and / or large airways of one or more lobes). In In certain embodiments, at least a portion of the liquid pharmaceutical composition is instilled into the small airways (z.e., <2 mm) of the lungs (e.g., the small airways of one or more lobes). In certain embodiments, the instilled volume is sufficient such that at least a portion of the liquid pharmaceutical composition is deposited into the small airways of the lungs (e.g., the small airways of one or more lobes).

[0052]

[0044] An “effective amount” of a composition described herein refers to an amount sufficient to elicit the desired biological response, i.e., clearance of mucus from the lungs. An effective amount may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, or pharmacodynamics of the particular composition, the condition being treated, the frequency of administration, and the species, age, and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is an amount sufficient for clearing mucus from the lungs.

[0053]

[0045] A “therapeutically effective amount” of a composition described herein is a volume sufficient to provide a therapeutic benefit, i.e., treating a disease such as a muco-obstructive lung disease. A therapeutically effective amount of a composition means a volume of the composition, alone or in combination with other therapies, which provides a therapeutic benefit. A “therapeutically effective amount” of a compound described herein refers to a therapeutically effective amount (by weight) of the compound. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for clearing mucus from the lungs of a subject and / or treating any of the lung conditions described herein.

[0046] An appropriate volume of the liquid pharmaceutical composition can be instilled into the lungs (e.g., as a single bolus dose). In certain embodiments, a volume of at least about 1 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject. In certain embodiments, a volume of about 1 mL to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose).

[0054]

[0047] In certain embodiments, about 1 mL to about 10000 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 10 mL to about 10 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 10 mL to about 1 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 10 mL to about 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 250 mL to about 750 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 400 mL to about 600 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose).

[0055]

[0048] In certain embodiments, about 1 L to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 2 L to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 5 L to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 10 L to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 15 L to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose).

[0056]

[0049] In certain embodiments, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 100 mL, about 150 mL, about 200 mL, about 250 mL, about 300 mL, about 350 mL, about 400 mL, about 450 mL, about 500 mL, about 550 mL, about 600 mL, about 650 mL, about 700 mL, about 750 mL, about 800 mL, about 850 mL, about 900 mL, about 950 mL, or about 1000 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose). In certain embodiments, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, or about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject (e.g., as a single bolus dose).

[0057]

[0050] In certain embodiments, the volume instilled may be large relative to the total lung volume, e.g., about 500 mL into a single lobe with a total air capacity of about 1000 mL. In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into a single lobe of the lungs of the subject. In certain embodiments, about 300 mL to about 500 mL of the liquid pharmaceutical composition is instilled into a single lobe of the lungs of the subject as a single bolus dose.

[0058]

[0051] The volume of solution instilled can correlate with the mucus burden in the subject. In certain embodiments, the volume instilled is a volume effective to rehydrate hyperconcentrated / dehydrated intrapulmonary mucus from values of about 12% to about 18% solids content to values within the normal range, e.g., about 2% solids content. In certain embodiments, an effective volume may require about 5-10 fold more instilled volume than the volume of the target intrapulmonary hyperconcentrated mucus. In some instances, the range of volumes of retained hyperconcentrated / dehydrated mucus in the lung in subjects with muco-obstructive disease can range from about 0.1 mL to about 100 mL. Consequently, a range of instilled therapeutically effective volumes can be used, e.g., ranging from mL volumes to liter volumes. For example, for a subject with one simple mucus plug with a volume of 1 mL of 12% hyperconcentrated mucus, about 5-10 mL of instilled solution may be effective. As another example, if the subject has 50 mL of hyperconcentrated mucus, then about 250-500 mL of instilled liquid may be appropriate to rehydrate the mucus to point of being clearable. Many severe patients may fall into the latter category, e.g., with a relatively large volume needed to address the relatively large mucus burden.

[0059]

[0052] The terms “bolus,” “bolus dose,” and “single bolus dose” are used interchangeably and refer a discrete amount of a pharmaceutical composition administered at once over a short period of time, i.e., in the same installation step.

[0060]

[0053] A method described herein can be performed once, or may be repeated one or more (e.g., 1, 2, 3, 4, 5, or more) times over varying intervals. In certain embodiments, the method is performed once. In certain embodiments, the method is repeated one or more (e.g., 1, 2, 3, 4, 5, or more) times over varying intervals.

[0061]

[0054] To aid in clearing mucus from the lungs, a method provided herein may further comprise a post-instillation step of subjecting the subject to a physical clearance aid such as a vibrating vest, Aerobika device, positive pressure device (e.g., intermittent positive pressure ventilation (IPPV)), or positional modification.

[0062]

[0055] A “subject” to which administration is contemplated refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease, disorder, or condition. In certain embodiments, the subject is a human.

[0063] Liquid Pharmaceutical Compositions

[0064]

[0056] In certain embodiments, methods provided herein comprise instilling into the lungs of a subject a liquid pharmaceutical composition comprising an osmolyte, ENaC inhibitor, and / or a mucolytic agent. A liquid pharmaceutical composition provided herein may also be referred to as a “lavage solution.” The terms “composition,” “formulation,” and “solution” are used interchangeably herein.

[0065]

[0057] The liquid pharmaceutical composition may comprise one or more additional agents such as one or more agent selected from epithelial sodium channel (ENaC) inhibitors, osmolytes, mucolytic agents, surfactants, DNases, anti-infectives, and anti-inflammatory agents.

[0066]

[0058] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution. In certain embodiments, the liquid pharmaceutical composition is an isotonic (e.g., isotonic aqueous Na gluconate) solution. Without wishing to be bound by any particular theory, isotonic solutions may be preferred as hypo-osmolal or hyper-osmolal solutions may cause cell swelling or cell shrinking, respectively, both of which may produce inflammation in the lungs.

[0067]

[0059] In certain embodiments, the liquid pharmaceutical composition has a pH of at or around physiological pH (i.e., a pH of about 7.4). In certain embodiments, the liquid pharmaceutical composition has a pH of from about 6.4 to about 8.4. In certain embodiments, the liquid pharmaceutical composition has a pH of from about 7.0 to about 8.0. In certain embodiments, the liquid pharmaceutical composition has a pH of from about 7.2 to about 7.6. In certain embodiments, the liquid pharmaceutical composition has a pH of from about 7.3 to about 7.5. In certain embodiments, the liquid pharmaceutical composition has a pH of about 7.4.

[0068]

[0060] In certain embodiments, the liquid pharmaceutical composition has a pH of about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, or about 8.4.

[0069]

[0061] Various aspects of the compositions are described herein. Any of the liquid pharmaceutical compositions described herein are also provided as aspects of the disclosure.

[0070] Osmolytes

[0071]

[0062] Liquid pharmaceutical compositions described herein may comprise an osmolyte. In certain embodiments, the osmolyte is not sodium chloride. In certain embodiments, the osmolyte has a longer retention time in the lungs than sodium chloride.

[0072]

[0063] “Osmolytes” according to the present disclosure are molecules or compounds that are osmotically active. “Osmotically active” molecules and compounds are membrane-impermeable (z.e., essentially non-absorbable) on airway or pulmonary epithelial surfaces. Suitable osmolytes include ionic osmolytes (z.e., salts (e.g., ionic sugars (e.g. sodium gluconate)), and non-ionic osmolytes (z.e., sugars (e.g., fructose, galactose, glucose, dextrose, lactose, maltose, xylose, sucrose), a reduced sugar (e.g., glycerol, erythritol, threitol, D-threitol, L-threitol, xylitol, ribitol, arabitol, D-arabitol, L-arabitol, D-xylitol, mannitol, sorbitol, galactitol, allitol, altritol, L- sorbitol, L-mannitol), sugar alcohols (e.g., mannitol, xylitol, sorbitol, lactitol, erythritol, glycerol, threitol, arabitol, ribitol, galactitol, fucitol, maltitol, isomalt) and organic osmolytes). Osmolytes suitable for use in the present disclosure may be in racemic form or in the form of an enantiomer, diastereomer, tautomer, polymorph, or pseudopolymorph.

[0073]

[0064] Examples of ionic osmolytes useful in the present disclosure include any salt of a pharmaceutically acceptable anion and a pharmaceutically acceptable cation. Preferably, either (or both) of the anion and cation are osmotically active and not subject to rapid active transport, in relation to the airway surfaces to which they are administered. Such compounds include but are not limited to anions and cations that are contained in FDA approved commercially marketed salts, see, e.g., Remington: The Science and Practice of Pharmacy, Vol. II, pg. 1457 (19thEd. 1995), and can be used in any combination as known in the art.

[0074]

[0065] Specific examples of pharmaceutically acceptable osmotically active anions include but are not limited to: acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate, edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (1,2-ethanedisulfonate), fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate (p-glycollamidophenylarsonate), hexylresorcinate, hydrabamine (A,A’-Di(dehydroabietyl) ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, nitrite, pamoate (embonate), pantothenate, phosphate or diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate (8- chlorotheophyllinate), triethiodide, and bicarbonate. Preferred anions include chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide, and phosphate.

[0075]

[0066] Specific examples of pharmaceutically acceptable osmotically active cations include but are not limited to: organic cations (e.g., benzathine ( / V’-dibcnzylcthylcncdiaminc), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methyl D-glucamine), procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium, N-methyl D-glycerol, and the like) and metallic cations (e.g., aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron, ammonium, and the like). Preferred organic cations include 3-carbon, 4- carbon, 5-carbon and 6-carbon organic cations. Preferred cations include sodium, potassium, choline, lithium, meglumine, D-lysine, ammonium, magnesium, and calcium.

[0076]

[0067] Specific examples of ionic osmolytes that may be used include but are not limited to, potassium chloride, choline chloride, choline iodide, lithium chloride, meglumine chloride, L- lysine chloride, D-lysine chloride, ammonium chloride, potassium sulfate, potassium nitrate, sodium gluconate, potassium gluconate, potassium iodide, ferric chloride, ferrous chloride, potassium bromide, and combinations thereof.

[0077]

[0068] Non-ionic osmolytes include sugars, sugar-alcohols, and organic osmolytes. Sugars and sugar-alcohols useful as osmolytes in the present disclosure include but are not limited to: 3- carbon sugars (e.g., glycerol, dihydroxy acetone), 4-carbon sugars (e.g., both the D and L forms of erythrose, threose, and erythrulose), 5-carbon sugars (e.g., both the D and L forms of ribose, arabinose, xylose, lyxose, psicose, fructose, sorbose, and tagatose), 6-carbon sugars (e.g., both the D and L forms of altose, allose, glucose, mannose, gulose, idose, galactose, and talose, and the D and L forms of allo-heptulose, allo-hepulose, gluco-heptulose, manno-heptulose, gulo- heptulose, ido-heptulose, galacto-heptulose, talo-heptulose), and sugar-alcohols thereof. Additional sugars useful in the practice of the present disclosure include raffinose, raffinose series oligosaccharides, and stachyose. Both the D and L forms of the reduced form of each sugar / sugar alcohol are also suitable for the present disclosure. For example, glucose, when reduced, becomes sorbitol; an osmolyte within the scope of the disclosure. Accordingly, sorbitol and other reduced forms of sugar / sugar alcohols (e.g., mannitol, dulcitol, arabitol) are suitable osmolytes for use in the present disclosure. In some embodiments, mannitol is a preferred nonionic osmolyte for use in combination with the compounds disclosed herein. In some embodiments, xylitol is a preferred non-ionic osmolyte for use in combination with the compounds disclosed herein.

[0078]

[0069] “Organic osmolytes” generally refers to molecules that control intracellular osmolality in the kidney. Such osmolytes may also be used in the lungs. See e.g., J. S. Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol. 268, F983-F996 (1995). Organic osmolytes include but are not limited to three major classes of compounds: polyols (polyhydric alcohols), methylamines, and amino acids. Suitable polyol organic osmolytes include but are not limited to inositol, myo-inositol, and sorbitol. Suitable methylamine organic osmolytes include but are not limited to choline, betaine, carnitine (L-, D- and DL forms), phosphorylcholine, lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and creatine phosphate. Suitable amino acid organic osmolytes include but are not limited to the D- and Informs of glycine, alanine, glutamine, glutamate, aspartate, proline, and taurine. Additional organic osmolytes suitable for use in the present disclosure include trehalose and sarcosine. Mammalian organic osmolytes are preferred, with human organic osmolytes being most preferred. However, certain organic osmolytes are of bacterial, yeast, and marine animal origin, and these compounds may also be employed.

[0079]

[0070] Osmolyte precursors may be used. An “osmolyte precursor” as used herein refers to a compound which is converted into an osmolyte by a metabolic step, either catabolic or anabolic. Examples of osmolyte precursors include but are not limited to glucose, glucose polymers, glycerol, choline, phosphatidylcholine, lyso-phosphatidylcholine and inorganic phosphates, which are precursors of polyols and methylamines. Precursors of amino acid osmolytes include proteins, peptides, and polyamino acids, which are hydrolyzed to yield osmolyte amino acids, and metabolic precursors which can be converted into osmolyte amino acids by a metabolic step such as transamination. For example, a precursor of the amino acid glutamine is poly-L- glutamine, and a precursor of glutamate is poly-L-glutamic acid.

[0080]

[0071] Chemically modified osmolytes or osmolyte precursors may also be employed in the formulations, uses, and regimens, described herein. Such chemical modifications involve linking the osmolyte, or precursor thereof, to an additional chemical group which alters or enhances the effect of the osmolyte or osmolyte precursor (e.g., inhibits degradation of the osmolyte molecule). Such chemical modifications have been utilized with drugs or prodrugs and are known in the art. (See, for example, U.S. Pat. Nos. 4,479,932 and 4,540,564; Shek, E. et al., J. Med. Chem. 19:113-117 (1976); Bodor, N. et al., J. Pharm. Sci. 67:1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26:313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986); each of which are incorporated herein by reference).

[0081]

[0072] In certain embodiments, the liquid pharmaceutical composition comprises an effective amount (e.g., therapeutically effective amount) of the osmolyte.

[0082]

[0073] In certain embodiments, the concentration of the osmolyte in the composition is from about 1% to about 10% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is from about 2.0% to about 5% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is from about 2.5% to about 5% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is from about 2.5% to about 4% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is from about 3.0% to about 3.5% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is from about 3.2% to about 3.3% (w / v). In certain embodiments, the concentration of the osmolyte in the composition is about 3.25% (w / v).

[0083]

[0074] In certain embodiments, the concentration of the osmolyte in the composition is about 2.5 %, about 2.6%, about 2.7%, about 2.75%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.25%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.75%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.25%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.75%, about 4.8%, about 4.9%, or about 5.0% (w / v).

[0084]

[0075] In certain embodiments, the osmolyte is a gluconate salt. In certain embodiments, the osmolyte is a sodium salt. In certain embodiment, the osmolyte is a sodium salt, provided that the sodium salt is not sodium chloride.

[0085]

[0076] In certain embodiments, the osmolyte is sodium gluconate. In certain embodiments, the liquid pharmaceutical composition is an isotonic sodium gluconate solution. In certain embodiments, the concentration of sodium gluconate in the composition is from about 1% to about 10% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is from about 2.0% to about 5% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is from about 2.5% to about 5% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is from about 2.5% to about 4% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is from about 3.0% to about 3.5% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is from about 3.2% to about 3.3% (w / v). In certain embodiments, the concentration of sodium gluconate in the composition is about 3.25% (w / v).

[0086]

[0077] In certain embodiments, the concentration of sodium gluconate in the composition is about 2.5 %, about 2.6%, about 2.7%, about 2.75%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.25%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.75%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.25%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.75%, about 4.8%, about 4.9%, or about 5.0% (w / v).

[0087]

[0078] In certain embodiments, the osmolyte is sodium chloride. In certain embodiments, the osmolyte is isotonic sodium chloride. In some embodiments, sodium chloride is used in combination with an ENaC inhibitor (e.g., Compound 2 or a pharmaceutically acceptable salt thereof) described herein. ENaC Inhibitors

[0088]

[0079] As described herein, the liquid pharmaceutical composition may comprise one or more epithelial sodium channel (ENaC) inhibitors. The epithelial protein that mediates the ratelimiting step of Na+and liquid absorption from pulmonary luminal surfaces is the epithelial Na+channel (“ENaC”). ENaC is positioned on the apical surface of the epithelium, z.e., the mucosal surface-environmental interface. ENaC inhibitors (e.g., small molecule ENaC inhibitors) are capable of directly preventing sodium transport through the ENaC channel pore, blocking subsequent extrusion to the basolateral Na-K-ATPase pump to effect transepithelial Na (and fluid) transport. In some instances, an ENaC inhibitor can be used to inhibit ENaC mediated Na+and liquid absorption. “Blocker” and “inhibitor” are used interchangeably herein.

[0089]

[0080] Examples of ENaC inhibitors can be found in, e.g., PCT Publication Nos. WO 2023 / 146892 and WO 2023 / 146896, the entire contents of which are incorporated herein by reference. For example, in certain embodiments, the ENaC inhibitor is the following: (Compound 1), or a pharmaceutically acceptable salt thereof. In certain embodiments, the ENaC inhibitor is the hydrochloride (HC1) salt of Compound 1.

[0090]

[0081] In certain embodiments, the ENaC inhibitor is the following: (Compound 2), or a pharmaceutically acceptable salt thereof. In certain embodiments, the ENaC inhibitor is the hydrochloride (HC1) salt of Compound 2.

[0091]

[0082] Additional examples of ENaC inhibitors can be found in, e.g., PCT Publication Nos. WO 2003 / 070182, WO 2003 / 070184, WO 2004 / 073629, WO 2005 / 025496, WO 2005 / 016879, WO

[0092] 2005 / 018644, WO 2006 / 022935, WO 2006 / 023573, WO 2006 / 023617, WO 2007 / 018640, WO 2007 / 146869, WO 2008 / 031028, and WO 2008 / 031048; and US Patent Nos. 6,858,614, 6,858,615, 6,903,105, 7,064,129, 7,186,833, 7,189,719, 7,192,958, 7,192,959, 7,192,960,

[0093] 7.241.766, 7,247,636, 7,247,637, 7,317,013, 7,332,496, 7,368,447, 7,368,450, 7,368,451, 7,375,102, 7,388,013, 7,399,766, 7,410,968, 7,807,834, 7,842,697, 7,868,010, 8,124,607, 8,669,262, 8,980,898, 9,029,382, 9,102,633, 9,593,084, 9,072,738, 7,956,059, 7,981,898, 8,058,278, 8,143,256, 8,163,758, 8,288,391, 7,745,442, 8,314,105, 8,324,218, and 9,856,224, the entire contents of each of which is incorporated herein by reference.

[0094]

[0083] Examples of other ENaC receptor blockers for use in the disclosure include but are not limited to amiloride and derivatives thereof such as those compounds described in US Patent No. 6858615; and PCT Publication Nos. WO 2003 / 070182, WO 2004 / 073629, WO 2005 / 018644, WO 2006 / 022935, WO 2007 / 018640, and WO 2007 / 146869, all to Parion Sciences, Inc., the entire contents of each of which is incorporated herein by reference.

[0095]

[0084] Other examples of ENaC blockers include, but are not limited to, amiloride, benzamil, and phenamil; and amiloride analogues as exemplified by US Pat. No. 6,858,614, US Pat. No. 6,858,615, US Pat. No. 6,903,105, US Pat. No. 6,995,160, US Pat. No. 7,026,325, US Pat. No.

[0096] 7,030,117, US Pat. No. 7,064,129, US Pat. No. 7,186,833, US Pat. No. 7,189,719, US Pat. No.

[0097] 7,192,958, US Pat. No. 7,192,959, US Pat. No. 7,241,766, US Pat. No. 7,247,636, US Pat. No.

[0098] 7,247,637, US Pat. No. 7,317,013, US Pat. No. 7,332,496, US Pat. No. 7,345,044, US Pat. No.

[0099] 7,368,447, US Pat. No. 7,368,450, US Pat. No. 7,368,451, US Pat. No. 7,375,107, US Pat. No.

[0100] 7.399.766, US Pat. No. 7,410,968, US Pat. No. 7,820,678, US Pat. No. 7,842,697, US Pat. No.

[0101] 7,868,010, and US Pat. No. 7,875,619, the entire contents of each of which is incorporated herein by reference.

[0102]

[0085] Additional non-examples of ENaC inhibitors can be found in, c. . , US Patent Nos. 9,938,256, 9,920,035, 9,932,324, and 10,155,749; US Patent Application Publication Nos. 2014 / 0323447, 2015 / 0018315, 2015 / 0011535, 2015 / 0018314, 2017 / 0050952, and 2017 / 0050992; and International Patent Application Publication Nos. WO 2011 / 028740, WO 2011 / 079087, WO 2008 / 135557, WO 2009 / 150137, WO 2009 / 074575, WO 2012 / 035158, WO 2017 / 0143705, WO 2018 / 096325, WO 2019 / 077340, and WO 2015 / 149527, the entire contents of each of which is incorporated herein by reference.

[0103]

[0086] In certain embodiments, the liquid pharmaceutical composition comprises an effective amount (e.g., therapeutically effective amount) of the ENaC inhibitor.

[0104]

[0087] In certain embodiments, the concentration of the ENaC inhibitor in the liquid pharmaceutical composition is from about 0.3 pg / mL to about 200 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 0.3 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 1 g / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 25 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 50 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 75 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 100 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 125 pg / mL to about 176 pg / mL. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 150 pg / mL to about 176 pg / mL.

[0105]

[0088] In certain embodiments, the concentration of the ENaC inhibitor in the liquid pharmaceutical composition is from about 1 pM to about 500 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pM to about 300 pM.

[0089] In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pM to about 200 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pM to about 100 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pM to about 50 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 10 pM to about 30 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is about 20 pM.

[0106]

[0090] In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 100 pM to about 300 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 150 pM to about 250 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 175 pM to about 225 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is from about 190 pM to about 210 pM. In certain embodiments, the concentration of the ENaC inhibitor in the composition is about 200 pM.

[0107]

[0091] In certain embodiments, the concentration of the ENaC inhibitor in the composition is about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, or about 300 pM.

[0092] In certain embodiments, as described herein, the ENaC inhibitor is Compound 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the liquid pharmaceutical composition is from about 1 pM to about 500 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 10 pM to about 300 pM.

[0108]

[0093] In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 10 pM to about 200 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 10 pM to about 100 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 10 pM to about 50 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 10 pM to about 30 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is about 20 pM.

[0109]

[0094] In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 100 pM to about 300 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 150 pM to about 250 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 175 pM to about 225 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is from about 190 pM to about 210 pM. In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is about 200 pM.

[0110]

[0095] In certain embodiments, the concentration of Compound 1 or pharmaceutically acceptable salt thereof in the composition is about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, or about 300 pM.

[0111] Mucolytic Agents

[0112]

[0096] As described herein, the liquid pharmaceutical composition may comprise one or more mucoactive agents, such as one or more mucolytic agents. The term “mucoactive agent” and the like refers to a class of chemical agents which aid in the clearance of mucus from the upper and / or lower airways of the lungs.

[0113]

[0097] Mucoactive agents include expectorants, mucolytics, mucoregulators, and mucokinetics. Exemplary mucoactive agents include hypertonic saline, iodide-containing compounds, glyceryl guaiacolate (guaifenesin), ion channel modifiers, anticholinergic agents, glucocorticoids, prednisolone, iodinated glycerol, domiodol, tricyclic nucleotides (e.g., uridine triphosphate and adenosine triphosphate), sodium citrate, potassium citrate, carbocysteine, potassium iodide, guaifenesin, tolu balsam, vasaka, ammonium chloride, macrolide antibiotics (e.g., erythromycin, azithromycin, clarithromycin, roxithromycin), acetylcysteine, acystelyn, ambroxol, bromhexine, carbocisteine, erdosteine, fudosteine, mecysteine, gelsolin, thymosin P4, non-destructive mucolytics (e.g., dextran and heparin), bronchodilators, tricyclic nucleotides, broxol and domase alfa, HC1, or any combination or subset thereof.

[0114]

[0098] “Mucolytic agent” refers to a compound that breaks up mucus, reducing its viscosity and making it easier to clear. In certain embodiments, the mucolytic agent is a mucus reducing agent. Mucin proteins provide the viscoelastic properties to mucus and are organized into high molecular weight polymers via the formation of covalent (disulfide) and non-covalent intermucin monomeric proteins bonds. Disruption of the covalent bonds with reducing agents is a well-established method to reduce the viscoelastic properties of mucus and can improve clearance. Reducing agents are well known to decrease mucus viscosity. Examples of reducing agents include sulfide containing molecules or phosphines capable of reducing protein di-sulfide bonds including, but not limited to, N-acetyl cysteine (NAC), Nacystelyn, carbocysteine, glutathione, dithiothreitol, thioredoxin containing proteins, and tris (2-carboxyethyl) phosphine. In certain embodiments, the mucolytic agent is cysteamine.

[0115]

[0099] Other non-limiting examples of mucolytic agents can be found in, e.g., US Patent Nos. 9,856,283, 9,346,753, 9,963,427, 10,526,283, 10,106,551, 10,968, 233, and 10,96,823, the entire contents of each of which is incorporated herein by reference. Other non-limiting examples of mucolytic agents can be found in, e.g., International Patent Application Publication Nos. WO 2016 / 123335, WO 2020 / 055916, WO 2021 / 231397, and WO 2021 / 231421, the entire contents of each of which is incorporated herein by reference. Other non-limiting examples of mucolytic agents can be found in, e.g., US Patent Application Publication No. US 2021 / 0230202, the entire contents of which is incorporated herein by reference.

[0116]

[0100] Reducing agents such as NAC are not well suited for aerosol administration and may be particularly well suited for the installation methods described herein. In certain embodiments, the mucolytic agent is NAC.

[0101] In certain embodiments, the mucolytic agent is:

[0117] (Compound 3), or a pharmaceutically acceptable salt thereof. In certain embodiments, the mucolytic agent is the HC1 salt of Compound 3.

[0118]

[0102] In certain embodiments, the mucolytic agent is selected from one of the following:

[0119]

[0120]

[0103] In certain embodiments, the liquid pharmaceutical composition comprises an effective amount (e.g., therapeutically effective amount) of the mucolytic agent.

[0121]

[0104] In certain embodiments, the concentration of the mucolytic agent in the liquid pharmaceutical composition is from about 1 mM to about 300 mM. In certain embodiments, the concentration of the mucolytic agent in the composition is from about 10 mM to about 200 mM. In certain embodiments, the concentration of the mucolytic agent in the composition is from about 50 mM to about 150 mM. In certain embodiments, the concentration of the mucolytic agent in the composition is from about 75 mM to about 125 mM. In certain embodiments, the concentration of the mucolytic agent in the composition is about 100 mM.

[0122]

[0105] In certain embodiments, the concentration of the mucolytic agent in the composition is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

[0106] In certain embodiments, the concentration of the mucolytic agent in the composition is from about 1% to about 30% (w / v). In certain embodiments, the concentration of the mucolytic agent in the composition is from about 10% to about 30% (w / v). In certain embodiments, the concentration of the mucolytic agent in the composition is from about 15% to about 25% (w / v). In certain embodiments, the concentration of the mucolytic agent in the composition is about 20% (w / v).

[0123]

[0107] In certain embodiments, the concentration of the mucolytic agent in the composition is about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% (w / v).

[0124]

[0108] As described herein, in certain embodiments, the mucolytic agent is Compound 3 or a pharmaceutically acceptable salt thereof. In certain embodiments, the concentration of Compound 3 or pharmaceutically acceptable salt thereof in the liquid pharmaceutical composition is from about 1 mM to about 300 mM. In certain embodiments, the concentration of Compound 3 or pharmaceutically acceptable salt thereof in the composition is from about 10 mM to about 200 mM. In certain embodiments, the concentration of Compound 3 or pharmaceutically acceptable salt thereof in the composition is from about 50 mM to about 150 mM. In certain embodiments, the concentration of Compound 3 or pharmaceutically acceptable salt thereof in the composition is from about 75 mM to about 125 mM. In certain embodiments, the concentration of Compound 3 or pharmaceutically acceptable salt thereof in the composition is about 100 mM.

[0125]

[0109] In certain embodiments, the concentration of Compound 3 or pharmaceutically acceptable salt thereof in the composition is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

[0126]

[0110] As described herein, in certain embodiments, the mucolytic agent is NAC. In certain embodiments, the concentration of NAC in the composition is from about 1% to about 30% (w / v). In certain embodiments, the concentration of NAC in the composition is from about 10% to about 30% (w / v). In certain embodiments, the concentration of NAC in the composition is from about 15% to about 25% (w / v). In certain embodiments, the concentration of NAC in the composition is about 20% (w / v).

[0127]

[0111] In certain embodiments, the concentration of NAC in the composition is about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% (w / v).

[0128] Chloride ( Cl') Salts

[0129]

[0112] Compositions described herein may comprise chloride (Cl’) ions in the form of chloride salt(s). Without wishing to be bound by any particular theory, the presence of chloride ions in a instillation solution can help prevent activation of vagal afferent nerves in the lungs which can help prevent cough. Therefore, an amount of a chloride salt may be included in the instillation solutions described herein.

[0130]

[0113] In certain embodiments, the concentration of the chloride salt is from about 1 mM to about 30 mM. In certain embodiments, the concentration of the chloride salt is from about 10 mM to about 30 mM. In certain embodiments, the concentration of the chloride salt is from about 15 mM to about 25 mM. In certain embodiments, the concentration of the chloride salt is from about 20 mM.

[0131]

[0114] In certain embodiments, the concentration of the chloride salt is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM.

[0132]

[0115] In certain embodiments, the chloride salt is sodium chloride. In embodiments where the liquid pharmaceutical composition comprises an osmolyte, wherein the osmolyte is not sodium chloride, the composition may comprise an amount of sodium chloride as a chloride ion source in addition to the non-NaCl osmolyte.

[0133]

[0116] In certain embodiments, the concentration of sodium chloride is from about 1 mM to about 30 mM In certain embodiments, the concentration of sodium chloride is from about 10 mM to about 30 mM. In certain embodiments, the concentration of sodium chloride is from about 15 mM to about 25 mM. In certain embodiments, the concentration of sodium chloride is from about 20 mM.

[0134]

[0117] In certain embodiments, the concentration of sodium chloride is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM.

[0135] Surfactants

[0136]

[0118] As described herein, the liquid pharmaceutical composition may further comprise one or more surfactants and / or detergents. In certain embodiments, the liquid pharmaceutical composition comprises a surfactant. Surfactants and detergents are spreading agents shown to decrease mucus viscoelasticity, thus improving mucus clearability. Examples of surfactants include dipalmitoyl phosphatidylcholine (DPPC), palmitic acid, palmitoyloleoylphosphatidylglycerol, surfactant-associated proteins (e.g. SP-A, B, or C), or animal derived (e.g. from cow or calf lung lavage or extracted from minced pig lung) or combinations thereof. See, e.g., US Pat. No. 7,897,577, US Pat. No. 5,876,970, US Pat. No. 5,614,216, US Pat. No. 5,100,806, and US Pat. No. 4,312,860. Examples of surfactant products include colfosceril palmitate, DPPC and egg phosphatidylglycerol, KL-4 surfactant, lusulptide, rSP-C surfactant, bovactant, poractant alfa, calfactant, modified bovine surfactant, Surface®, nonionic alcohol ethoxylate surfactant, and beractant. Examples of detergents include, but are not limited to, Tween-80 and triton-X 100.

[0137]

[0119] In certain embodiments, the liquid pharmaceutical composition comprises an effective amount (e.g., therapeutically effective amount) of the surfactant.

[0138] Proteases and. Other Components

[0139]

[0120] In some embodiments, a composition described herein comprises a protease (e.g., a protease that effects mucins). In certain embodiments, the protease is bromelain. See, e.g., Pillai et al. Adv Respir Med. 2023 Apr 14;91(2): 146-163. In certain embodiments, the protease is mucin specific protease (SteC). In certain embodiments, the protease is described in International Patent Application Publication No. WO 2020 / 097386, the entire contents of which is incorporated herein by reference.

[0140]

[0121] As described herein, the liquid pharmaceutical composition may further comprise one or more addition agents. In certain embodiments, the liquid pharmaceutical composition comprises one or more anti-infectives (e.g., one or more antibiotics). In certain embodiments, the liquid pharmaceutical composition comprises one or more anti-inflammatory agents. In certain embodiments, the liquid pharmaceutical composition comprises one or more DNases.

[0141] Instillation Solutions - Certain Embodiments

[0142]

[0122] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising an osmolyte and an ENaC inhibitor. In certain embodiments, the composition is an aqueous solution comprising about 2.5% to about 5% (w / v) of an osmolyte; and about 10 pM to about 300 pM of an ENaC inhibitor (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor).

[0143]

[0123] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising an osmolyte, an ENaC inhibitor, and a chloride salt. In certain embodiments, the composition is an aqueous solution comprising about 2.5% to about 5% (w / v) of an osmolyte; about 10 pM to about 300 pM of an ENaC inhibitor (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor); and about 10 mM to about 30 mM of a chloride salt.

[0144]

[0124] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising sodium gluconate and an ENaC inhibitor. In certain embodiments, the composition is an aqueous solution comprising about 2.5% to about 5% (w / v) of sodium gluconate; and about 10 pM to about 300 pM of an ENaC inhibitor (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor). In certain embodiments, the composition is an aqueous solution comprising about 3.25% (w / v) of sodium gluconate; and about 10 pM to about 300 pM of an ENaC inhibitor (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor).

[0145]

[0125] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising sodium gluconate, an ENaC inhibitor, and a chloride salt. In certain embodiments, the composition is an aqueous solution comprising about 2.5% to about 5% (w / v) of sodium gluconate; about 10 pM to about 300 pM of an ENaC inhibitor (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor); and about 10 mM to about 30 mM of a chloride salt. In certain embodiments, the composition is an aqueous solution comprising about 3.25% (w / v) of sodium gluconate; about 10 pM to about 300 pM of an ENaC inhibitor (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor); and about 10 mM to about 30 mM of a chloride salt.

[0146]

[0126] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising sodium gluconate and Compound 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition is an aqueous solution comprising about 2.5% to about 5% (w / v) of sodium gluconate; and about 10 pM to about 300 pM of Compound 1 or a pharmaceutically acceptable salt thereof (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of Compound 1 or a pharmaceutically acceptable salt thereof). In certain embodiments, the composition is an aqueous solution comprising about 3.25% (w / v) of sodium gluconate; and about 10 pM to about 300 pM of Compound 1 or a pharmaceutically acceptable salt thereof (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of an ENaC inhibitor). In certain embodiments, the composition is an aqueous solution comprising about 3.25% (w / v) of sodium gluconate; and about 20 pM or about 200 pM of Compound 1 or a pharmaceutically acceptable salt thereof.

[0147]

[0127] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising sodium gluconate, Compound 1 or a pharmaceutically acceptable salt thereof, and sodium chloride. In certain embodiments, the composition is an aqueous solution comprising about 2.5% to about 5% (w / v) of sodium gluconate; about 10 pM to about 300 pM of Compound 1 or a pharmaceutically acceptable salt thereof (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 |aM, of Compound 1 or a pharmaceutically acceptable salt thereof); and about 10 mM to about 30 mM of sodium chloride. In certain embodiments, the composition is an aqueous solution comprising about 3.25% (w / v) of sodium gluconate; about 10 pM to about 300 pM of Compound 1 or a pharmaceutically acceptable salt thereof (e.g., about 10 pM to about 30 pM, or about 150 pM to about 250 pM, of Compound 1 or a pharmaceutically acceptable salt thereof); and about 10 mM to about 30 mM of sodium chloride. In certain embodiments, the composition is an aqueous solution comprising about 3.25% (w / v) of sodium gluconate; about 20 pM or about 200 pM of Compound 1 or a pharmaceutically acceptable salt thereof; and about 20 mM of sodium chloride.

[0148]

[0128] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising a mucolytic agent. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 10 mM to about 200 mM of a mucolytic agent. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 100 mM of a mucolytic agent. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 10 mM to about 200 mM of Compound 3 or a pharmaceutically acceptable salt thereof. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 100 mM of Compound 3 or a pharmaceutically acceptable salt thereof. The composition may further comprise a chloride salt (e.g., about 10 mM to about 30 mM (e.g., about 20 mM) of chloride salt such as sodium chloride)

[0149]

[0129] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 10% to about 30% (w / v) of a mucolytic agent. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 20% (w / v) of a mucolytic agent. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 10% to about 30% (w / v) of NAC. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 20% (w / v) of NAC.

[0150]

[0130] In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising a mucolytic agent and a chloride salt. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 10% to about 30% (w / v) of a mucolytic agent; and about 10 mM to about 30 mM of a chloride salt. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 20% (w / v) of a mucolytic agent; and about 20 mM of a chloride salt. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 10% to about 30% (w / v) of NAC; and about 10 mM to about 30 mM of a sodium chloride. In certain embodiments, the liquid pharmaceutical composition is an aqueous solution comprising about 20% (w / v) of NAC; and about 20 mM of sodium chloride.

[0151] Lung Conditions, Methods of Treatment and Uses

[0152]

[0131] Provided herein are methods of clearing mucus from the lungs. As used herein, “clearing” refers to reduction of the amount of mucus in the lungs of a subject, such as complete or partial elimination of excess mucus and / or one or more mucus plugs from the lungs. In certain embodiments, the mucus is one or more mucus plugs. In certain embodiments, the mucus in the lungs of the subject is cleared by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.

[0153]

[0132] As described herein, in certain embodiments, a method provided herein may further comprise a post-instillation step of subjecting the subject to a physical clearance aid such as a vibrating vest, Aerobika device, positive pressure device (e.g., intermittent positive pressure ventilation (IPPV)), or positional modification.

[0154]

[0133] In certain embodiments, the subject has a lung condition, such as a mucus obstructive lung condition (z.e., “muco-obstructive lung condition” or MOLD). In certain embodiments, the subject has reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), asthma, primary ciliary dyskinesia (PCD), bronchiectasis, bronchiectasis with nasal polyposis (BENP, Woakes syndrome), bronchiectasis due to conditions other than cystic fibrosis (non-CF bronchiectasis), acute bronchitis, chronic bronchitis, immunodeficiency syndrome, post- viral mucus congestion, cystic fibrosis (CF), idiopathic pulmonary fibrosis, pneumonia, panbronchiolitis, transplant-associate bronchiolitis, or ventilator-associated tracheobronchitis. In certain embodiments, the subject is intubated. In certain embodiments, the subject is intubated post-surgery. In certain embodiments, the subject is treated in an intensive care unit (ICU).

[0155]

[0134] In certain embodiments, the subject has asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), primary ciliary dyskinesia (PCD), non-CF bronchiectasis, Job’s syndrome (autosomal dominant STAT3 deficiency).

[0156]

[0135] Also provided herein are methods of treating any of the aforementioned lung conditions. Also provided herein are pharmaceutical compositions, including the liquid pharmaceutical compositions described herein (z.e., “lavage solutions”) for clearing mucus from the lungs of a subject or treating any of the aforementioned lung conditions in a subject. Also provided herein are uses of pharmaceutical compositions, including the liquid pharmaceutical compositions described herein (z.e., “lavage solutions”) as medicaments (e.g., for clearing mucus from the lungs of a subject or treating any of the aforementioned lung conditions in a subject)

[0157]

[0136] The terms “condition,” “disease,” and “disorder” are used interchangeably herein. The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.

[0158] Additional Embodiments

[0159]

[0137] Additional embodiments are provided according to the following numbered Embodiments: Embodiment 1. A method of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an osmolyte, wherein the osmolyte is not sodium chloride.

[0160] Embodiment 2. The method of Embodiment 1, wherein the osmolyte has a longer retention time in the lungs than sodium chloride.

[0161] Embodiment 3. The method of Embodiment 1 or 2, wherein the osmolyte is a gluconate salt.

[0162] Embodiment 4. The method of any one of Embodiments 1-3, wherein the osmolyte is sodium gluconate.

[0163] Embodiment 5. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition is an aqueous solution.

[0164] Embodiment 6. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition is isotonic.

[0165] Embodiment 7. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition is an isotonic sodium gluconate solution.

[0166] Embodiment 8. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition further comprises an epithelial sodium channel (ENaC) inhibitor.

[0167] Embodiment 9a. The method of Embodiment 8, wherein the ENaC inhibitor is Compound 1, or a pharmaceutically acceptable salt thereof.

[0168] Embodiment 9b. The method of Embodiment 8, wherein the ENaC inhibitor is Compound 2, or a pharmaceutically acceptable salt thereof.

[0169] Embodiment 10. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition further comprises a mucolytic agent.

[0170] Embodiment 11. The method of Embodiment 10, wherein the mucolytic agent is N- acetyl cysteine (NAC).

[0171] Embodiment 12. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition further comprises a surfactant.

[0172] Embodiment 13. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition further comprises an antibiotic.

[0173] Embodiment 14. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition further comprises an anti-inflammatory agent.

[0174] Embodiment 15a. The method of any one of the preceding Embodiments, wherein at least a portion of the liquid pharmaceutical composition is instilled into the small airways of the lungs.

[0175] Embodiment 15b. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition is instilled in sufficient volume to flood the small airways of the lungs.

[0176] Embodiment 16. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition is instilled into the lungs of the subject via bronchoscope.

[0177] Embodiment 17. The method of any one of the preceding Embodiments, wherein the liquid pharmaceutical composition is instilled into the lungs of the subject via endotracheal tube or tracheostomy.

[0178] Embodiment 18. The method of any one of the preceding Embodiments, wherein 10 mL to 10000 mL, inclusive, of the liquid pharmaceutical composition is instilled into the lungs of the subject.

[0179] Embodiment 19. The method of any one of the preceding Embodiments, wherein approximately 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject.

[0180] Embodiment 20. The method of any one of the preceding Embodiments, wherein 10 mL to 10000 mL, inclusive, of the liquid pharmaceutical composition is instilled into the lungs of the subject as a single bolus dose.

[0181] Embodiment 21. The method of any one of the preceding Embodiments, wherein approximately 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject as a single bolus dose.

[0182] Embodiment 22. The method of any one of the preceding Embodiments, wherein the mucus is a mucus plug.

[0183] Embodiment 23. The method of any one of the preceding Embodiments, wherein the subject has a mucus obstructive lung condition.

[0184] Embodiment 24. The method of any one of the preceding Embodiments, wherein the subject has asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), primary ciliary dyskinesia (PCD), non-CF bronchiectasis, or Job syndrome.

[0185] Embodiment 25. The method of any one of the preceding Embodiments, where the subject is intubated post-surgery. Embodiment 26. The method of any one of the preceding Embodiments further comprising subjecting the subject to a vibrating vest, Aerobika device, or positive pressure device.

[0186] Embodiment 27. The method of any one of the preceding Embodiments, wherein the subject is a human.

[0187] EXAMPLES

[0188]

[0138] In order that the present disclosure may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.

[0189] Clearance of Mucus from the Lungs

[0190]

[0139] A liquid pharmaceutical composition (z.e., “lavage solution”) can be instilled in the lung through a flexible bronchoscope to hydrate mucus and enable clearance of mucus obstructions in patients with severe mucus obstructive lung conditions. In a variety of diseases and conditions, mucus accumulates in the small airways of the lung first. Mucus in large airways can be cleared intermittently by cough but mucus in both large and especially smaller airways can form plugs and completely block air movement to and from regions distal to the site of the mucus plug. With the advent of techniques to visualize the mucus plugs, there is increasing awareness of the impact of mucus burden on respiratory disease. Using a flexible bronchoscope, the lavage solution can be instilled in each lung lobe that has significant mucus obstruction to mechanically displace the mucus plugs. The lavage solution must remain in the lung to hydrate the mucus plugs to render them clearable by natural homeostatic mechanisms.

[0191]

[0140] Isotonic saline (0.9% NaCl in water) has been used to lavage the lung, but it is rapidly absorbed (approximately 60 min) from the lung surface (Berthiaume Y, Matthay MA. “Alveolar edema fluid clearance and acute lung injury.” Re spir Physiol Neurobiol. 2007; 159(3):350-9; Bastarache JA, et al. “Alveolar fluid clearance is faster in women with acute lung injury compared to men.” J Crit Care. 2011;26(3):249-56; Kylstra JA, et al. “Volume-controlled lung lavage in the treatment of asthma, bronchiectasis, and mucoviscidosis.” Am Rev Respir Dis.

[0192] 1971 ; 103(5):651 -65). Hyperconcentrated mucus (12% solid content verses normal 2% solid content (Boucher RC. “Muco-obstructive lung diseases.” N Engl J Med. 2019 May 16) can take approximately 4 hours to rehydrate into the clearable range. Therefore, as described herein, an epithelial sodium channel (ENaC) blocker (e.g., Compound 1 or 2) can be added to the aqueous solution to slow sodium, and thereby water, absorption from the lung, thus retaining the isotonic solution in the lung for sufficient time to rehydrate mucus suitable for restoration of clearance. As certain ENaC inhibitors, such as Compound 1, are not soluble in a NaCl-containing solution, isotonic sodium gluconate can be used, with the result being that sodium absorption from the lung is still slowed, resulting in retention of the isotonic solution on the lung surface. This is one advantage to using sodium gluconate as an osmolyte in these methods.

[0193] Extending Residence Time in the Lungs

[0194]

[0141] Heretofore, there has not been a requirement to extend the residence time of isotonic fluid instilled into the lung. Typically, isotonic fluid has been instilled into the lung to improve sampling of lung secretions for diagnostic purposes, e.g., a diagnostic bronchoalveolar lavage. However, due to the slow rehydrating properties of hyperconcentrated mucus, a property of the very long (multiple microns) (“fuzzy”) mucin molecules that confer viscoelastic properties to mucus, there is a need to prolong the contact time between instilled isotonic fluid and hyperconcentrated mucus to allow mucus to rehydrate to levels of hydration commensurate with restoration of mucus clearance. Mucin polymers in hyperconcentratated mucus are very long, rough / fuzzy ropes heavily interpenetrating one another in a compact “nest”-like structure. Pulling apart individual ropes to disentangle the nest is best achieved by pulling the ends of the rope slowly over long periods of time. In the case of mucus, without wishing to be bound by any particular theory, this explains the contact duration of instilled fluid and hyperconcentrated mucus to the times (e.g., multiple hours) required to hydrate / dissolve hyperconcentrated mucus.

[0195]

[0142] The short duration of retention of saline instilled into the lung (<lhr) reflects that active absorption of NaCl and water (saline) from the lung. This process is rate limited by the transport of Na from the luminal liquid into the cell via the epithelial Na channel (ENaC) followed by extrusion of Na across the basolateral barrier by the Na-K-ATPase. CP follows Na+to preserve electroneutrality via transcellular transport via the CFTR CP channel and / or via ion selective pores in the paracellular space.

[0196]

[0143] Described herein in certain embodiments is a dual, additive strategy to slow the absorption of instilled isotonic fluid into the lung to provide sufficient time (e.g., multiple hours in some embodiments) to rehydrate hyperconcentrated mucus to restore mucus clearance from the lung. First, addition of a poorly absorbable, i.e., long residence time ENaC inhibitor, can block absorption of Na+and liquid from the lung for prolonged periods to achieve this goal. Compound 1 is an example of such an ENaC inhibitor. Second, inclusion of a cell impermeant anion can retard Na+ / fluid absorption by limiting the availability of the required anion for electroneutral NaCl and fluid absorption. Gluconate is an example of such an impermeant anion.

[0144] In addition, agents that can accelerate the process of disentanglement of hyperconcentrated mucins / mucus “nests” can add to the efficacy of instilled fluids designed to clear mucus from the lungs. In one embodiment, agents that render mucus molecules shorter can be used (e.g., mucolytic agents). Examples of this strategy include thiol reducing agents that cleave the S-S bonds that produce the long mucin chains comprised of single mucin subunits (hence the name mucin polymer) and agents that proteolytically cleave the mucin protein backbone. A second strategy is to add surfactants with detergent like activity to the instilled solution.

[0197] Example 1: Clearance of Mucus in Humans

[0198]

[0145] An exemplary product for mucus clearance in humans can be provided as a sterile 500 mL isotonic solution of sodium gluconate containing 1 pg of Compound 1 or Compound 2 per 1 mL of solution. An effective volume of fluid can be instilled through a flexible bronchoscope (FIGs. 1A-1C) in the regions of the lung that are heavily burdened with mucus obstructions.

[0199] Example 2: Formulation Preparation

[0200]

[0146] Preparation of the instillation solutions used in Examples 3-6 are described here.

[0201] Example 2A: 200 pM Compound. 1 in 3.25% Sodium Gluconate (wt / v) and 20 mM Sodium Chloride Formulation

[0202]

[0147] Dissolve 3.25 grams (g) of sodium gluconate and 116 milligrams (mg) of sodium chloride in 100 mL of deionized water. Weigh 22 mg of Compound 1 HC1 (80% wt / wt, 17.6 mg net) and dissolve in the gluconate solution. Eilter the solution through a 0.2 p filter. Characterization of the formulation showed a final osmolality of 308 mOsm / Kg, a pH = 6.7, and a concentration of 200 pM by UV / Vis.

[0203] Example 2B: 20 pM Compound 1 in 3.25% Sodium Gluconate (wt / v) and 20 mM Sodium Chloride Formulation

[0204]

[0148] A 20 pM formulation of Compound 1 was prepared using an analogous procedure described for the 200 pM formulation (Example 2A).

[0205] Example 2C: 200 pM Compound 2 in 3.25% Sodium Gluconate (wt / v) and 20 mM Sodium Chloride Formulation

[0206]

[0149] A 200 pM formulation of Compound 2 was prepared using an analogous procedure described for the 200 pM formulation Compound 1 (Example 2A) by substituting in Compound 2 into the procedure. Example 2D: 100 pM Compound 3 in Aqueous Solution Formulation

[0207]

[0150] The solid hydrochloride salt of Compound 3 was brought to room temperature prior to use. A stock solution of approximately 160 mM was prepared by dissolving 163.8 mg of Compound 3 in 2 mL of water. High concentration stocks of Compound 3 are visibly out of solution. To solubilize Compound 3 stock, the pH was adjusted from 2.99 to 7.13 by addition of 13.75 pL ION NaOH. Subsequently, the pH was adjusted to the final pH of 7.08 by addition of 11 pL IN HC1. The active thiol concentration in the Compound 3 stock solution was determined using (5,5'-dithio-bis- [2-nitrobenzoic acid]) DTNB. Briefly, when combined with DTNB, thiol- containing reagents produce a quantifiable fluorescent readout at 412 nm proportional to the concentration of active drug in the reaction. The amount of active drug present in the compound stock solution was calculated using a derivation of Beer’s Law (A=sbC). Compound 3 stock solution had an active concentration of 143.7 mM and was then diluted to a final concentration of 100 mM in water for delivery. The final osmolality of the 100 mM Compound 3 dosing solution was 309 mOsm / kg.

[0208] Example 2E: N -Acetyl Cysteine and 20 mM NaCl Formulations

[0209]

[0151] N-Acetyl Cysteine (NAC) formulations of compounds were prepared using an analogous procedure described for the 100 pM formulation of Compound 3 (Example 2D) substituting in commercially available 20% NAC and with the addition of 20 mM NaCl.

[0210] Example 3: In Vivo Cmpd 1 and Cmpd 2 + / - Sodium Gluconate Instillation Study to Assess Pulmonary Fluid Retention in Wild- Type Mice

[0211]

[0152] To gain an understanding for how Compound 1 in isotonic Na Gluconate can impact retention of instilled fluid levels in vivo, cohorts of male and female wild-type mice, approximately 8 to 11 weeks in age, were administered a test solution dose (100 pL) by direct instillation to the left lobe of the mouse (FIG. 3A). Treatment groups (n=6) included PBS and 200 pM Cmpd 1 (176 pg / mL) in isotonic Na Gluconate (3.25%). Mice were euthanized either immediately after dosing (t = 0 hours) or 4 hours post-dose and the left lobe isolated. The left lobe was immediately weighed (wet weight) prior to being dried in an oven for 24 hour and a dry weight being obtained. The percentage of retention of was calculated by subtracting the dry weight from the wet weight, dividing by the wet weight and then multiplying by 100.

[0212]

[0153] Instilled fluid retention decreased over the 4-hour study duration for both PBS-treated and Cmpd 1 in isotonic Na Gluconate-treated wild-type mice (FIG. 3C). However, more fluid was retained in the left lobe of mice dosed with Cmpd 1 in isotonic Na Gluconate at 4 hours postdose than in PBS control mice (FIG. 3C). Cmpd 1 in isotonic Na Gluconate was associated with retention of fluid in the left lobe after instillation, for at least 4 hours after lung instillation. Data for Cmpd 2 is shown in FIG. 3B.

[0213] Example 4: p-ENaC Transgenic Mouse

[0214]

[0154] The dominant model for MOLD is the C57BL / 6N P-ENaC transgenic (P-ENaC-Tg) mouse (Mall et al., 2008; Livraghi-Butrico et al., 2018). The P-ENaC subunit overexpressing mouse exhibits the full spectrum of COPD pulmonary disease, including intrapulmonary accumulation of hyperconcentrated mucus, and mucus plugging (characterized predominately by the Muc5b mucin), airway epithelial remodeling, polymorphonuclear cell inflammation, and emphysema. This model has achieved widespread use for studies of disease pathophysiology and drug interventions.

[0215]

[0155] Cohorts of male and female 8-week-old P-ENaC mice were administered a test solution dose (100 pL) by direct instillation to the left lobe of the mouse. Treatment groups (n = 6-12) included naive (non-instrumented) control, PBS, isotonic vehicle, and a test article(s) instillation in single or multiple concentrations. Mice were euthanized 24 hours after dosing. The left lobe was immersion fixed, embedded, and stained with hematoxylin and eosin (H&E) and Alcian Blue Periodic Acid Schiff (AB-PAS) to allow histopathological analysis and morphometric quantification of AB-PAS positive volume density (i.e., mucus glycoconjugate content) using Visiopharm image analysis software. Images of the AB-PAS-stained slides were acquired via an Olympus VS 120 Slide Scanner at 20x and then imported into Visiopharm for quantification of mucus volume density. An optimized threshold was selected to accurately classify both intraepithelial and intraluminal AB-PAS-positive material. Length of basal lamina and area of positive AB-PAS stain for the epithelium and lumen were then obtained from the main axial airway in the proximal, intermediate, and distal section for each sample Volume density (nL / mm2) was calculated as: (area of AB-PAS+ stain mm2 / ((perimeter mm) x 4 / K)).

[0216] Compound 1 (20 M; 200 pM)

[0217]

[0156] The intrapulmonary mucus burden was not decreased by PBS alone compared with sham (FIGs. 4A-4B). Mucus burden exhibited trends toward clearance of approximately 50% (nonsignificant) with 3.25% Na Gluconate ± 20 pM Cmpd 1 vs. naive control P-ENaC mice (FIGs. 4A-4B). Notably, the mucus burden was eliminated (significant) with 200 pM Cmpd 1 in 3.25% Na Gluconate. Given the mechanism of action of Cmpd 1, and without being bound by any particular theory, the results suggest this Cmpd 1 concentration in Na Gluconate solution was effective in slowing sodium, and consequently water, absorption from the lung, providing sufficient time for the instilled fluid to rehydrate abnormal mucus, resulting in efficient mucus clearance from the left lobe of P-ENaC mice. Compound 2 (200 pM)

[0218]

[0157] The intrapulmonary mucus burden was not significantly decreased by PBS alone compared with sham control (FIGs. 5A-5B). Mucus burden exhibited trends toward clearance of approximately 50% with PBS + 200 pM Cmpd 2 vs. naive control P-ENaC mice (2). Notably, 3.25% Na Gluconate decreased the mucus burden more than PBS alone and the addition 200 pM Cmpd 2 in 3.25% Na Gluconate virtually eliminated the mucus burden (FIGs. 5A-5B).

[0219] Compound 3 (lOOmM)

[0220]

[0158] The intrapulmonary mucus burden was slightly decreased by PBS alone compared with sham (FIG. 7). Mucus burden decreases with statistical significance (p<0.05 by ANOVA vs No Rx) with treatment of 100 mM Compound 3 in aqueous solution (FIG. 7).

[0221] Example 5: House Dust Mite Challenged P-ENaC Transgenic Mouse

[0222]

[0159] To develop an asthma-COPD overlap syndrome-like mouse model (pulmonary mucus plugs dominated by the asthma associated Muc5ac mucin), C57BL / 6N P-ENaC transgenic mice were sensitized with 2 doses of HDM administered by intraperitoneal injection (dO and d7), then challenged with 3 doses of HDM administered by oropharyngeal instillation (dl4, dl5 and dl6). This approach produced a P-ENaC mouse model with asthma Muc5ac mucin goblet cell metaplasia, consistent with intrapulmonary plugs populated by Muc5ac and not Muc5b (FIGs. 6A-6C).

[0223]

[0160] Cohorts of male and female 8-week-old HDM-P-ENaC mice were administered a single dose of solution (100 pL) by direct instillation to the left lobe of the mouse. Treatment groups (n= 6) included a naive control, isotonic Na Gluconate (3.25%), and 200 pM (176 pg / mL) Cmpd 1 in the presence of 3.25% Na Gluconate. Mice were euthanized approximately 24 hours after dosing. The left lobe was immersion fixed, embedded, and stained with H&E and AB-PAS to allow histopathological analysis and morphometric quantification of AB-PAS positive volume density (i.e., mucus glycoconjugates content) using Visiopharm image analysis software. Images of the AB-PAS-stained slides were acquired via an Olympus VS 120 Slide Scanner at 20x and then imported into Visiopharm for quantification of mucus volume density. An optimized threshold was selected to accurately classify both intraepithelial and intraluminal AB-PAS-positive material. Length of basal lamina and area of positive AB-PAS stain for the epithelium and lumen were then obtained from the main axial airway in the proximal, intermediate, and distal section for each sample Volume density (nl / mm2) was calculated as: (area of AB-PAS+ stain mm2 / ((perimeter mm) x 4 / K)). Compound 1 (200 pM)

[0224]

[0161] In the HDM challenged P-ENaC mouse model, the mucus burden decreased with 3.25% Na Gluconate but achieved a significant approximately 50% clearance in the presence of 200 pM Cmpd 1 and 3.25% Na Gluconate compared with the naive control (FIG. 6D).

[0225] Compound 1, and Compound 1 and NAC

[0226]

[0162] In this study, the intrapulmonary mucus burden showed a slight decrease with PBS when compared with no treatment (no Rx; FIG. 6D) but was insignificant. The mucus burden then exhibited a further decrease associated with clearance with administration of 3.25% Na Gluconate + 200 pM Cmpd 1 and with 2.1% Na Gluconate (was decreased upon 50 mM NAC addition to match the tonicity of the 3.25% Na Gluconate) + 200 pM Cmpd 1 + 50 mM NAC vs. no Rx control in HDM challenged P-ENaC mice (FIG. 6D). Both the Cmpd 1 and Cmpd 1 plus NAC treatment groups showed a statistically significant decrease (p<0.05) in mucus burden vs the no Rx treatment group, with addition of NAC providing a numerical improvement over Cmpd 1 and Na gluconate alone.

[0227] Example 6: Effect of Compound 3 on mucus burden of beta ENaC mouse

[0228]

[0163] Compound 3, in an isotonic CP containing vehicle, was effective in reducing intrapulmonary mucus burden in beta ENaC mice as compared to sham or PBS along treatments (FIG. 7).

[0229] EQUIVALENTS AND SCOPE

[0230]

[0164] The word “about” when immediately preceding a numerical value means a range, e.g., of plus or minus 10% of that value. For example, “about 10” means 9 to 11, unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For instance in a list of numerical values such as “about 10, about 11, about 12, ...”, “about 11” means a range extending to less than half the interval(s) between the preceding and subsequent values, i.e., more than 10.5 to less than 11.5. Additionally, when a value is provided herein is immediately preceded by the word “about,” the exact value is also provided herein as an embodiment of the disclosure.

[0231]

[0165] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0232]

[0166] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0233]

[0167] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0234]

[0168] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising sodium gluconate.

2. The method of claim 1, wherein the liquid pharmaceutical composition is an aqueous solution.

3. The method of claim 1 or 2, wherein the liquid pharmaceutical composition is an isotonic solution.

4. The method of any one of claims 1-3, wherein the concentration of sodium gluconate is from about 2.5% to about 5% (w / v).

5. The method of any one of claims 1-3, wherein the concentration of sodium gluconate is about 3.25% (w / v).

6. A method of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an osmolyte, wherein the osmolyte is not sodium chloride.

7. The method of claim 6, wherein the liquid pharmaceutical composition is an aqueous solution.

8. The method of claim 6 or 7, wherein the osmolyte has a longer retention time in the lungs than sodium chloride.

9. The method of any one of claims 6-8, wherein the osmolyte is a sodium salt.

10. The method of any one of claims 6-9, wherein the osmolyte is a gluconate salt.

11. The method of any one of claims 6-10, wherein the liquid pharmaceutical composition is isotonic.

12. The method of any one of claims 6-11, wherein the concentration of the osmolyte is from about 2.5% to about 5% (w / v).

13. The method of any one of claims 6-11, wherein the concentration of the osmolyte is about 3.25% (w / v).

14. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises an epithelial sodium channel (ENaC) inhibitor.

15. A method of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising an epithelial sodium channel (ENaC) inhibitor.

16. The method of claim 15, wherein the liquid pharmaceutical composition further comprises an osmolyte.

17. The method of claim 15, wherein the osmolyte is sodium chloride or sodium gluconate.

18. The method of any one of claims 15-17, wherein the liquid pharmaceutical composition is an aqueous solution.

19. The method of any one of claims 15-18, wherein the liquid pharmaceutical composition is an isotonic solution.

20. The method of any one of claims 14-19, wherein the ENaC inhibitor is:(Compound 1),or a pharmaceutically acceptable salt thereof.

21. The method of claim 20, wherein the ENaC inhibitor is the hydrochloride (HC1) salt of Compound 1.

22. The method of any one of claims 14-19, wherein the ENaC inhibitor is:(Compound 2), or a pharmaceutically acceptable salt thereof.

23. The method of any one of claims 14-22, wherein the concentration of the ENaC inhibitor is from about 0.3 pg / mL to about 176 pg / mL.

24. The method of any one of claims 14-22, wherein the concentration of the ENaC inhibitor is from about 10 pM to about 300 pM.

25. The method of any one of claims 14-22, wherein the concentration of the ENaC inhibitor is from about 10 pM to about 30 pM.

26. The method of any one of claims 14-22, wherein the concentration of the ENaC inhibitor is about 20 pM.

27. The method of any one of claims 14-22, wherein the concentration of the ENaC inhibitor is from about 150 pM to about 250 pM.

28. The method of any one of claims 14-22, wherein the concentration of the ENaC inhibitor is about 200 pM.

29. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition is an aqueous solution comprising sodium gluconate and an ENaC inhibitor.

30. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition is an aqueous solution comprising sodium gluconate and Compound 1 or a pharmaceutically acceptable salt thereof.

31. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises a chloride salt.

32. The method of claim 31, wherein the chloride salt is sodium chloride.

33. The method of claim 31 or 32, wherein the concentration of the chloride salt is from about 10 mM to about 30 mM.

34. The method of any one of claims 31-33, wherein the concentration of the chloride salt is about 20 mM.

35. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises a mucolytic agent.

36. The method of claim 35, wherein the mucolytic agent is N-acetyl cysteine (NAC).

37. A method of clearing mucus from the lungs of a subject in need thereof, the method comprising instilling into the lungs of the subject an effective amount of a liquid pharmaceutical composition comprising a mucolytic agent.

38. The method of claim 37, wherein the liquid pharmaceutical composition is an aqueous solution.

39. The method of claim 37 or 38, wherein the liquid pharmaceutical composition is an isotonic solution.

40. The method of any one of claims 37-39, wherein the mucolytic agent is:(Compound 3), or a pharmaceutically acceptable salt thereof.

41. The method of claim 40, wherein the mucolytic agent is the hydrochloride (HC1) salt of Compound 3.

42. The method of any one of claims 37-41, wherein the mucolytic agent is NAC.

43. The method of any one of claims 37-42, wherein the concentration of the mucolytic agent is from about 10 mM to about 200 mM.

44. The method of any one of claims 37-43, wherein the concentration of the mucolytic agent is about 100 mM.

45. The method of any one of claims 37-44, wherein the liquid pharmaceutical composition further comprises a chloride salt.

46. The method of claim 45, wherein the chloride salt is sodium chloride.

47. The method of claim 45 or 46, wherein the concentration of the chloride salt is from about 10 mM to about 30 mM.

48. The method of any one of claims 45-47, wherein the concentration of the chloride salt is about 20 mM.

49. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises a surfactant.

50. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises an antibiotic.

51. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises an anti-inflammatory agent.

52. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition further comprises a protease that effects mucins, optionally bromelain or mucin specific protease (SteC).

53. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition has a pH of from about 6.4 to about 8.4.

54. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition has a pH of from about 7.2 to about 7.6.

55. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition has a pH of about 7.4.

56. The method of any one of the preceding claims, wherein at least a portion of the liquid pharmaceutical composition is instilled into the small and / or large airways of the lungs.

57. The method of any one of the preceding claims, wherein at least a portion of the liquid pharmaceutical composition is instilled into the small airways of the lungs.

58. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition is instilled in sufficient volume to flood the small airways of the lungs.

59. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition is instilled into the lungs of the subject via bronchoscope.

60. The method of any one of the preceding claims, wherein the liquid pharmaceutical composition is instilled into the lungs of the subject via endotracheal tube or tracheostomy.

61. The method of any one of the preceding claims, wherein the liquid pharmaceuticalcomposition is instilled into the lungs of the subject via a double lumen endotracheal tube.

62. The method of any one of the preceding claims, wherein a volume of about 1 mL to about 20 L of the liquid pharmaceutical composition is instilled into the lungs of the subject.

63. The method of any one of the preceding claims, wherein a volume of about 10 mL to about 10 L of the liquid pharmaceutical composition is instilled into the lungs of the subject.

64. The method of any one of the preceding claims, wherein a volume of about 500 mL of the liquid pharmaceutical composition is instilled into the lungs of the subject.

65. The method of any one of the preceding claims, wherein the volume of the liquid pharmaceutical composition is administered as a single bolus dose.

66. The method of any one of the preceding claims, wherein the mucus is one or more mucus plugs.

67. The method of any one of the preceding claims, wherein the subject has a mucus obstructive lung condition.

68. The method of any one of the preceding claims, wherein the subject has asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), primary ciliary dyskinesia (PCD), non-CF bronchiectasis, Job’s syndrome, Woakes syndrome, or immunodeficiency syndrome.

69. The method of any one of the preceding claims, where the subject is intubated.

70. The method of any one of the preceding claims, where the subject is treated in an intensive care unit (ICU).

71. The method of any one of the preceding claims further comprising subjecting the subject to a vibrating vest, Aerobika device, or positive pressure device.

72. The method of any one of the preceding claims further comprising subjecting the subject to positional modification.

73. The method of any one of the preceding claims, wherein the subject is a human.

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