WO2017151725A1

WO2017151725A1 - Formulations of an s-nitrosoglutathione reductase inhibitor

Info

Publication number: WO2017151725A1
Application number: PCT/US2017/020129
Authority: WO
Inventors: Chris DELANY; Rajendrakumar Dasharath KALE; Sherif GABRIEL; Peter Bove
Original assignee: Nivalis Therapeutics Inc
Current assignee: Nivalis Therapeutics Inc
Priority date: 2016-03-03
Filing date: 2017-03-01
Publication date: 2017-09-08
Anticipated expiration: 2018-09-03

Abstract

The present invention provides direct blend formulations comprising 3-chloro-4-(6- hydroxyquinolin-2-yl)benzoic acid. Additionally, the present invention is directed to a method of treating or lessening the severity of cystic fibrosis in a patient, comprising the step of stabilizing the CFTR protein both in the cell and at the cell membrane by administering to said patient an effective amount of an inhibitor of S-nitrosoglutathione reductase (GSNOR).

Description

FORMULATIONS OF AN S-NITROSOGLUTATHIONE REDUCTASE INHIBITOR

RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Serial No. 62/303,218, filed March 3, 2016, entitled "Formulations of an S-Nitrosoglutathione Reductase Inhibitor" and United States Provisional Application Serial No. 62/314,189, filed March 28, 2016, entitled "Methods for the Treatment of Cystic Fibrosis", each of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to pharmaceutical compositions comprising 3- chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound 1). The invention also provides pharmaceutically acceptable compositions comprising solid forms of Compound 1 and methods of using the compositions in the treatment of various disorders. The present invention is also directed to a method of treating or lessening the severity of cystic fibrosis in a patient, comprising the step of stabilizing the CFTR protein both in the cell and at the cell membrane by administering to said patient an effective amount of an inhibitor of S- nitrosoglutathione reductase (GSNOR).

BACKGROUND

[0003] Nitric oxide (NO) is one of the few gaseous signaling molecules known in biological systems, and plays an important role in controlling various biological events. For example, the endothelium uses NO to signal surrounding smooth muscle in the walls of arterioles to relax, resulting in vasodilation and increased blood flow to hypoxic tissues. NO is also involved in regulating smooth muscle proliferation, platelet function, and

neurotransmission, and plays a role in host defense. Although NO is highly reactive and has a lifetime of a few seconds, it can both diffuse freely across membranes and bind to many molecular targets. These attributes make NO an ideal signaling molecule capable of controlling biological events between adjacent cells and within cells.

[0004] NO is a free radical gas, which makes it reactive and unstable, thus NO is short lived in vivo, having a half-life of 3-5 seconds under physiologic conditions. In the presence of oxygen, NO can combine with thiols to generate a biologically important class of stable NO adducts called S-nitrosothiols (SNO's). This stable pool of NO has been postulated to act as a source of bioactive NO and as such appears to be critically important in health and disease, given the centrality of NO in cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA, 89:7674-7677 (1992)). Protein SNO's play broad roles in the function of cardiovascular, respiratory, metabolic, gastrointestinal, immune, and central nervous system (Foster et al., Trends in Molecular Medicine, 9 (4): 160- 168, (2003)). One of the most studied SNO's in biological systems is S-nitrosoglutathione (GSNO) (Gaston et al., Proc. Natl. Acad. Sci. USA 90: 10957-10961 (1993)), an emerging key regulator in NO signaling since it is an efficient trans-nitrosating agent and appears to maintain an equilibrium with other S- nitrosated proteins (Liu et al., Nature, 410:490-494 (2001)) within cells. Given this pivotal position in the NO-SNO continuum, GSNO provides a therapeutically promising target to consider when NO modulation is pharmacologically warranted.

[0005] S-nitrosoglutathione (GSNO) is a key regulator of nitric oxide (NO) homeostasis and cellular S-nitrosothiol (SNO) levels, and studies have focused on examining endogenous production of GSNO and SNO proteins, which occurs downstream from the production of the NO radical by the nitric oxide synthetase (NOS) enzymes. More recently there has been an increasing understanding of enzymatic catabolism of GSNO which has an important role in governing available concentrations of GSNO and consequently available NO and SNO's.

[0006] Central to this understanding of GSNO catabolism, researchers have recently identified a highly conserved S-nitrosoglutathione reductase (GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al., (2001)). GSNOR is also known as glutathione- dependent formaldehyde dehydrogenase (GSH-FDH), alcohol dehydrogenase 3 (ADH-3) (Uotila and Koivusalo, Coenzymes and Cofactors., D. Dolphin, ed. pp. 517-551 (New York, John Wiley & Sons, (1989)), and alcohol dehydrogenase 5 (ADH-5). Importantly GSNOR shows greater activity toward GSNO than other substrates (Jensen et al., (1998); Liu et al., (2001)) and appears to mediate important protein and peptide denitrosating activity in bacteria, plants, and animals. GSNOR appears to be the major GSNO-metabolizing enzyme in eukaryotes (Liu et al., (2001)). Thus, GSNO can accumulate in biological compartments where GSNOR activity is low or absent (e.g., airway lining fluid) (Gaston et al., (1993)).

[0007] GSNO specifically has been implicated in physiologic processes ranging from the drive to breathe (Lipton et al., Nature, 413: 171-174 (2001)) to regulation of the cystic fibrosis transmembrane regulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001)), to regulation of vascular tone, thrombosis, and platelet function (de Belder et al., Cardiovasc Res.; 28(5):691-4 (1994)), Z. Kaposzta, et al., Circulation; 106(24): 3057 - 3062, (2002)) as well as host defense (de Jesus-Berrios et al., Curr. Biol., 13: 1963-1968 (2003)). Other studies have found that GSNOR protects yeast cells against nitrosative stress both in vitro (Liu et al., (2001)) and in vivo (de Jesus-Berrios et al., (2003)).

[0008] Collectively, data suggest GSNO as a primary physiological ligand for the enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizes GSNO and consequently reduces available SNO's and NO in biological systems (Liu et al., (2001)), (Liu et al., Cell, 116(4), 617-628 (2004)), and (Que et al., Science, 308, (5728): 1618-1621 (2005)). As such, this enzyme plays a central role in regulating local and systemic bioactive NO. Since

perturbations in NO bioavailability has been linked to the pathogenesis of numerous disease states, including cystic fibrosis, hypertension, atherosclerosis, thrombosis, asthma, gastrointestinal disorders, inflammation, and cancer, agents that regulate GSNOR activity are candidate therapeutic agents for treating diseases associated with NO imbalance.

[0009] Nitric oxide (NO), S-nitrosoglutathione (GSNO), and S-nitrosoglutathione reductase (GSNOR) regulate normal lung physiology and contribute to lung pathophysiology. Under normal conditions, NO and GSNO maintain normal lung physiology and function via their modulatory effects on the cystic fibrosis transmembrane regulator (CFTR), antiinflammatory and bronchodilatory actions. Lowered levels of these mediators in pulmonary diseases such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD) may occur via up-regulation of GSNOR enzyme activity. These lowered levels of NO and GSNO, and thus lowered anti-inflammatory capabilities, are key events that contribute to pulmonary diseases and which can potentially be reversed via GSNOR inhibition.

[0010] S-nitrosoglutathione (GSNO) has been shown to promote repair and/or

regeneration of mammalian organs, such as the heart (Lima et al., 2010), blood vessels (Lima et al., 2010) skin (Georgii et al., 2010), eye or ocular structures (Haq et al., 2007) and liver (Prince et al., 2010). S-nitrosoglutathione reductase (GSNOR) is the major catabolic enzyme of GSNO. Inhibition of GSNOR is thought to increase endogenous GSNO.

[0011] Inflammatory bowel diseases (IBD's), including Crohn's and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal (GI) tract, in which NO, GSNO, and GSNOR can exert influences. Under normal conditions, NO and GSNO function to maintain normal intestinal physiology via anti-inflammatory actions and maintenance of the intestinal epithelial cell barrier. In IBD, reduced levels of GSNO and NO are evident and likely occur via up-regulation of GSNOR activity. The lowered levels of these mediators contribute to the pathophysiology of IBD via disruption of the epithelial barrier via dysregulation of proteins involved in maintaining epithelial tight junctions. This epithelial barrier dysfunction, with the ensuing entry of micro-organisms from the lumen, and the overall lowered anti-inflammatory capabilities in the presence of lowered NO and GSNO, are key events in IBD progression that can be potentially influenced by targeting GSNOR.

[0012] Cystic fibrosis (CF) is one of the most common lethal genetic diseases in

Caucasians. Approximately one in 3,500 children in the US is born with CF each year. It is a disease that affects all racial and ethnic groups, but is more common among Caucasians. An estimated 30,000 American adults and children have CF, and the median predicted age of survival is approximately 41 years (CFF Registry Report 2012, Cystic Fibrosis Foundation, Bethesda, MD). CF is an autosomal recessive hereditary disease caused by a mutation in the gene for the cystic fibrosis transmembrane regulator (CFTR) protein. The CFTR protein is located on the apical membrane and is responsible for chloride transport across epithelial cells on mucosal surfaces. CF is diagnosed by the level of chloride in sweat because patients with CF have elevated sweat chloride due to the primary defect in CFTR. More than 1,000 disease-associated mutations have been discovered in the CFTR gene with the most common mutation being a deletion of the amino acid phenylalanine at position 508 (F508del). The F508del mutation is present in approximately 86% of CF patients. Approximately 47% of CF patients are homozygous and have two copies of the F508del mutation, and

approximately 39% are heterozygous and have one copy. In both homozygous and heterozygous patients, lung disease is the most critical manifestation, characterized by airway obstruction, infection and inflammation that allow bacteria to grow unfettered and impair the lung's immune system. More than 90% of all CF patients die of respiratory failure. In the pancreas, damage caused by CF leads to diabetes, while the build-up of mucus prevents the release of digestive enzymes leading to poor nutrient absorption.

[0013] In patients with CF, decreased CFTR activity is due in part to reduced levels of S- nitrosoglutathione (GSNO), which is regulated by the enzyme S-nitrosoglutathione reductase (GSNOR). GSNO is the human body's most abundant low molecular weight S-nitrosothiol, or SNO. While SNOs are normally present in the human airway, concentrations tend to be reduced in CF patients (Grasemann 1998). In the CF patient's lung, the depleted GSNO levels adversely affect CFTR activity.

[0014] GSNO has been identified as a positive modulator of CFTR. GSNOR is the primary catabolizing enzyme of GSNO. Inhibition of GSNOR may improve F508del-CFTR function via nitrosation of chaperone proteins, prevention of CFTR proteosomal degradation, promotion of CFTR maturation, and maintenance of epithelial tight junctions. Currently there is no curative treatment for CF; therefore, new therapies are needed for the disease.

[0015] NO, GSNO, and GSNOR can also exert influences on disorders of the

gastrointestinal (GI) tract, such as inflammatory bowel disease (IBD) (including Crohn's and ulcerative colitis) and possibly cystic fibrosis gastrointestinal disease. Under normal conditions, NO and GSNO function to maintain normal intestinal physiology via antiinflammatory actions and maintenance of the intestinal epithelial cell barrier.

[0016] Acetaminophen overdoses are the leading cause of acute liver failure (ALF) in the United States, Great Britain and most of Europe. More than 100,000 calls to the U.S. Poison Control Centers, 56,000 emergency room visits, 2,600 hospitalizations, nearly 500 deaths are attributed to acetaminophen in this country annually. Approximately, 60% recover without needing a liver transplant, 9% are transplanted and 30% of patients succumb to the illness. The acetaminophen-related death rate exceeds by at least three-fold the number of deaths due to all other idiosyncratic drug reactions combined (Lee, Hepatol Res 2008; 38 (Suppl. 1):S3- S8).

[0017] Liver transplantation has become the primary treatment for patients with fulminant hepatic failure and end- stage chronic liver disease, as well as certain metabolic liver diseases. Thus, the demand for transplantation now greatly exceeds the availability of donor organs. It has been estimated that more than 18,000 patients are currently registered with the United Network for Organ Sharing (UNOS) and that an additional 9,000 patients are added to the liver transplant waiting list each year, yet less than 5,000 cadaveric donors are available for transplantation.

[0018] Currently, there is a great need in the art for treatments for medical conditions where there is a need for increased NO bioactivity. In addition, there is a significant need for novel compounds, compositions, and methods for preventing, ameliorating, or reversing NO- associated disorders.

[0019] The compound 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid is disclosed in International PCT Publication WO2012/048181 ("the ' 181 application") as an inhibitor of GSNOR and thus as a useful treatment for NO related diseases such as cystic fibrosis, asthma, COPD, IBD, etc. Form A of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid, which is a substantially crystalline hemi-hydrate form known as Compound 1 Form A, is disclosed in U.S. Provisional Application No. 62/216,765, entitled "SOLID FORMS OF AN S-NITROSOGLUTATHIONE REDUCTASE INHIBITOR", filed on September 10, 2015 by Jian Qiu, and PCT application PCT/US2016/050974 filed September 9, 2016. Anhydrous form, Compound 1 Form B is disclosed in the same applications, US 62/216,765, filed on September 10, 2015 by Jian Qiu, and PCT/US2016/050974 filed September 9, 2016. All applications are incorporated in their entirety by reference herein. A need remains, however, for pharmaceutical compositions comprising Compound 1 that are readily prepared and that are suitable for use as therapeutics.

SUMMARY OF THE INVENTION

[0020] The invention includes pharmaceutical compositions and pharmaceutical preparations comprising 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound 1) which has the structure below:

[0021] Compound 1 is active in a variety of assays and therapeutic models demonstrating selective and reversible inhibition of the s-nitrosoglutathione reductase enzyme. Notably, Compound 1 demonstrates efficacy in asthma, COPD, cystic fibrosis, and IBD models (described in the ' 181 application and international application PCT/US2015/054728). Accordingly, Compound 1 is useful for treating one or more disorders associated with activity of GSNOR.

[0022] In one aspect, the invention provides a pharmaceutical composition comprising:

[0023] a. Compound 1

[0024] b. a disintegrant

[0025] c. a filler

[0026] d. a lubricant

[0027] e. a binder.

[0028] In one embodiment, the composition includes two or more types of fillers.

[0029] In one aspect, the invention provides a pharmaceutical composition comprising:

[0030] a. Compound 1

[0031] b. a disintegrant

[0032] c. a filler

[0033] d. a lubricant

[0034] e. a glidant. [0035] In one embodiment, the composition includes two or more types of fillers. In another embodiment, the composition includes two or more types of glidants.

[0036] In other embodiments, Compound 1 is in substantially one of its crystalline solid forms, as described in U.S. Provisional Application No. 62/216,765, entitled "SOLID FORMS OF AN S -NITROS OGLUTATHIONE REDUCTASE INHIBITOR", filed on September 10, 2015 by Jian Qiu, and PCT application PCT/US2016/050974 filed September 9, 2016. In one embodiment, Compound 1 is in substantially crystalline hemi-hydrate Form A (Compound 1 Form A). In one embodiment, Compound 1 is in substantially crystalline anhydrous Form B (Compound 1 Form B). It is understood that the term "Compound 1," as used throughout, includes, amongst other forms, including non-crystalline forms, the following solid state forms: Compound 1 Form A and Compound 1 Form B. When amounts of Compound 1 are disclosed, it is intended to mean amount of active Compound 1. For example, a formulation containing Compound 1, if in anhydrous form B, is about 100% active. Additionally, for example, a formulation containing Compound 1, if in semi-hydrate form A, is about 97% active, and about 3% water (i.e. if 100 mg of Compound 1 semi- hydrate Form A is weighed out, once the water is taken into account, it contains about 97 mg of active Compound 1.)

[0037] In some embodiments, the dosage form of the pharmaceutical composition comprises about 95 mg to about 100 mg of Compound 1.

[0038] In general, the pharmaceutical compositions described herein, are useful for treating or lessening the severity of a variety of diseases or disorders as described in detail herein. In one embodiment the diseases or disorders are selected from pulmonary disorders and inflammatory disorders. In one embodiment the diseases or disorders are selected from cystic fibrosis, asthma, chronic obstructive pulmonary disorder, and inflammatory bowel disease.

[0039] In one aspect, the invention provides a pharmaceutical composition comprising the following components: Compound 1, Lactose monohydrate (Fast- Flow), HPC

(hydroxypropyl cellulose), and Magnesium Stearate. In another aspect of the invention, the composition additionally contains Microcrystalline Cellulose, and / or Dibasic calcium phosphate, and / or Croscarmellose sodium.

[0040] In another aspect, the invention provides a pharmaceutical composition comprising the following components: Compound 1, Lactose monohydrate (Fast- Flow), Dibasic calcium phosphate (anhydrous), Pregelatinized starch, Colloidal silicon dioxide, and Magnesium Stearate. [0041] In another aspect, the invention provides a pharmaceutical composition in the form of a capsule (containing about 95 - 100 mg of Compound 1) that comprises Compound 1 and one or more pharmaceutically acceptable excipients, for example, a filler, a

disintegrant, a binder, a glidant, and a lubricant and any combination thereof, where the capsule has a dissolution of at least about 60% in about 30 minutes. In another embodiment, the dissolution rate is at least about 75% in about 30 minutes. In another embodiment, the dissolution rate is at least about 80% in about 30 minutes.

[0042] The pharmaceutical compositions of the invention can be processed into a tablet form, capsule form, pouch form, lozenge form, or other solid form that is suited for oral administration. Thus in some embodiments, the pharmaceutical compositions are in capsule form.

[0043] The present invention also relates to methods for treating patients with cystic fibrosis, an autosomal recessive hereditary disease caused by a mutation in the gene CFTR. Mutations in the CFTR protein result in loss of CFTR activity at the surface of epithelial cells leading to abnormal ion transport, dehydration of the airway surface, mucosal obstruction of exocrine glands, and an altered inflammatory response, especially in the lungs. Multiple organ systems are involved, most notably the respiratory and gastrointestinal (GI) systems. Pulmonary problems are characterized by airway obstruction, impaired mucociliary clearance, inflammation, and infection. CF is diagnosed by the levels of chloride in sweat and people with CF have elevated sweat chloride levels. Interventions that modulate CFTR activity use sweat chloride as a clinical biomarker of effect. Reduction in sweat chloride levels indicates direct modulation of CFTR. Therefore, the ultimate goal of CFTR modulator therapy is to maximize and maintain CFTR function, thereby restoring chloride transport.

[0044] Preservation of S-nitrosoglutathione (GSNO), through inhibition of its primary catabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), may provide a novel therapeutic strategy in CF. GSNO, and GSNOR regulate normal lung physiology and contribute to lung pathophysiology. Under normal conditions, GSNO maintains normal lung physiology and function via its anti-inflammatory and bronchodilatory effects. Lowered levels of the mediator GSNOR in pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis may occur via up-regulation of GSNOR enzyme activity. In CF, lowered levels of GSNO adversely affect CFTR function and inflammation, which are key factors in the pathophysiology of CF and which can potentially be reversed via GSNOR inhibition. [0045] The present invention provides methods for treating or lessening the severity of CF by stabilizing the CFTR protein at the cell membrane for a prolonged period of time, comprising the step of administering to a patient in need an effective amount of an S- nitrosoglutathione reductase ("GSNOR") inhibitor. In some embodiments, the GSNOR inhibitor is a compound of Formula 1 as shown in the detailed description. In some embodiments, the GSNOR inhibitor is Compound 1, or 3-chloro-4-(6-hydroxyquinolin-2- yl)benzoic acid. In some embodiments, the GSNOR inhibitor is administered concurrently with, prior to, or subsequent to, one or more secondary active agents. In one embodiment, the secondary active agent(s) of the invention are selected from CFTR correctors and CFTR potentiators. In one embodiment, the secondary active agent is the CFTR corrector VX-809. In another embodiment, the secondary active agent is the CFTR corrector VX-661. In another embodiment, the secondary active agent is the CFTR potentiator VX-770. In another embodiment, the GSNOR inhibitor is administered with VX-809 and VX-770. In another embodiment, the GSNOR inhibitor is administered with VX-661 and VX-770.

[0046] In another embodiment, the GSNOR inhibitor 3-chloro-4-(6-hydroxyquinolin-2- yl)benzoic acid is administered with VX-809 and VX-770. In another embodiment, the GSNOR inhibitor 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid is administered with VX- 661 and VX-770.

[0047] The invention encompasses pharmaceutically acceptable salts, stereoisomers, prodrugs, metabolites, and N-oxides of the described compounds.

[0048] Also encompassed by the invention are methods that include administering a therapeutically effective amount of a GSNOR inhibitor for the treatment of cystic fibrosis administered with one or more of the secondary active agents VX-809, VX-661 and VX-770, and additionally can be administered with one or more palliative agents.

[0049] The compositions of the present invention can be prepared in any suitable pharmaceutically acceptable dosage form.

[0050] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publicly available publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

[0051] Both the foregoing summary and the following detailed description are exemplary and explanatory and are intended to provide further details of the compositions and methods as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0052] Figure: 1: CFBE41o- cells stably expressing HRP-tagged F508del-CFTR were treated with a CFTR corrector (VX-809 or VX-661, 3 μΜ, 24 h) alone or in the presence of a GSNORi (100 μΜ, 24 h). Relative fluorescent units (RFU) was plotted for each treatment.

[0053] Figure 2A and 2B: CFBE41o- cells stably expressing HRP-tagged F508del-CFTR were treated with a CFTR corrector (VX-809 or VX-661, 3 μΜ, 24 h) alone or in the presence of a GSNORi (100 μΜ, 24 h): Figures 2A and 2B show the % of CFTR remaining at the plasma membrane for VX-809 treated (Figure 2A), or VX-661 treated cells (Figure 2B)

[0054] Figures 3: CFBE41o- cells were treated with a CFTR corrector (VX-809 or VX- 661) + potentiator (VX-770) in addition to the GSNOR inhibitor 3-chloro-4-(6- hydroxyquinolin-2-yl)benzoic acid. Relative fluorescent units (RFU) was plotted for each treatment.

[0055] Figure 4A and 4B: CFBE41o- cells were treated with a CFTR corrector (VX-809 or VX-661) + potentiator (VX-770) in addition to the GSNOR inhibitor 3-chloro-4-(6- hydroxyquinolin-2-yl)benzoic acid. Figures 4A and 4B show the % of CFTR remaining at the plasma membrane for the VX-809 + VX-770 treatment groups (Figure 4A), and for the VX-661 + VX-770 treatment groups (Figure 4B).

DETAILED DESCRIPTION

[0056] In accord with the present invention, GSNOR has been shown to function in vivo and in vitro to metabolize GSNO. Based on this, it follows that inhibition of this enzyme potentiates bioactivity of GSNO in diseases in which activity of this enzyme is increased and GSNO levels are depleted, such as CF. Dysregulation of this enzyme and depleted GSNO levels in CF contribute to the instability of CFTR, its degradation, and lack of function.

[0057] CF is a lethal genetic disease affecting 70,000 people worldwide, including approximately 30,000 in the U.S. (Ramsey et al. 2011, Cystic Fibrosis Foundation Patient Registry Report 2013). Due to advancements in the management of CF and the development of novel drugs that target the defective CFTR protein, the predicted median age of death has risen to approximately 41 years of age, however, the actual median age at death still remains at approximately 27 years (Cystic Fibrosis Foundation Patient Registry Report 2013).

[0058] CF is an autosomal recessive hereditary disease caused by a mutation in the gene for the cystic fibrosis transmembrane regulator (CFTR) protein. Mutations in the CFTR protein result in loss of CFTR activity at the surface of epithelial cells leading to abnormal ion transport, dehydration of secretions, mucosal obstruction of exocrine glands, and an altered inflammatory response, especially in the lungs. Instability of the mutant CFTR protein contributes to its lack of function and is increasingly recognized as a novel target for new drug therapies targeted toward CF (Arora et al., Biochemistry. 2014; 53(25): 4169-79, Clancy, Sci Transl Med. 2014. 6(246): 246fs27). CFTR aids the regulation of epithelial salt and water transport in multiple organs, including the lung, pancreas, liver, and intestinal tract. Clinical manifestations of CF include abnormal sweat electrolytes, chronic and progressive respiratory disease, exocrine pancreatic dysfunction, and infertility; however, it is lung disease that is the primary cause of morbidity and mortality. In the lung, the loss of CFTR mediated CI^" secretion is believed to cause airway surface dehydration due to both a decrease in CFTR-mediated CI^" and fluid secretion and a secondary increase in epithelial Na⁺ channel (ENaC)-mediated Na⁺ and fluid absorption. This imbalance results in dehydration of the airway surface, and likely contributes to the deleterious cascade of mucus accumulation, infection, inflammation, and destruction that characterizes CF lung disease. The

accumulation of mucus leads to plugging in the passageways in the lung and other organs, such as the pancreas.

[0059] Over 1900 mutations in CFTR alleles are known to cause CF (Sosnay et al., Nat Genet. 2013 45(10)). Despite this large number, relatively few mutations account for the majority of disease-causing CFTR alleles in humans. The most prevalent disease causing mutation is F508del-CFTR (a Class II mutation). This mutation accounts for approximately 70% of all CFTR alleles, and as high as 86% in northern European Caucasians (Riordan et al., Annu. Rev. Biochem. 2008. 77: 15.1-15.26). Of these, approximately 47% of patients have two copies of the F508del mutation (homozygous) and approximately 39% have one copy of the mutation (heterozygous). Among the heterozygous population, the G551D mutation is the most common mutation found on the second allele, a Class III mutation present in approximately 4% of heterozygous patients (Cystic Fibrosis Foundation Patient Registry, 2013 Annual Data Report. Bethesda, Maryland. ©2014 Cystic Fibrosis

Foundation, www.cff.org). Phenotypic studies have shown that mutations, such as F508del and G551D (Classes II and III) are associated with more severe disease, including pancreatic insufficiency and more rapid progression of lung disease, compared to CF associated with other mutations.

[0060] The biogenesis of mature, functional CFTR is a multi-stepped and complex process because the recently transcribed protein must fold properly and undergo

glycosylation steps as it moves from the endoplasmic reticulum (ER) to the Golgi complex and is eventually transported to the cell surface. Once at the cell surface, the channel must be able to properly activate and open, allowing anion transport to occur.

[0061] The cell's quality control mechanisms, ER-associated degradation (ERAD), rapidly degrade and remove any misfolded proteins. The F508del mutation causes protein misfolding, resulting in efficient (i.e., rapid) ERAD and minimal protein expression at the plasma membrane. Further decreases in function are caused by abnormal activation of the small amount of protein that reaches the membrane and reduced residence time in the apical membrane (Boinot et al., J Pharmacol Exp Ther. 2014. 350:624-634, Odolczyk et al., EMBO Molecular Medicine. 2013. 5: 1484-1501). Thus the complexity of restoring the function of F508-del CFTR will require multiple approaches. Since the median predicted survival age is currently about 37 years, there is a large medical need for more efficacious therapies that address the underlying defect of CF.

[0062] To address this need, there has been increased interest in small-molecule therapies that increase CFTR function because such an approach could address the consequences of CFTR dysfunction as well as slow the progression of the disease. Such therapies are broadly classified as CFTR modulators and include CFTR activators, potentiators, correctors, and antagonists. CFTR activators act on their own to stimulate CFTR-mediated ion transport and include agents that increase cAMP levels, such as b-adrenergic agonists, adenylate cyclase activators, and phosphodiesterase inhibitors. CFTR potentiators act in the presence of endogenous or pharmacological CFTR activators to increase the channel gating activity of cell-surface localized CFTR, resulting in enhanced ion transport. CFTR correctors act by increasing the delivery and amount of functional CFTR protein to the cell surface, resulting in enhanced ion transport. Additionally, compounds with a new mechanism of action, GSNOR inhibitors, have been identified that have been shown to modulate CFTR activity (PCT/US2015/054728). Depending on the molecular consequence of the mutation and disease severity, CFTR stabilizers (such as the GSNOR inhibitors of the present invention), amplifiers, activators, potentiators, and correctors may be coadministered to maximize clinical efficacy or therapeutic window, if needed. [0063] The ultimate goal of CFTR modulation therapy in CF patients is to maximize and maintain CFTR function, thereby restoring fluid transport across epithelial cells. Currently two CFTR modulators have been approved for use in the US and Europe. The first of these was ivacaftor (VX-770), a CFTR "potentiator" that initially targeted the F508del/G551D heterozygous population. It was designed to increase the time that activated CFTR channels remain open and functional. Ivacaftor has demonstrated significant clinical benefit in a specific group of heterozygous CF patients. Despite the significant improvements in lung function achieved with ivacaftor in these patients, current strategies are aimed at providing even greater improvements to target the F508del-CFTR allele.

[0064] Approximately half of all CF patients are homozygous for the F508del-CFTR mutation, a trafficking mutation, and maximizing CFTR function in these patients is challenging. Most of the abnormal CFTR made at the ribosome does not fold correctly and is rapidly degraded before reaching the apical cell surface. Furthermore, the small amount of F508del-CFTR that does reach the cell surface is not stable, and is retrieved from the membrane and degraded much more rapidly than normal CFTR. Lumacaftor (VX-809) is an FDA-approved CFTR "corrector" that targets the misfolded protein (i.e., improves the folding in certain respects). Despite strong pre-clinical results, when used alone in clinical trials, lumacaftor does not improve lung function. Moderate improvements in lung function have been observed, however, with the combination of lumacaftor and ivacaftor in the F508del-CFTR homozygous patient population (Wainwright et al, N Engl J Med. 2015. 373(18): 1783-4).

[0065] Two clinical trials in patients homozygous for F508del-CFTR (Wainwright et al, 2015) have demonstrated that the approach of combining a corrector (lumacaftor) and a potentiator (ivacaftor), although promising based on pre-clinical predictions (Van Goor et al., Proc Natl Acad Sci USA. 2011. 108(46): 18843-8), resulted in only moderate improvements in terms of absolute change in percent predicted FEVi (ppFEVl). The pooled effect on ppFEVi from the two trials showed a mean absolute improvement of approximately 2.8% in both treatment arms being tested (Wainwright et al., 2015). Analysis of key secondary endpoints confirmed a beneficial effect of lumacaftor/ivacaftor on CF pulmonary

exacerbations (pooled reduction of ~ 35%), increased BMI (+0.26 kg/m²), and a statistically significant but modest improvement in a validated symptom score (CFQ-R). On the basis of these results, the combination of lumacaftor and ivacaftor (ORKAMBI™) was approved for marketing in the US on July 2, 2015 for F508del-CFTR CF patients, 12 years of age and older. However, as the improvements were modest, there remains a need for further advancement in treatment of patients homozygous for F508del-CFTR. Specifically, several investigators have suggested that the disconnect between the pre-clinical observations and clinical efficacy may be due to the decreased length of time that F508del-CFTR spends at the membrane after being treated with lumacaftor and ivacaftor. Treatment with a GSNOR inhibitor that stabilizes the F508del-CFTR may lead to improved outcomes.

[0066] Recently, two independent research laboratories reported in vitro data showing that the combination treatment produced a negative effect on "CFTR stability" in F508del- CFTR HAE cells (Cholon et al., Sci Transl Med. 2014. 6(246): 246ra96; Veit, et al., Sci Transl Med. 2014. 6(246) :246ra97). When primary HAE cells were co-treated with both lumacaftor and ivacaftor for 24 or 48 hours, a decrease in forskolin-stimulated CFTR- mediated current was observed, as well as an increase in the rate of loss of CFTR activity after forskolin stimulation, compared to treatment with lumacaftor alone. The data suggests an additional therapeutic approach with a different mode of action, especially one that may improve stability, as evidenced by an increased amount of CFTR protein at the cell surface for a prolonged period of time, may be required to produce additional significant

improvements in clinical outcomes.

[0067] In the lungs of CF patients, the lack of chloride transport and accompanying water across the airway epithelium and excessive sodium reabsorption leads to dehydrated airway surface fluid, impaired mucociliary clearance, infection and inflammation. Increasing the amount of F508-CFTR that reaches the plasma membrane, or otherwise improving its function, including increasing the amount of CFTR protein at the plasma membrane for a prolonged period of time, offers the potential to improve the hydration of the airway surface fluid and reverse part of the underlying pathophysiology.

[0068] Inhibitors of S-nitrosoglutathione reductase (GSNOR), the primary catabolizing enzyme of S-nitrosoglutathione (GSNO), may provide a novel therapeutic strategy in cystic fibrosis (CF). GSNO has been identified as a potential modulator of CFTR (Zaman et al., 2001); however, attempts to deliver GSNO exogenously are fraught with difficulties related to formulation, intracellular delivery, and inconsistency of results. GSNOR inhibitors on the other hand are distinguished by their ability to consistently demonstrate preservation of intracellular GSNO and potent bronchodilatory and anti-inflammatory effects in animal models of COPD and asthma. [0069] Increasing the amount of F508-CFTR function, through stabilizing the protein (or increasing the amount of the protein) at the cell membrane for a prolonged period of time, offers the potential to improve the hydration of the airway surface fluid and reverse part of the underlying pathophysiology. It is believed that GSNOR inhibition can increase CFTR mediated chloride transport. Mechanisms by which GSNOR inhibitors may improve F508del-CFTR function include nitrosation of chaperone proteins potentially improving the stability of the misfolded protein allowing it to move beyond a stalled folding intermediate(s) (Coppinger et al., PLoS One. 2012;7(5):e37682), prevention of CFTR proteosomal degradation, promotion of CFTR maturation, and maintenance of epithelial tight junctions.

[0070] The potential benefits of GSNOR inhibitors in CF extend beyond their potential to affect chloride and water transport and to increase the airway surface fluid level. They may also affect what appears to be a primary defect in local mucosal immunity. Cohen and Prince have noted that even in the absence of clinically apparent viral or bacterial infection, there is often evidence of inflammation in CF airways, as evidenced by polymorphonuclear neutrophil (PMN) accumulation and excessive concentrations of interleukin-8 (IL-8) and free proteases, accompanied by over-activated nuclear factor kappa B (NFKB) and ineffective antioxidant transport (Cohen and Prince (Nat Med. 2012; 18(4): 509-19)).

[0071] The anti-inflammatory properties of GSNOR inhibition have been demonstrated in several in vitro and in vivo models. Of particular relevance to cystic fibrosis are the mouse models of COPD (cigarette smoke and elastase/papain) in which cellular influx was prevented or reversed, and epithelial cell damage was minimized. The relevance of these models to CF lung disease lies in their common inflammatory manifestations of NFKB activation, neutrophilic infiltration, and elastase-mediated lung injury. GSNOR inhibition has been shown to down regulate the activity of transcription factor NFKB by nitrosation of NFKB regulatory proteins. GSNOR inhibition, therefore, offers a novel mechanism for targeting inflammatory pathways in CF.

[0072] The combination of improved CFTR function through stabilizing the protein both in the cell and at the cell membrane and anti-inflammatory effects arising from GSNOR inhibition may lead to clinical improvement in CF patients, which may be preceded by measurable changes in FEVi, sweat chloride, NPD, and inflammatory biomarkers in serum and airway secretions, sputum and/or bronchoalveolar lavage fluid (BALF). Other measurements of clinical improvement may be intestinal current measurements and weight gain. [0073] International PCT Publication WO2012/048181 ("the ' 181 application"), with the filing date of October 7, 2011, the entirety of which is hereby incorporated herein by reference, describes certain quinolone compounds which selectively and reversibly inhibit the activity of S-nitrosoglutathione reductase (GSNOR). Such compounds include compounds of Formula 1, or a pharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, or N-oxide thereof:

wherein m is selected from the group consisting of 0, 1, 2, or 3;

Ri is independently selected from the group consisting of chloro, fluoro, bromo, cyano, and methoxy;

R₂b and R_2c are independently selected from the group consisting of hydrogen, halogen, C1-C3 alkyl, fluorinated C1-C3 alkyl, cyano, C1-C3 alkoxy, and N(CH₃)₂;

X is selected from the group consisting of

n is selected from the group consisting of 0, 1, and 2;

R₃ is independently selected from the group consisting of halogen, Ci-C₃ alkyl, fluorinated Ci-C₃ alkyl, cyano, hydroxy, Ci-C₃ alkoxy, and NR₄R₄' where R₄ and R₄' are independently selected from the group consisting of Ci-C₃ alkyl, or R₄ when taken together with R₄' form a ring with 3 to 6 members; and

A is selected from the group consisting of O

O' H N -^ N H N-S N-N H H N-0 >

[0074] In a further aspect of the invention, Ri is independently selected from the group consisting of chloro, fluoro, and bromo; R₃ is independently selected from the group consisting of halogen, Ci-C₃ alkyl, fluorinated Ci-C₃ alkyl, cyano, Ci-C₃ alkoxy, and NR₄R₄' where R₄ and R₄' are independently selected from the group consisting of Ci-C₃ alkyl, or R₄ when taken together with R₄' form a ring with 3 to 6 members; and

X is selected from the group consisting of

[0075] In a further aspect of the invention, R₃ is independently selected from the group consisting of halogen, Ci-C₃ alkyl, fluorinated Ci-C₃ alkyl, cyano, Ci-C₃ alkoxy, and NR₄R₄' where R₄ and R₄' are methyl, or alternatively together with the said N form the ring aziridin- 1-yl or morpholino.

[0076] In a further aspect of the invention, m is selected from the group consisting of 0 and 1 ; R₂b and R_2c are independently selected from the group consisting of hydrogen, chloro, fluoro, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from the group consisting of 0 and 1 ; and R₃ is independently selected from the group consisting of fluoro, chloro, bromo, methyl, trifluoromethyl, cyano, hydroxy, methoxy, and N(CH₃)₂.

[0077] In a further aspect of the invention, X is

[0078] In a further aspect of the invention, A is COOH.

[0079] In a further aspect of the invention, suitable compounds of Formula I include, but are not limited to:

4-(6-hydroxy-3-methylquinolin-2-yl)benzoic acid;

2-(4-(lH-tetrazol-5-yl)phenyl)-3-methylquinolin-6-ol; 4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-(lH-tetrazol-5-yl)phenyl)quinolin-6-ol;

1 -(6-hydroxyquinolin-2-yl)piperidine-4-carboxylic acid;

(lr,4r)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

(ls,4s)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

3- chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2-fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-(2H-tetrazol-5-yl)phenyl)-4-chloroquinolin-6-ol;

3- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(2H)-one;

3- fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4- (6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;

5- (6-hydroxyquinolin-2-yl)thiophene-2-carboxylic acid;

4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylic acid;

4-(3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(4-chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(3-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

3-(2-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(4H)-one;

3- (3-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(4H)-one;

4- (4-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

2- (2-chloro-4-(2H-tetrazol-5-yl)phenyl)quinolin-6-ol;

5- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,3,4-oxadiazol-2(3H)-one;

3- (dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4- (4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(6-hydroxyquinolin-2-yl)-3-methylbenzoic acid;

4-(3-chloro-6-hydroxyquinolin-2-yl)-3-fluorobenzoic acid;

3- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-thiadiazol-5(2H)-one;

4- (6-hydroxyquinolin-2-yl)-3-(trifluoromethyl)benzoic acid;

4- (6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)benzoic acid;

2-(4-carboxyphenyl)-6-hydroxyquinoline 1 -oxide;

5- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,3,4-thiadiazol-2(3H)-one;

5-(4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-3(2H)-one;

(lr,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid; (ls,4s)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid; 3-chloro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

2- (5-(2H-tetrazol-5-yl)thiophen-2-yl)quinolin-6-ol;

5-(4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-thiadiazol-3(2H)-one;

3- fluoro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

1- (6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylic acid;

4- (5-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

(lr,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylic acid;

(ls,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylic acid;

4-(5-bromo-6-hydroxyquinolin-2-yl)benzoic acid;

3- bromo-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4- (4-(dimethylamino)-6-hydroxyquinolin-2-yl)benzoic acid;

4-(4-fluoro-6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;

3- cyano-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-carboxy-2-chlorophenyl)-6-hydroxyquinoline 1 -oxide;

4- (4-amino-6-hydroxyquinolin-2-yl)benzoic acid;

4-(3-cyano-6-hydroxyquinolin-2-yl)benzoic acid;

4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(8-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

3- hydroxy-4-(6-hydroxyquinolin-2-yl)benzoic acid; and

3-fluoro-4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid.

[0080] In some embodiments, the GSNOR inhibitor is selected from the group consisting of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid, 3-fluoro-4-(6-hydroxyquinolin-2- yl)benzoic acid, and 4-(6-hydroxyquinolin-2-yl)-3-methylbenzoic acid.

[0081] In some embodiments, the GSNOR inhibitor is 3-chloro-4-(6-hydroxyquinolin-2- yl)benzoic acid, or Compound 1 :

1

[0082] Compound 1, 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid, is designated as compound number 8 in the '181 application and the synthesis of Compound 1 is described in detail at Example 8 of the ' 181 application. [0083] Compound 1 is active in a variety of assays and therapeutic models demonstrating selective and reversible inhibition of s-nitrosoglutathione reductase (GSNOR). Notably, Compound 1 demonstrates efficacy in asthma, COPD, cystic fibrosis, and IBD models (described in international PCT Publication WO2012/048181 and PCT application

PCT/US2015/054728). Accordingly, Compound 1 is useful for treating one or more disorders associated with activity of GSNOR.

[0084] The present invention provides methods and pharmaceutical compositions that are useful in treating or lessening the severity of cystic fibrosis in a patient by increasing the amount of the CFTR protein at the plasma membrane for a prolonged time by administering to said patient an effective amount of a GSNOR inhibitor, administered alone or with one or more secondary active agents. The GSNOR inhibitor of the pharmaceutical composition can be administered concurrently with, prior to, or subsequent to, one or more secondary active agents. In some embodiments the GSNOR inhibitor is a compound of Formula 1. In some embodiments, the GSNOR inhibitor is Compound 1.

[0085] In one embodiment, the present invention provides methods of treating or lessening the severity of cystic fibrosis in a patient by increasing the amount of CFTR protein at the plasma membrane for a prolonged period of time by administering to said patient a therapeutically effective amount of a GSNOR inhibitor with the corrector VX-809, either with our without the potentiator VX-770. In another embodiment, the present invention provides methods of treating or lessening the severity of cystic fibrosis in a patient by increasing the amount of CFTR protein at the plasma membrane for a prolonged period of time by administering to said patient a therapeutically effective amount of a GSNOR inhibitor with the corrector VX-661, either with our without the potentiator VX-770. In some embodiments Compound 1 is administered with VX-809, with or without the potentiator VX- 770. In some embodiments Compound 1 is administered with VX-661, with or without the potentiator VX-770.

[0086] In some embodiments, the increase of the amount of CFTR protein at the plasma membrane versus control is for at least 1.5 hours. In some embodiments, the increase of the amount of the CFTR protein at the plasma membrane versus control is for at least 3 hours. In some embodiments the amount of CFTR protein at the plasma membrane at 1.5 hours after treatment with GSNOR inhibitor is increased by at least 10% (i.e. at least 20%, at least 30%, at least 40%, at least 50%) versus control. In some embodiments the amount of CFTR protein at the plasma membrane at 3 hours after treatment with GSNOR inhibitor is increased by at least 10% (i.e. greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50%) versus control. In some embodiments the GSNOR inhibitor is a compound of Formula 1. In some embodiments, the GSNOR inhibitor is Compound 1.

[0087] In some embodiments, the method includes a therapeutically effective amount of GSNOR inhibitor administered with VX-809 whereby the amount of CFTR at the plasma membrane at 3 hours post treatment is increased by at least 20 % (i.e. greater than 20%, or greater than 30%) versus treatment with VX-809 alone. In some embodiments, the method includes a therapeutically effective amount of GSNOR inhibitor administered with VX-661 whereby the amount of CFTR at the plasma membrane at 3 hours post treatment is increased by at least 30 % (i.e. greater than 40%, or greater than 50%) versus treatment with VX-661 alone. In some embodiments the GSNOR inhibitor is a compound of Formula 1. In some embodiments, the GSNOR inhibitor is Compound 1.

[0088] In some embodiments, the method includes a therapeutically effective amount of GSNOR inhibitor administered with VX-809 and VX-770 whereby the amount of CFTR at the plasma membrane at 3 hours post treatment is increased by at least 20% (i.e. greater than 20%, greater than 30%, greater than 40%, or greater than 50%) versus treatment with VX- 809 and VX-770. In some embodiments, the method includes a therapeutically effective amount of GSNOR inhibitor administered with VX-661 and VX-770 whereby the amount of CFTR at the plasma membrane at 3 hours post treatment is increased by at least 20% (i.e. greater than 20%, greater than 30%, greater than 40%, or greater than 50%) versus treatment with VX-661 and VX-770. In some embodiments the GSNOR inhibitor is a compound of Formula 1. In some embodiments, the GSNOR inhibitor is Compound 1.

[0089] Pharmaceutical Compositions of Compound 1

[0090] The invention also provides pharmaceutical compositions, pharmaceutical formulations, and solid dosage forms comprising Compound 1, which may be in substantially crystalline form. In some embodiments, Compound 1 is in crystalline Form A (Compound 1 Form A), disclosed in U.S. Provisional Application US 62/216,765, entitled "SOLID FORMS OF AN S -NITROS OGLUTATHIONE REDUCTASE INHIBITOR", filed on September 10, 2015 by Jian Qiu, and in PCT application PCT/US2016/050974, filed September 9, 2016 with same name and inventor. In some embodiments, Compound 1 is in crystalline Form B (Compound 1 Form B), also disclosed in US 62/216,765 and PCT/US2016/050974. In some embodiments of this aspect, the amount of Compound 1 that is present in the dosage form of the pharmaceutical composition is about 95 to about 100 mg. In some embodiments of this aspect, weight/weight relative percent of Compound 1 that is present in the pharmaceutical composition is from about 23 wt % to about 37 wt %. In some embodiments of this aspect, weight/weight relative percent of Compound 1 that is present in the pharmaceutical composition is from about 24 percent to about 35 percent. In some embodiments of this aspect, weight/weight relative percent of Compound 1 that is present in the pharmaceutical composition is from about 25 percent to about 27 percent. In some embodiments of this aspect, weight/weight relative percent of Compound 1 that is present in the pharmaceutical composition is from about 32 percent to about 34 percent. In these and other embodiments, Compound 1 is present as substantially pure Compound 1. "Substantially pure" means greater than ninety percent pure; preferably greater than 95 percent pure; more preferably greater than 99 percent pure (i.e., not mixed with other crystalline forms of Compound 1).

[0091] Thus in one aspect, the invention provides a pharmaceutical composition comprising:

[0092] a. Compound 1

[0093] b. a disintegrant

[0094] c. at least one filler

[0095] d. a lubricant, and

[0096] e. a dry binder.

[0097] In another aspect, the invention provides a pharmaceutical composition comprising:

[0098] a. Compound 1

[0099] b. a disintegrant

[00100] c. at least one filler

[00101] d. a lubricant, and

[00102] e. a glidant.

[00103] In one embodiment of this aspect, the dosage form of the pharmaceutical composition comprises about 95 to about 100 mg of Compound 1. In one embodiment, the composition comprises from about 23 wt % to about 37 wt % of Compound 1 by weight of the composition.

[00104] In some embodiments, the pharmaceutical composition comprises Compound 1, a disintegrant, at least one filler, a lubricant, and a dry binder. In this embodiment, the composition comprises from about 23 wt % to about 29 wt % of Compound 1 by weight of the composition. In another embodiment, this composition comprises from about 25 wt % to about 27 wt % of Compound 1 by weight of the composition.

[00105] In some embodiments, the pharmaceutical composition comprises Compound 1, a disintegrant, at least one filler, a lubricant, and a glidant. In this embodiment, the composition comprises from about 30 wt % to about 37 wt % of Compound 1 by weight of the composition. In another embodiment, this composition comprises about 31 wt % to about 35 wt % of Compound 1 by weight of the composition. In another embodiment, this

composition comprises about 32 wt % to about 34 wt % of Compound 1 by weight of the composition.

[00106] The concentration of Compound 1 in the composition depends on several factors such as the amount of pharmaceutical composition needed to provide a desired amount of Compound 1, the desired dissolution profile of the pharmaceutical composition, and the bulk density of compound 1.

[00107] Fillers suitable for the invention are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, the hardness, the chemical stability, the physical stability, or the biological activity of the pharmaceutical composition. The term diluent is often used interchangeably with fillers. Exemplary fillers include: celluloses, modified celluloses, (e.g. sodium carboxymethyl cellulose, ethyl cellulose hydroxymethyl cellulose, hydroxypropylcellulose, powdered cellulose), cellulose acetate, microcrystalline cellulose, calcium phosphates, dibasic calcium phosphate (DCP, or dicalcium phosphate), starches (e.g. corn starch, potato starch), sugars (confectioner's sugar, compressible sugar, dextrates, dextrin, dextrose, sucrose, lactose, mannitol, sorbitol, or the like), or any combination thereof.

[00108] Thus, in one embodiment, the pharmaceutical composition comprises a total weight percent of filler in an amount of at least at least 40 wt % (e.g., at least about 45 wt %, at least about 50 wt %, or at least about 60 wt %) by weight of the composition. In this embodiment, the pharmaceutical composition can contain one, two or three different fillers. For example, the pharmaceutical composition comprises from about 40 wt % to about 70 wt % of total amount of filler (e.g., about 45 wt % to about 70 wt %, or about 45 wt % to about 57 wt %), by weight of the composition. In one embodiment, the pharmaceutical

composition comprises one or more fillers in an amount each of at least 8 wt % (e.g., at least about 10 wt %, at least about 20 wt %,at least about 30 wt %, or at least about 40 wt %) by weight of the composition. In another example, the pharmaceutical composition comprises about 15 wt % to about 70 wt % (e.g. about 25 wt % to about 50 wt %, or about 30 wt % to about 45 wt %, or about 33 wt % to about 44 wt %) of lactose by weight of the composition. In yet another example, the pharmaceutical composition comprises from about 5 wt % to about 35 wt % (e.g., from about 8 wt % to about 30 wt %, from about 8 wt % to about 20 wt%) of DCP (dibasic calcium phosphate), by weight of the composition. In yet another example, the pharmaceutical composition comprises about 15 wt % to about 50 wt % (e.g., about 18 wt to about 48 wt %) of Microcrystalline Cellulose, PH102, by weight of the composition.

[00109] Disintegrants suitable for the invention enhance the dispersal of the

pharmaceutical composition and are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the chemical stability, the physical stability, the hardness, or the biological activity of the pharmaceutical composition.

Exemplary disintegrants include croscarmellose sodium, pregelatinized starch, sodium starch glycolate, or a combination thereof.

[00110] Thus, in one embodiment, the pharmaceutical composition comprises disintegrant in an amount of about 20 wt % or less (e.g., about 15 wt % or less,) by weight of the composition. For example, the pharmaceutical composition comprises from about 0.5 wt % to about 16 wt % (e.g., from about 1 wt % to about 15 wt %, or from about 8 wt % to about 16 wt %, or from about 9 wt % to about 15 wt %) of disintegrant, by weight of the composition. In another example, the pharmaceutical composition comprises about 15 wt % or less (e.g., about 15 wt %, or about 13 wt %, or about 11 wt %, or about 10 wt %, or about 9 wt %) of pregelatinized starch, by weight of the composition. In another example, the pharmaceutical composition comprises about 5 wt % or less (e.g., about 2 wt %, or about 1 wt %) of croscarmellose sodium, by weight of the composition.

[00111] Binders suitable for the invention enhance the tablet strength of the

pharmaceutical composition and are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the chemical stability, the physical stability, or the biological activity of the pharmaceutical composition. Exemplary binders include polyvinylpyrrolidone, dibasic calcium phosphate, sucrose, corn (maize) starch, modified cellulose (e.g., hydroxymethyl cellulose, hydroxypropyl cellulose), or any combination thereof.

[00112] Thus, in one embodiment, the pharmaceutical composition comprises a binder in an amount of about 10 wt % or less (e.g., about 8 wt % or less, about 6 wt % or less) by weight of the composition. In one example, the pharmaceutical composition comprises about 10 wt % or less (e.g., about 8 wt %, or about 6 wt %) of hydroxypropyl cellulose (HPC), by weight of the composition.

[00113] Glidants suitable for the invention enhance the flow properties of the

pharmaceutical composition and are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, the hardness, the chemical stability, the physical stability, or the biological activity of the pharmaceutical composition. Exemplary glidants include colloidal silicon dioxide, talc, or a combination thereof.

[00114] Thus, in one embodiment, the pharmaceutical composition comprises a glidant in an amount of 2 wt % or less (e.g., 1.75 wt % or less, 1.25 wt % or less, 1.0 wt % or less) by weight of the composition. For example, the pharmaceutical composition comprises from about 0.05 wt % to about 2 wt % (e.g., from about 0.07 wt % to about 1.5 wt % or from about 1.0 wt % to about 1.2 wt %) of glidant, by weight of the composition. In another example, the pharmaceutical composition comprises about 2 wt % or less (e.g., about 1.75 wt % or less, about 1.5 wt % or less, or about 1.25 wt % or less) of colloidal silicon dioxide, by weight of the composition. In yet another example, the pharmaceutical composition comprises from about 0.05 wt % to about 2 wt % (e.g., from about 0.7 wt % to about 1.5 wt % or from about 1.0 wt % to about 1.2 wt %) of colloidal silicon dioxide, by weight of the composition.

[00115] In some embodiments, the pharmaceutical composition can include an oral solid pharmaceutical dosage form which can comprise a lubricant that can prevent adhesion of a powder blend to a surface (e.g., a surface of a mixing bowl, a compression die, punch or tamping pin). A lubricant can also reduce interparticle friction within the granulate and improve the compression and ejection of compressed pharmaceutical compositions from a die press. The lubricant is also compatible with the ingredients of the pharmaceutical

composition, i.e., they do not substantially reduce the solubility, the hardness, or the biological activity of the pharmaceutical composition. Exemplary lubricants include magnesium stearate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, hydrogenated vegetable oil or any combination thereof.

[00116] In one embodiment, the pharmaceutical composition comprises a lubricant in an amount of 5 wt % or less (e.g., 4.0 wt % or less, or 3.00 wt % or less, or 2.0 wt % or less, or 1.0 wt % or less) by weight of the composition. For example, the pharmaceutical composition comprises from about 5 wt % to about 0.10 wt % (e.g., from about 3 wt % to about 0.5 wt % or from about 2 wt % to about 1 wt %) of lubricant, by weight of the composition. In another example, the pharmaceutical composition comprises 5 wt % or less (e.g., 4.0 wt % or less, 3.0 wt % or less, or 2.0 wt % or less, or 1.0 wt % or less) of magnesium stearate, by weight of the composition. In yet another example, the pharmaceutical composition comprises from about 5 wt % to about 0.10 wt % (e.g., from about 3 wt % to about 0.5 wt % or from about 2.0 wt % to about 1.0 wt %) of magnesium stearate, by weight of the composition.

[00117] Pharmaceutical compositions of the invention can optionally comprise one or more colorants, flavors, and/or fragrances to enhance the visual appeal, taste, and/or scent of the composition. Suitable colorants, flavors, or fragrances are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, the chemical stability, the physical stability, the hardness, or the biological activity of the pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a colorant, a flavor, and/or a fragrance.

[00118] The compound N91115 (3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid) is disclosed in International PCT Publication WO2012/048181 ("the ' 181 application") as an inhibitor of GSNOR and thus as a useful treatment for NO related diseases such as cystic fibrosis, asthma, COPD, IBD, etc. Form A of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid, which is a substantially crystalline hemi-hydrate form known as N91115 Form A, is disclosed in PCT application Serial No. PCT/US2016/050974, filed on September 9, 2016. All applications are incorporated in their entirety by reference herein.

[00119] It has been found that N91115 can exist in a variety of solid forms. Such forms include polymorphs and amorphous forms. The solid forms can be solvates, hydrates and unsolvated forms of N91115. All such forms are contemplated by the present invention. In certain embodiments, the present invention provides N91115 as a mixture of one or more solid forms of N91115.

[00120] As used herein, the term "polymorph" refers to the different crystal structures (of solvated or unsolvated forms) in which a compound can crystallize.

[00121] As used herein, the term "solvate" refers to a solid form with either a stoichiometric or non-stoichiometric amount of solvent (e.g., a channel solvate). For polymorphs, the solvent is incorporated into the crystal structure. Similarly, the term "hydrate" refers to a solid form with either a stoichiometric or non-stoichiometric amount of water. The term "hemi-hydrate" refers to a solid form with about 1 equivalent of water relative to 2 equivalents of anhydrous N91115 in the crystal structure. For polymorphs, the water is incorporated into the crystal structure.

[00122] As used herein, the term "about", when used in reference to a degree 2-theta value refers to the stated value + 0.3 degree 2-theta. In certain embodiments, "about" refers to + 0.2 degree 2-theta or + 0.1 degree 2-theta. In certain embodiments, "about" refers to + 0.2 degree 2-theta.

[00123] In certain embodiments, N91115 is a crystalline solid. In other embodiments, N91115 is a crystalline solid substantially free of amorphous N91115. As used herein, the term "substantially free of amorphous N91115" means that the compound contains no significant amount of amorphous N91115. In certain embodiments, at least about 90% by weight of crystalline N91115 is present, or at least about 95% by weight of crystalline N91115 is present. In still other embodiments of the invention, at least about 97%, 98% or 99% by weight of crystalline N91115 is present.

[00124] In certain embodiments, N91115 is a hydrate polymorphic form. In one embodiment, the present invention provides a hemi-hydrate polymorphic form of N91115 referred to herein as Form A. Form A of N91115 is also known as Form III of N91115.

[00125] According to another embodiment, Form A of N91115 is characterized by one or more peaks in its powder X-ray diffraction pattern selected from those at about 12.92, about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta (°2Θ). In some embodiments, Form A of N91115 is characterized by two or more peaks in its powder X-ray diffraction pattern selected from those at about 12.92, about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In certain embodiments, Form A of N91115 is characterized by three or more peaks in its powder X-ray diffraction pattern selected from those at about 12.92, about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. For example, in one embodiment, Form A of N91115 is characterized by a peak in its powder X-ray diffraction pattern at about 12.92. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92 and one or more additional peaks selected from those at about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92 and two or more additional peaks selected from those at about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92 and three or more additional peaks selected from those at about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92 and four or more additional peaks selected from those at about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92 and about 18.01, and optionally one or more additional peaks selected from about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92, about 18.01, and about 22.63, and optionally one or more additional peaks selected from about 18.86, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92, about 18.01, about 22.63, and about 24.88, and optionally one or more additional peaks selected from about 18.86, about 23.00, and about 23.72. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.92, about 18.01, about 18.86, about 22.63, about 23.00, about 23.72, and about 24.88 degrees 2-theta. In another embodiment, Form A of N91115 is characterized by having one or more peaks in its X-ray powder diffraction pattern selected from the group consisting of at about 8.32, 8.97, 12.92, 14.56, 15.14, 18.01, 18.86, 22.63, 23.00, 23.72 and 24.88 degrees 2- theta. In particular embodiments, Form A of N91115 is characterized by all or substantially all of the peaks in its X-ray powder diffraction pattern selected from those at about 8.32, 8.97, 12.92, 14.56, 15.14, 18.01, 18.86, 22.63, 23.00, 23.72 and 24.88 degrees 2-theta. In an exemplary embodiment, Form A of N91115 is characterized by all or substantially all of the peaks in its X-ray powder diffraction pattern selected from those at about:

^°2Θ 16.70 24.49

8.32 18.01 24.88

8.97 18.86 25.18

9.39 19.57 25.37

10.85 19.85 25.73

11.15 20.42 26.01

11.38 21.06 26.29

12.16 21.62 26.64

12.92 21.80 27.12

13.44 22.18 27.87

14.14 22.63 28.27

14.56 23.00 28.97

15.14 23.72 29.46

16.43 23.91

[00126] According to another embodiment, Form A of N91115 is characterized by one or more peaks in its powder X-ray diffraction pattern selected from those at about 12.9, about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In some embodiments, Form A of N91115 is characterized by two or more peaks in its powder X-ray diffraction pattern selected from those at about 12.9, about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In certain embodiments, Form A of N91115 is characterized by three or more peaks in its powder X-ray diffraction pattern selected from those at about 12.9, about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. For example, in one embodiment, Form A of N91115 is characterized by a peak in its powder X-ray diffraction pattern at about 12.9. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9 and one or more additional peaks selected from those at about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9 and two or more additional peaks in its powder X-ray diffraction pattern selected from those at about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9 and three or more additional peaks in its powder X-ray diffraction pattern selected from those at about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2- theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9 and four or more additional peaks in its powder X-ray diffraction pattern selected from those at about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9 and about 18.0, and optionally one or more additional peaks selected from about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9, about 18.0, and about 22.6, and optionally one or more additional peaks selected from about 18.9, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9, about 18.0, about 22.6, and about 24.9, and optionally one or more additional peaks selected from about 18.9, about 23.0, and about 23.7. In another example, Form A of N91115 is characterized by peaks in its powder X-ray diffraction pattern at about 12.9, about 18.0, about 18.9, about 22.6, about 23.0, about 23.7, and about 24.9 degrees 2-theta. In another embodiment, Form A of N91115 is characterized by having one or more peaks in its X-ray powder diffraction pattern selected from the group consisting of at about 8.3, 9.0, 12.9, 14.6, 15.1, 18.0, 18.9, 22.6, 23.0, 23.7 and 24.9 degrees 2-theta. In particular embodiments, Form A of N91115 is characterized by all or substantially all of the peaks in its X-ray powder diffraction pattern selected from those at about 8.3, 9.0, 12.9, 14.6, 15.1, 18.0, 18.9, 22.6, 23.0, 23.7 and 24.9 degrees 2-theta. In an exemplary embodiment, Form A of N91115 is characterized by all or substantially all of the peaks in its X-ray powder diffraction pattern selected from those at about:

8.3

9.0

9.4

10.9

11.2

11.4

12.2

12.9

13.4

14.1

14.6

15.1

16.4

16.7

18.0

18.9

19.6

19.9

20.4

21.1

21.6

21.8

22.2

22.6

23.0

23.7

23.9

24.5

24.9

25.2

25.4

25.7

26.0

26.3

26.6

27.1

27.9

28.3

29.0

29.5 [00127] In some embodiments, the pharmaceutical composition includes or can be made into a capsule.

[00128] In some embodiments, the pharmaceutical composition includes or can be made into tablets and the tablets can be coated with a colorant and optionally labeled with a logo, other image and/or text using a suitable ink. In still other embodiments, the pharmaceutical composition includes or can be made into tablets and the tablets can be coated with a colorant, waxed, and optionally labeled with a logo, other image and/or text using a suitable ink. Suitable colorants and inks are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, the chemical stability, the physical stability, the hardness, or the biological activity of the pharmaceutical composition. The suitable colorants and inks can be any color and are water based or solvent based. In one embodiment, tablets made from the pharmaceutical composition can be coated with a colorant and then labeled with a logo, other image, and/or text using a suitable ink.

[00129] The pharmaceutical compositions of the invention can be processed into a tablet form, capsule form, pouch form, lozenge form, or other solid form that is suited for oral administration. Thus in some embodiments, the pharmaceutical compositions are in capsule form.

[00130] Capsule formulations can be obtained by filling the powder blend or granulated pharmaceutical formulations mentioned hereinbefore in conventional capsules, for instance, hard or soft capsules.

[00131] Pharmaceutical Compositions of Formula 1

[00132] Pharmaceutical compositions of the invention include a GSNOR inhibitor of Formula 1 and at least one pharmaceutically acceptable carrier. In one embodiment, the GSNOR inhibitor pharmaceutical composition can be administered as a monotherapy. In another embodiment, the pharmaceutical composition of the invention includes a GSNOR inhibitor in combination with one or more secondary active agents. In another embodiment, the pharmaceutical composition of the invention includes a GSNOR inhibitor in combination with one or more palliative care agents.

[00133] In another embodiment, the GSNOR inhibitor of the pharmaceutical composition can be administered concurrently with, prior to, or subsequent to, one or more secondary active agents and/or palliative care agents.

[00134] The invention encompasses pharmaceutical compositions comprising the compositions described herein and at least one pharmaceutically acceptable carrier. Suitable carriers are described in "Remington: The Science and Practice, Twentieth Edition," published by Lippincott Williams & Wilkins, which is incorporated herein by reference. Pharmaceutical compositions according to the invention may also comprise one or more non- inventive compound active agents.

[00135] The compounds of the pharmaceutical compositions of Formula 1 can be employed in combination therapies, that is, the compounds and pharmaceutically acceptable compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired secondary active agents or medical procedures. The particular combination of therapies (secondary agents or procedures) to employ in a combination regimen will take into account compatibility of the desired agents and/or procedures and the desired therapeutic effect to be achieved. The therapies employed may achieve a desired effect for the same disorder (for example, a pharmaceutical composition may be administered concurrently with one or more secondary agents used to treat the same disorder), or they may achieve different effects (such as control adverse effects).

[00136] In one embodiment the secondary active agent of the pharmaceutical combination is selected from a compound or therapy that modulates CFTR function. In one embodiment, the secondary active agent(s) are selected from CFTR correctors and/or CFTR potentiators. In one embodiment, the secondary active agents include a CFTR potentiator and a CFTR corrector. In one embodiment, the secondary active agent is selected from one or more CFTR amplifiers.

[00137] In one embodiment, the pharmaceutical composition may be used with any single or combination of palliative agents including mucolytic agents, bronchodilators, antibiotics, anti-infective agents, anti-inflammatory agents, nutritional agents, or other palliative agents known to manage CF.

[00138] The compounds of the pharmaceutical combination of the invention can be utilized in any pharmaceutically acceptable dosage form, including, but not limited to injectable dosage forms, liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, dry powders, tablets, capsules, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, etc. Specifically, the compounds of the invention described herein can be formulated: (a) for administration selected from the group consisting of oral, pulmonary, intravenous, intra-arterial, intrathecal, intra- articular, rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal,

intraperitoneal, local, buccal, nasal, and topical administration; (b) into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, ointments, creams, tablets, sachets, and capsules; (c) into a dosage form selected from the group consisting of lyophilized formulations, dry powders, fast melt formulations, controlled release

formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination thereof.

[00139] Preferably, the pharmaceutically acceptable dosage form is administered orally. Compound 1 has been formulated and delivered orally via capsule (US provisional application 62/216,771, US provisional application 62/303,218, and PCT application

PCT/US2016/051009).

[00140] For respiratory infections or pulmonary exacerbations of CF, an inhalation formulation can be used to achieve high local concentrations. Formulations suitable for inhalation include dry power or aerosolized or vaporized solutions, dispersions, or suspensions capable of being dispensed by an inhaler or nebulizer into the endobronchial or nasal cavity of infected patients to treat upper and lower respiratory bacterial infections.

[00141] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can comprise one or more of the following components: (1) a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; (2) antibacterial agents such as benzyl alcohol or methyl parabens; (3) antioxidants such as ascorbic acid or sodium bisulfite; (4) chelating agents such as ethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates, or phosphates; and (5) agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[00142] Pharmaceutical compositions suitable for injectable use may comprise sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The pharmaceutical composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. [00143] The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol or sorbitol, and inorganic salts such as sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the

composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00144] Sterile injectable solutions can be prepared by incorporating the active reagent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating at least one compound of the invention into a sterile vehicle that contains a basic dispersion medium and any other required ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and freeze-drying, both of which yield a powder of a compound of the invention plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00145] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed, for example, in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compound of the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

[00146] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g. , a gas such as carbon dioxide, a nebulized liquid, or a dry powder from a suitable device. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active reagents are formulated into ointments, salves, gels, or creams as generally known in the art. The reagents can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00147] In one embodiment, the compounds of the invention are prepared with carriers that will protect against rapid elimination from the body. For example, a controlled release formulation can be used, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[00148] Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00149] Additionally, suspensions of the compounds of the invention may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery.

Optionally, the suspension may also include suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[00150] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the compound of the invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the compound of the invention and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals.

[00151] Pharmaceutical compositions of compounds of Formula 1 can comprise one or more pharmaceutical excipients. Examples of such excipients include, but are not limited to binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Exemplary excipients include: (1) binding agents which include various celluloses and cross-linked polyvinylpyrrolidone,

microcrystalline cellulose, such as Avicel^® PH101 and Avicel^® PH102, silicified

microcrystalline cellulose (ProSolv SMCC™), gum tragacanth and gelatin; (2) filling agents such as various starches, lactose, lactose monohydrate, and lactose anhydrous; (3)

disintegrating agents such as alginic acid, Primogel, corn starch, lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof; (4) lubricants, including agents that act on the flowability of a powder to be compressed, include magnesium stearate, colloidal silicon dioxide, such as Aerosil^® 200, talc, stearic acid, calcium stearate, and silica gel; (5) glidants such as colloidal silicon dioxide; (6) preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of

parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride; (7) diluents such as pharmaceutically acceptable inert fillers, such as

microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing; examples of diluents include microcrystalline cellulose, such as Avicel^® PH101 and Avicel^® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose^® DCL21 ; dibasic calcium phosphate such as Emcompress^®; mannitol; starch; sorbitol; sucrose; and glucose; (8) sweetening agents, including any natural or artificial sweetener, such as sucrose, saccharin sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9) flavoring agents, such as peppermint, methyl salicylate, orange flavoring, Magnasweet^® (trademark of MAFCO), bubble gum flavor, fruit flavors, and the like; and (10) effervescent agents, including effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[00152] Definitions [00153] As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[00154] As used herein, the term "bioactivity" indicates an effect on one or more cellular or extracellular process (e.g., via binding, signaling, etc.) which can impact physiological or pathophysiological processes.

[00155] As used herein, N-oxide, or amine oxide, refers to a compound derived from a tertiary amine by the attachment of one oxygen atom to the nitrogen atom, R₃N⁺-0^~. By extension the term includes the analogous derivatives of primary and secondary amines.

[00156] As utilized herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils.

[00157] A "pharmaceutically acceptable salt" or "salt" of a compound of the invention is a product of the disclosed compound that contains an ionic bond, and is typically produced by reacting the disclosed compound with either an acid or a base, suitable for administering to a subject. A pharmaceutically acceptable salt can include, but is not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Li, Na, and K, alkali earth metal salts such as Mg or Ca, or organic amine salts.

[00158] A "pharmaceutical composition" is a formulation comprising the disclosed combination in a form suitable for administration to a subject. A pharmaceutical composition of the invention is preferably formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, oral and parenteral, e.g. , intravenous, intradermal, subcutaneous, inhalation, topical, transdermal, transmucosal, and rectal administration.

[00159] As used herein, a "secondary active agent" is a compound or therapy that increases CFTR function. In one embodiment, a secondary active agent is selected from the group consisting of CFTR correctors and CFTR potentiators. In one embodiment, a secondary active agent is selected from the group consisting of CFTR correctors, potentiators, or amplifiers as well as gene therapy directed toward CF.

[00160] As used herein, a "CFTR corrector" is a compound that promotes maturation and delivery of CFTR proteins to the apical surface. Examples of CFTR correctors include but are not limited to VX-809 (3-{ 6-{ [l-(2,2- difluoro- l,3-benzodioxol-5- yl)cyclopropanecarbonyl]amino}-3-methylpyridin-2-yl}benzoic acid), VX-661 (l-(2,2- difluoro-l,3-benzodioxol-5-yl)-N-[l-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy- l, l- dimethylethyl)-lH-indol-5-yl]-cyclopropanecarboxamide), compounds of

PCT/US2014/038385 and compounds of PCT/US2015/021841.

[00161] VX-809 has also recently been classified as a "CFTR conformational stabilizer" by the FDA in a Summary Review of Regulatory Action dated June 25, 2015, but continues to be known in the art and literature as a CFTR corrector, and is treated as such herein.

[00162] As used herein, a "CFTR potentiator" is a compound that activates apical CFTR by increasing the open time of the channel. An example of a CFTR potentiator includes but is not limited to VX-770 (N-(2,4-Di-ieri-butyl-5-hydroxyphenyl)-4-oxo-l,4- dihydroquinoline-3-carboxamide).

[00163] As used herein, "gene therapy" is any therapy directed toward the genetic defect in CF.

[00164] As used herein a "CFTR amplifier" is any compound that increases CFTR activity.

[00165] A "palliative care agent" is an agent for the management of CF other than a secondary active agent that may include a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a nutritional agent, or other agent known to manage the symptoms of CF, collectively termed herein as palliative care.

[00166] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[00167] As used herein the term "therapeutically effective amount" generally means the amount necessary to ameliorate at least one symptom of a disorder to be prevented, reduced, or treated as described herein. The phrase "therapeutically effective amount" as it relates to the GSNOR inhibitors of the present invention shall mean the GSNOR inhibitor dosage that provides the specific pharmacological response for which the GSNOR inhibitor is

administered in a significant number of subjects in need of such treatment. It is emphasized that a therapeutically effective amount of a GSNOR inhibitor that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a

therapeutically effective amount by those of skill in the art.

[00168] The phrase "therapeutically effective amount" as it relates to the secondary active agent of the present invention shall mean the dosage that provides the specific

pharmacological response for which the secondary active agent is administered in a significant number of subjects in need of such treatment.

[00169] As used herein, the term "GSNOR inhibitor" or "s-nitrosoglutathione reductase inhibitor" or "GSNORi" means a compound which inhibits the enzyme S-nitrosoglutathione reductase and demonstrates activity in the enzyme assay described in WO2012/048181 (the '181 application) (e.g., GSNOR Assays as described in detail in Example 58 of the ' 181 application).

[00170] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[00171] Kits Comprising the Compositions of the Invention

[00172] The present invention also encompasses kits comprising the compositions of the invention. Such kits can comprise, for example, (1) at least one compound of the invention; and (2) at least one pharmaceutically acceptable carrier, such as a solvent or solution.

Additional kit components can optionally include, for example: (1) any of the

pharmaceutically acceptable excipients identified herein, such as stabilizers, buffers, etc., (2) at least one container, vial, or similar apparatus for holding and/or mixing the kit

components; and (3) delivery apparatus, such as an inhaler, nebulizer, syringe, etc.

[00173] Methods of Treatment

[00174] The invention encompasses methods of preventing the progression of or treating cystic fibrosis through the use of one or more of the disclosed pharmaceutical compositions. The methods comprise administering a therapeutically effective amount of a GSNOR inhibitor in combination with one or more secondary agent(s) to a patient in need. The GSNOR inhibitor can be administered concurrently with, prior to, or subsequent to, one or more secondary active agents.

[00175] In one embodiment, the method is a method of treating or lessening the severity of cystic fibrosis in a patient by stabilizing the CFTR protein at the cell membrane, comprising the step of administering to said patient an effective amount of the pharmaceutical composition described herein.

[00176] In one embodiment, the method is a method of treating or lessening the severity of cystic fibrosis in a patient by increasing the amount of the CFTR protein at the plasma membrane for a prolonged period of time comprising the step of administering to said patient an effective amount of a GSNOR inhibitor pharmaceutical composition administered alone or with one or more secondary active agents. The GSNOR inhibitor of the pharmaceutical composition can be administered concurrently with, prior to, or subsequent to, one or more secondary active agents. In some embodiments the GSNOR inhibitor is a compound of Formula 1. In some embodiments, the GSNOR inhibitor is Compound 1.

[00177] The GSNOR inhibitor of the pharmaceutical composition of the invention used in the methods of treatment according to the invention can be a pharmaceutically acceptable salt, a stereoisomer, a prodrug, a metabolite, or an N-oxide thereof.

[00178] The methods of the present invention can be pharmaceutical compositions of the invention employed in combination therapies, that is, the pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired secondary active agents or medical procedures. The particular combination of therapies (secondary agents or procedures) to employ in a combination regimen will take into account compatibility of the desired agents and/or procedures and the desired therapeutic effect to be achieved.

[00179] The patient can be a human patient with any disease causing mutation of CF. In one embodiment, the patient has at least one copy of the F508del mutation. In one embodiment, the patient is a F508del-CFTR homozygous patient. In one embodiment the patient is a F508del-CFTR homozygous patient. As used herein, the terms patient and subject may be used interchangeably.

[00180] As used herein, "treating" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. More specifically, "treating" includes reversing, attenuating, alleviating, minimizing, suppressing, or halting at least one deleterious symptom or effect of a disease (disorder) state, disease progression, disease causative agent (e.g., bacteria or viruses), or other abnormal condition. Treatment is continued as long as symptoms and/or pathology ameliorate.

[00181] F. Uses

[00182] GSNOR inhibitors and compositions described herein are generally useful for the treatment of diseases wherein there is a need for increased NO bioactivity. GSNOR inhibitors of the invention, are active in a variety of assays and therapeutic models demonstrating selective and reversible inhibition of s-nitrosoglutathione reductase. Notably, GSNOR inhibitors of Formula 1 demonstrate efficacy in asthma, COPD, cystic fibrosis, and IBD models (disclosed in the international PCT Publication WO2012/048181 and PCT application PCT/US2015/054728). Accordingly, GSNOR inhibitors of Formula 1 are useful for treating one or more disorders associated with activity of GSNOR.

[00183] In subjects in which treatment with a GSNOR inhibitor is indicated, modulation of GSNOR may be achieved, for example, by administering one or more of the GSNOR inhibitors of the disclosed compositions that disrupts or down-regulates GSNOR function, or decreases GSNOR levels.

[00184] The present invention provides a method for treating or lessening the severity of a subject with cystic fibrosis.

[00185] In one embodiment the method of the invention is a method of treating a subject afflicted with any mutation of CF in any age group. Such a method comprises administering to a subject a therapeutically effective amount of a GSNOR inhibitor as a monotherapy or in combination with one or more secondary active agents.

[00186] In other embodiments, the subject of the invention is afflicted with at least one copy of the F508del mutation.

[00187] In other embodiments, the subject of the invention is afflicted with two copies of the F508del mutation.

[00188] Pharmaceutical compositions of the invention are capable of treating and/or slowing the progression of cystic fibrosis. For approximately 90% of patients with CF, death results from progressive respiratory failure associated with impaired mucus clearance and excessive overgrowth of bacteria and fungi in the airways (Gibson et al., 2003, Proesmans et al., 2008). GSNOR inhibitors are capable of preserving endogenous s-nitrosothiol (SNO) pools via inhibiting GSNO catabolism and therefore positively modulate CFTR. GSNOR inhibitors are also distinguished by their ability to demonstrate preservation of GSNO, potent bronchodilatory and anti-inflammatory effects in animal models of COPD (porcine pancreatic elastase) (Blonder et al., ATS 2011 abstract reference) and asthma. Pharmaceutical compositions of the invention are capable of treating and/or slowing the progression of CF. In this embodiment, appropriate amounts of compounds of the pharmaceutical compositions are an amount sufficient to treat and/or slow the progression of CF and can be determined without undue experimentation by preclinical and/or clinical trials.

[00189] The therapeutically effective amount for the treatment of a subject is the amount that causes amelioration of the disorder being treated or protects against a risk associated with the disorder. For cystic fibrosis, a therapeutically effective amount is an amount effective in reducing sweat chloride, improving or preventing the decline in lung function, decreasing the frequency of infective pulmonary exacerbations, improving nutritional status and body weight or improving overall symptoms.

[00190] In some embodiments, as a consequence of the increased amount of CFTR protein at the plasma membrane for a prolonged period of time, the therapeutically effective amount or dosing schedule of the GSNOR inhibitor and / or secondary active agents may be favorably altered to maximize any one or more of the positive effects listed above.

[00191] GSNOR inhibitors and compositions thereof, according to the method of the present invention, may be administered using any amount and any route of administration effective for treating or lessening the severity of the disorder or disease. The exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular mutation of the disease, the particular agent, its mode of administration, and the like. GSNOR inhibitors of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

[00192] Uses in an Apparatus [00193] The compositions of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer, prodrug, metabolite, or N-oxide thereof, can be applied to various apparatus in circumstances when the presence of such compounds would be beneficial. Such apparatus can be any device or container, for example, implantable devices in which a compound of the invention can be used to coat a surgical mesh or cardiovascular stent prior to implantation in a patient. The compounds of the invention can also be applied to various apparatus for in vitro assay purposes or for culturing cells.

[00194] The compounds of the compositions of the present invention or a

pharmaceutically acceptable salt thereof, or a stereoisomer, a prodrug, a metabolite, or an N- oxide thereof, can also be used as an agent for the development, isolation or purification of binding partners to compounds of the invention, such as antibodies, natural ligands, and the like. Those skilled in the art can readily determine related uses for the compounds of the present invention.

[00195] Method of Making Compound 1

[00196] Compound 1 is used as the starting point for the other solid state forms and can be prepared as was described in WO 2012/048181.

[00197] Compound 1 Form A was prepared as described in U.S. Provisional Application US 62/216,765, entitled "SOLID FORMS OF AN S -NITROS OGLUTATHIONE

REDUCTASE INHIBITOR", filed on September 10, 2015 by Jian Qiu, and in PCT application PCT/US2016/050974, filed September 9, 2016 with same name and inventor.

[00198] Compound 1 Form B was prepared as described in U.S. Provisional Application US 62/216,765, entitled "SOLID FORMS OF AN S -NITROS OGLUTATHIONE

[00199] Methods for Making the Pharmaceutical Compositions

[00200] In some embodiments, solid forms, including powders comprising the active agent Compound 1 and the included pharmaceutically acceptable excipients (e.g. filler,

disintegrant, glidant, lubricant, or any combination thereof) can be subjected to direct blending, meaning mixing followed by encapsulation occurs without compression, compaction, or liquid addition and is often used synonymously with dry blending. In some embodiments, the blend comprising the active agent Compound 1 and the included pharmaceutically acceptable excipients (e.g. filler, disintegrant, glidant, lubricant, or any combination thereof) can be encapsulated to provide capsules.

[00201] Formulations as described herein may be produced using one or more mixing and/or dry granulations steps. Dry granulation can be carried out by a mechanical process, which transfers energy to the mixture without any use of any liquid substances (neither in the form of aqueous solutions, solutions based on organic solutes, or mixtures thereof) in contrast to wet granulation processes, also contemplated herein. In some aspects, the mechanical process requires compaction such as the one provided by roller compaction. An example of an alternative method for dry granulation is slugging.

[00202] Brief Manufacturing Procedure

[00203] In some embodiments, the method for producing a pharmaceutical composition comprises providing an admixture of a solid forms, e.g. an admixture of powdered and/or liquid ingredients, the admixture comprising Compound 1 and one or more excipients selected from for example, a glidant, a lubricant, a disintegrant, and a filler; mixing the admixture until the admixture is substantially homogenous. In some embodiments, the admixture can then be compressed or compacted into a granular form. Then the granular composition comprising Compound 1 can be compressed into tablets or formulated into capsules as described above or in the Examples below. Pharmaceutical formulations, for example a capsule as described herein, can be made using the granules prepared

incorporating Compound 1 in addition to the selected excipients described herein.

[00204] In some embodiments, the admixture is mixed by stirring, blending, shaking, or the like using hand mixing, a mixer, a blender, any combination thereof, or the like. When ingredients or combinations of ingredients are added sequentially, mixing can occur between successive additions, continuously throughout the ingredient addition, after the addition of all of the ingredients or combinations of ingredients, or any combination thereof. The admixture is mixed until it has a substantially homogenous composition.

[00205] In some embodiments, the manufacturing of Compound 1 capsules involves three major unit operations: blending, roller compaction, and encapsulation.

[00206] In some embodiments, the manufacturing of Compound 1 capsules involves two major unit operations: direct blending and encapsulation.

[00207] In some instances, roller compactors such as the Freund Vector TF-Labo may be used. The blend may be roller compacted in ribbons and milled into granules using a Freund Vector TF-Labo (for example, Roller Pressure: 6 MPa, Roller Speed: 2 rpm, Feed Screw Speed: 40 rpm, Screen: 18 Mesh). The roller compacted granules may be blended with extra- granular excipients such as fillers and lubricant using a V-shell blender. The blending time may be up to 5 minutes, for example 5, 3 or 1 minute(s).

[00208] Compound 1 and excipients may be screened prior to or after weigh-out.

Appropriate screen sizes are mesh 14 to mesh 35. Compound 1 may be pre-blended with one or more of the excipients to simplify screening.

[00209] Blending: Compound 1 and excipients may be added to the blender in different order. The blending may be performed in a Turbula blender, bin blender or v-shell blender. The components may be blended for up to 52 minutes without lubricant followed by additional blending with lubricant for up to up to 5 minutes, for example 4, 2, or 1 minute(s).

[00210] Encapsulation: The powder blend may be encapsulated using either semi- automated or fully automated capsule filling machines. For example, the T.M.P. Millan MS3/6N, the Bosch GFK-705, or the Bosch GKF-1500.

[00211] Screening/Weighing: Compound 1 and excipients may be screened prior to or after weigh-out. Possible screen sizes are mesh 14, mesh 20, mesh 30, or mesh 35.

Compound 1 may be pre-blended with one or more of the excipients to simplify screening.

[00212] Dosage forms prepared as above can be subjected to in vitro dissolution evaluations according to Test 711 "Dissolution" in United States Pharmacopeia, United States Pharmacopeial Convention, Inc., Rockville, Md., ("USP"), to determine the rate at which the active substance is released from the dosage forms.

[00213] The content of active substance and the impurity levels are typically measured by techniques such as high performance liquid chromatography (HPLC).

[00214] In some embodiments, the invention includes use of packaging materials such as containers and closures of high-density polyethylene (HDPE), low-density polyethylene (LDPE) and or polypropylene and/or glass, glassine foil, aluminum pouches, and blisters or strips composed of aluminum or high-density polyvinyl chloride (PVC), optionally including a desiccant, polyethylene (PE), polyvinylidene dichloride (PVDC), PVC/PE/PVDC, PVC/Aclar and the like.

[00215] Due to the practical limitations of weighing, measuring, dispensing, blending, filling and analytical operations used in the preparation of the capsules of the present invention, the quantities of each of the various components of the final formulations can vary as much as 5 %. To reflect these possible variations, when referring to specific amounts of any given component in a dosage unit of the present invention (e.g., 100 mg of Compound 1) the term "about" is used herein. [00216] Methods for Administering the Pharmaceutical compositions

[00217] In one aspect, the pharmaceutical compositions of the invention can be administered to a patient once daily or about every twenty-four hours. Alternatively, the pharmaceutical compositions of the invention can be administered to a patient twice daily or about every twelve hours. These pharmaceutical compositions are administered as oral formulations in a unit dosage containing about 100 mg of Compound 1. In some instances, a dose of Compound 1 is a single capsule containing about 100 mg. In some instances, a dose of about 200 mg of Compound 1, may comprise two capsules of the invention each containing about 100 mg of Compound 1. In some instances, a dose of about 400 mg of Compound 1, may comprise four capsules of the invention each containing about 100 mg of Compound 1.

[00218] It will also be appreciated that the compound and pharmaceutically acceptable compositions and formulations of the invention can be employed in combination therapies; that is, Compound 1 and pharmaceutically acceptable compositions thereof can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, for example, cystic fibrosis, or condition, are known as "appropriate for the disease or condition being treated."

[00219] In one embodiment, the additional therapeutic agent is selected from one or more CFTR modulators other than Compound 1 of the invention, and one or more antiinflammatory agents.

[00220] Dosing Administration Methods / Schedule

[00221] In another aspect, the invention relates to a method of treating pulmonary disorders or inflammatory disorders in a subject comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition provided by the invention. In another aspect, the invention relates to a method of treating a CFTR mediated disease in a subject comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition provided by the invention. In another aspect, the invention relates to a method of treating cystic fibrosis in a subject comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition provided by the invention. In another aspect, the invention relates to a method of treating asthma in a subject comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition provided by the invention. In another aspect, the invention relates to a method of treating COPD in a subject comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition provided by the invention. In another aspect, the invention relates to a method of treating IBD in a subject comprising administering to a subject in need thereof an effective amount of the

pharmaceutical composition provided by the invention.

[00222] In another embodiment, the pharmaceutical composition is administered to the subject once a day. In another embodiment, the pharmaceutical composition is administered to the subject twice a day. In one embodiment, when the pharmaceutical composition is a capsule according to Tables 1-14, dosing is once a day. In another embodiment, when the pharmaceutical composition is a capsule according to Tables 1-14, dosing is twice a day. In another embodiment, more than one capsule is dosed at a time.

EXAMPLES

[00223] The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all references to a publicly available document, including a U.S. patent, are specifically incorporated by reference.

[00224] Exemplary Oral Pharmaceutical Formulations Comprising Compound 1

[00225] Method 1 - Method 4 described below were used to prepare example capsules of the invention.

[00226] Method 1; Process for preparation of Capsules of Examples 1-5 described below

[00227] 1. Manually screen all excipients and Compound 1 with through a 30 mesh screen after dispensing.

[00228] 2. Mix Compound 1 with Lactose, Microcrystalline Cellulose, PH102 or Dibasic calcium phosphate , and/or Croscarmellose sodium and hydroxypropyl cellulose (HPC), at the speed 47 rpm for 10 min on Turbula mixer.

[00229] 3. Add Magnesium Stearate (Intragranular) to Step 2) mixture and continue mixing for 2 min at the speed of 47 rpm.

[00230] 4. Roller compact the above mixture to form dry granule (Roller Pressure: 6 MPA, Roller Speed: 2 rpm, Feed Screw Speed: 40 rpm, Screen: 18 Mesh).

[00231] 5. Add Magnesium Stearate (Extragranular) to the dry granule and mix for 2 min at the speed of 47 rpm.

[00232] 6. Encapsulation using semi-automatic capsule filling machine, T.M.P. Millan MS3/6N into empty hypromellose capsules.

[00233] Method 2: Process for preparation of Capsules of Examples 6-13 described below.

[00234] 1) Manually screen all excipients and Compound 1 through a 30 mesh screen after dispensing.

[00235] 2) Mix Compound 1 with Lactose, Dibasic calcium phosphate , and Pregelatinized starch, at the speed 47 rpm for 10 min on Turbula mixer.

[00236] 3) Add Colloidal silicon dioxide to Step 2) mixture, and continue mixing for 6 min at the speed of 47 rpm.

[00237] 4) Add Magnesium Stearate to Step 3) mixture and mix for 2 min at the speed of 47 rpm.

[00238] 5) Encapsulation using semi-automatic capsule filling machine, T.M.P. Millan

MS3/6N, into empty hypromellose capsules.

[00239] Method 3: Additional method of capsule preparation

[00240] 1) Screen Compound 1 and all excipients through 35 Mesh after dispensing.

[00241] 2) Mix Compound 1 with Lactose and Dibasic calcium phosphate, Pregelatinized starch in a 10 L Bin for 25 min using a Bin Blender (20 rpm).

[00242] Add Colloidal silicon dioxide to Step 2) mixture and continue mixing for 15 min at the speed of 20 rpm.

[00243] Add Magnesium stearate to Step 3) mixture and continue mixing for 3 min at the speed of 20 rpm.

[00244] Encapsulation using automatic capsule filling machine, Bosch 705, into empty hypromellose capsules.

[00245] Method 4: Additional Method of capsule preparation suitable for larger scale

[00246] 1) Sequential addition of Lactose Monohydrate, Compound 1, Dibasic Calcium Phosphate, and Pregelatinized Starch, then screening all excipients through Mesh 14 after dispensing.

[00247] 2) Blend for 20 minutes at 25 rpm in a 5 cubic foot V-Shell Blender.

[00248] 3) Add Colloidal silicon dioxide (screened through 20 Mesh sieve) to Step 2) mixture

[00249] 4) Blend for 10 min at 25 rpm.

[00250] 5) Add Magnesium stearate (screened through 20 Mesh sieve) to Step 4) mixture

[00251] 6) Blend for 3 min at 25 rpm

[00252] 7) Encapsulation using automatic filling machine, Bosch GKF-1500 encapsulator, into empty hypromellose capsules

[00253] In Capsules 1-5 described in Examples 1-5 below, grades/brands of excipients were: Lactose Monohydrate: Fast Flo, Foremost; Microcrystalline Cellulose, PH102; Dibasic calcium phosphate : Anhydrous, Innophos; Croscarmellose Sodium: FMC, USA, Magnesium Stearate: Macron, USA; HPC (hydroxypropyl cellulose): Ashland; Colloidal Silicon Dioxide: Compendial, Evonic, Germany.

[00254] Capsules 1-5 had a total fill weight of 380 mg in a size 1 hypromellose capsule with a fill volume of about 0.5 ml.

[00255] In Capsules 6-13 described in Examples 6-13, grades/brands of excipients were: Lactose Monohydrate: Fast Flo, Foremost; Dibasic calcium phosphate (DCP): Anhydrous, Innophos; Pregelatinized starch: Colorcon; Colloidal Silicon Dioxide: Compendial, Evonic, Germany; Magnesium Stearate: Macron, USA.

[00256] Capsules 6-14 had a total fill weight of 300 mg in a size 1 hypromellose capsule with a fill volume of about 0.5 ml.

[00257] The capsules described in Examples 1-13 were prepared comprising 100 mg of API (i.e. Compound 1 Form A), per capsule. Because the hemi-hydrate form A was used, when 100 mg was weighed out, the amount of active Compound 1 was about 97 mg per capsule for Capsules 1-13.

[00258] Example 1: Capsule 1.

[00259] A capsule was prepared with the components and amounts listed in Table 1 and was prepared by Method 1.

[00260] Table 1: Capsule 1 Capsule 1 Components mg/unit % (w/w)

Compound 1, Form A 100.0 26.3

Lactose monohydrate 174.6 45.9

Microcrystalline Cellulose 75.0 19.7

Croscarmellose Sodium 3.8 1.0

HPC 22.8 6.0

Mg Stearate, intragranular 1.9 0.5

Mg Stearate, extragranular 1.9 0.5

[00261] Example 2: Capsule 2.

[00262] A capsule was prepared with the components and amounts listed in Table 2 and was prepared by Method 1.

[00263] Table 2: Capsule 2

[00264] Example 3: Capsule 3.

[00265] Capsule 3 was prepared with the components and amounts listed in Table 3 and was prepared by Method 1.

[00266] Table 3: Capsule 3

[00267] Example 4: Capsule 4.

[00268] Capsule 4 was prepared with the components and amounts listed in Table 4 and was prepared by Method 1. [00269] Table 4: Capsule 4

[00270] Example 5: Capsule 5.

[00271] Capsule 5 was prepared with the components and amounts listed in Table 5 and was prepared by Method 1.

[00272] Table 5: Capsule 5

[00273] Example 6: Capsule 6.

[00274] Capsule 6 was prepared with the components and amounts listed in Table 6 and was prepared by Method 2.

[00275] Table 6: Capsule 6

[00276] Example 7: Capsule 7. [00277] Capsule 7 was prepared with the components and amounts listed in Table 7 and was prepared by Method 2.

[00278] Table 7: Capsule 7

[00279] Example 8: Capsule 8.

[00280] Capsule 8 was prepared with the components and amounts listed in Table 8 and was prepared by Method 2.

[00281] Table 8: Capsule 8

[00282] Example 9: Capsule 9.

[00283] Capsule 9 was prepared with the components and amounts listed in Table 9 and was prepared by Method 2.

[00284] Table 9: Capsule 9

[00285] Example 10: Capsule 10. [00286] Capsule 10 was prepared with the components and amounts listed in Table 10 and was prepared by Method 2.

[00287] Table 10: Capsule 10

[00288] Example 11: Capsule 11.

[00289] Capsule 11 was prepared with the components and amounts listed in Table 10 and was prepared by Method 2.

[00290] Capsule 11 was also prepared following Method 3.

[00291] Table 11: Capsule 11

[00292] Example 12: Capsule 12.

[00293] Capsule 12 was prepared with the components and amounts listed in Table 12 and was prepared by Method 2.

[00294] Table 12: Capsule 12

[00295] Example 13: Capsule 13.

[00296] Capsule 13 was prepared with the components and amounts listed in Table 13 and was prepared by Method 2.

[00297] Table 13: Capsule 13

[00298] Example 14: Capsule 14.

[00299] Capsule 14 was prepared with the components and amounts listed in Table 11, however the amount of compound 1, form A that was weighed out was corrected for the hemi-hydrate form, to give a total amount of active compound of 100 mg. Because the amount of Compound 1, form A weighed out was greater than 100 mg, the amount of lactose monohydrate was adjusted to keep constant the total fill weight of 300 mg per capsule. Capsule 14 was prepared by Method 4.

[00300] Table 14: Capsule 14

* Compound 1, form A (the hemi-hydrate) was weighed out taking into account the water (i.e. an amount of greater than 100 mg of compound 1 form A is contained in each capsule, but which is equivalent to about 100 mg of active compound 1).

[00301] Example 15: Assays:

[00302] Compound 1 demonstrates efficacy in asthma, COPD, cystic fibrosis, and IBD models (described in the ' 181 application and in PCT/US2015/054728). [00303] Dissolution assay

[00304] In vitro dissolution testing is often used to guide the optimization of drug release from various formulations. This method characterizes how the active pharmaceutical ingredient is extracted out of a solid dosage form such as a capsule. The in vitro dissolution profile can indicate the efficiency of in vivo dissolution. A desired dissolution profile might be to have about 80% dissolution in 30 minutes. It is also desired for dissolution profiles to be similar to the dissolution profiles of lower dosage strength capsules.

[00305] Dissolution method for Tables 15 and 16: The dissolution procedure for capsules uses USP Apparatus 2 with sinkers in 0.05N Potassium Phosphate, pH 6.8 dissolution medium. Dissolution samples are analyzed with a reversed phase HPLC method using a Phenomenex Kinetex C18, 150 x 3.0 mm, 2.6 μιη column or equivalent. The mobile phase (isocratic elution) consists of 70% 0.1% FA in water and 30% acetonitrile (v/v). The flow rate is 0.4 mL/minute and the column temperature is maintained at 30°C. Detection is by UV at 268 nm.

[00306] Table 15 below includes the dissolution profiles of Capsules of Examples 6, 7, 8, and 9.

[00308] Table 16 below includes the dissolution profiles of Capsules of Examples 10, 11, 12, and 13.

[00309] Table 16: Dissolution profiles of Capsules of Examples 10-13.

[00310] Dissolution method for Table 17: The dissolution procedure for capsules uses USP Apparatus 2 with sinkers in 0.05N Potassium Phosphate, pH 6.8 dissolution medium, and 1% Tween 20 is added. Dissolution samples are analyzed with a reversed phase HPLC method using a Phenomenex Kinetex C18, 150 x 3.0 mm, 2.6 μιη column or equivalent. The mobile phase (isocratic elution) consists of 70% 0.1% FA in water and 30% acetonitrile (v/v). The flow rate is 0.5 mL/minute and the column temperature is maintained at 30°C. Detection is by UV at 268 nm.

[00311] Table 17 below includes the dissolution profiles of Capsules of Example 14.

[00312] Table 17: Dissolution results of capsules of Example 14

[00313] Example 16: Assessing CFTR Plasma Membrane (PM) stability with the addition of the GSNOR inhibitor 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

[00314] CFBE410- cells stably expressing HRP-tagged F508del-CFTR under the control of a tetracycline transactivator have been engineered as previously described (Phuan et al. Mol Pharmacol. 2014 Jul; 86(1):42-51). This cell model was well-characterized and has been described in detail (Veit G et al, Sci Transl Med. 2014 Jul 23; 6(246)). For optimal measurements of CFTR plasma membrane stability, CFBE41o- cells cells with induced expression of F508del-CFTR were grown for 3 days at 37°C followed by 48 h low- temperature rescue at 26°C. Cells were treated as described below. De novo protein synthesis was inhibited by treating with cycloheximide (CHX, 100 μg/ml) and CFTR plasma membrane removal was accelerated by chase at 37°C for 1.5 or 3 h. HRP-tagged CFTR PM density was measured in a VICTOR Light plate reader (PerkinElmer) and % CFTR PM remaining was calculated for cells treated.

[00315] In Figure 1, cells were treated with a CFTR corrector (VX-809 or VX-661, 3 μΜ, 24 h) alone or in the presence of a GSNORi (100 μΜ, 24 h). Cells treated with CFTR corrector (VX-809 or VX-661) + the GSNOR inhibitor 3-chloro-4-(6-hydroxyquinolin-2- yl)benzoic acid (GSNORi*) showed a significant increase in the amount of CFTR at the plasma membrane (plasma membrane CFTR expression) at 3h (37% and 77% respectively), demonstrating enhanced protein stability at the cell surface compared to CFTR corrector alone. Relative fluorescent units (RFU, Figure 1) was plotted for each treatment, as well as the % of CFTR remaining at the plasma membrane for VX-809 treated (Figure 2A), or VX- 661 treated cells (Figure 2B).

[00316] In Figure 3, cells treated with CFTR corrector (VX-809 or VX-661) + the CFTR potentiator (VX-770) showed a significant decrease in CFTR plasma membrane expression at 3h compared to corrector treatment alone, suggesting a negative effect of the combination (both the VX-809 + VX-770 combination and the VX661 and VX-770 combination) in vitro. However, when cells were treated with a CFTR corrector (VX-809 or VX-661) + potentiator (VX-770) in addition to the GSNOR inhibitor 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid, we observed a significant increase in the amount of CFTR at the plasma membrane over corrector + potentiator treatment alone at 3 h (61% and 56% respectively). This data supports the hypothesis of improved CFTR plasma membrane stability with the addition of GSNOR inhibitor treatment. Relative fluorescent units (RFU, Figure 3) was plotted for each treatment as well as the % of CFTR remaining at the PM for the VX-809 + VX-770 treatment groups (Figure 4A), and for the VX-661 + VX-770 treatment groups (Figure 4B). The PM densities (RFU) were determined by cell surface ELISA. The data represents the average and standard deviation from 3 separate experiments. Statistical comparison of values were performed using the student t-test.

[00317] Summary: F508del-CFTR plasma membrane turnover (up to 3 h) was

significantly reduced when CFBE41o- (expressing r 50S<ie/-CFTR-HRP) were treated with the GSNOR inhibitor 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid alone or when added to CFTR corrector (VX-809 or VX-661) or a corrector plus potentiator (VX-770), demonstrating increased F508del-CFTR plasma membrane stability.

Claims

We claim:

1. A formulation comprising

(a) about 30-37 % w/w of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound 1) or a pharmaceutically acceptable salt thereof;

(b) about 45-57% w/w of total filler;

(c) about 8-16% w/w of a disintegrant;

(d) about 0.5-3% w/w of a lubricant;

(e) about 0.7-1.5% w/w of a glidant.

2. The formulation according to claim 1, wherein compound 1 is included in the range of

31- 35%.

3. The formulation according to claim 1, wherein compound 1 is included in the range of

32- 34%.

4. The formulation according to claim 1, wherein a dosage unit of the formulation

includes about 95 to 100 mg of compound 1.

5. The formulation according to claim 1, wherein a dosage unit of the formulation

includes about 100 mg of compound 1.

6. The formulation according to claim 1, wherein the filler is selected from the group consisting of celluloses, sodium carboxymethyl cellulose, ethyl cellulose

hydroxymethyl cellulose, hydroxypropylcellulose, powdered cellulose, cellulose acetate, microcrystalline cellulose, calcium phosphates, dibasic calcium phosphate (DCP), starches, confectioner's sugar, compressible sugar, dextrates, dextrin, dextrose, sorbitol, mannitol, lactose, sucrose, and any combination thereof.

7. The formulation according to claim 1, wherein the formulation includes at least two fillers selected from the group consisting of celluloses, sodium carboxymethyl cellulose, ethyl cellulose hydroxymethyl cellulose, hydroxypropylcellulose, powdered cellulose, cellulose acetate, microcrystalline cellulose, calcium phosphates, dibasic calcium phosphate (DCP), starches, confectioner's sugar, compressible sugar, dextrates, dextrin, dextrose, sorbitol, mannitol, lactose, sucrose, and any combination thereof.

8. The formulation according to claim 1, wherein the disintegrant is selected from the group consisting of croscarmellose sodium, pregelatinized starch, sodium starch glycolate, and any combination thereof.

9. The formulation according to claim 1, wherein the lubricant is selected from the group consisting of magnesium stearate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, hydrogenated vegetable oil, and any combination thereof.

10. The formulation according to claim 1, wherein the glidant is selected from the group consisting of colloidal silicon dioxide, talc, and any combination thereof.

11. A process for preparing a pharmaceutical composition according to claim 1

comprising the steps of:

(a) screening 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound 1) or a pharmaceutically acceptable salt thereof, a filler, a disintegrant, a binder, and a glidant through an appropriate size mesh;

(b) mixing the 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound 1) or a pharmaceutically acceptable salt thereof, the filler, the disintegrant, the binder, and the glidant from step (a)

(c) lubricating the resulting mixture from step (b); and

(d) encapsulating the resulting mixture into a solid oral dosage form.

12. A capsule containing about 95 to 100 mg of compound 1 and in a formulation

containing a) about 30-37 % w/w of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (Compound 1) or a pharmaceutically acceptable salt thereof; b) about 45-57% w/w of a filler;

c) about 8-16% w/w of a disintegrant;

d) about 0.5-3% w/w of a lubricant;

e) about 0.7-1.5% w/w of a glidant.

13. A method of treating or lessening the severity of cystic fibrosis in a patient by administering a CFTR corrector and a CFTR potentiator, wherein the improvement comprises the administration of a GSNOR inhibitor that increases the amount of CFTR protein at the plasma membrane for a prolonged amount of time.

14. The method of claim 13 wherein the corrector is VX-809 and the potentiator is VX-

770.

The method of claim 13 wherein the corrector is VX-661 and the potentiator is VX-

770.

The method of claim 13, wherein the GSNOR inhibitor comprises compounds of

Formula 1 wherein m is selected from the group consisting of 0, 1, 2, or 3;

R₂b and R_2c are independently selected from the group consisting of hydrogen, halogen, C1-C3 alkyl, fluorinated C1-C3 alkyl, cyano, C1-C3 alkoxy, and N(CH₃)₂; X is selected from the group consisting of

n is selected from the group consisting of 0, 1, and 2;

R₃ is independently selected from the group consisting of halogen, Ci-C₃ alkyl, fluorinated Ci-C₃ alkyl, cyano, Ci-C₃ alkoxy, and NR₄R₄' where R₄ and R₄' are independently selected from the group consisting of Ci-C₃ alkyl, or R₄ when taken together with R₄' form a ring with 3 to 6 members; and

A is selected from the group consisting of

17. The method of claim 16 wherein

Ri is independently selected from the group consisting of chloro, fluoro, and bromo; X is selected from the group consisting of

R₂b and R_2c are independently selected from the group consisting of hydrogen, chloro, fluoro, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from the group consisting of 0 and 1; and R₃ is independently selected from the group consisting of fluoro, chloro, bromo, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂.

19. The method of claim 18 wherein X is

20. The method of claim 19 wherein A is COOH.

21. The method of claim 13, wherein the GSNOR inhibitor is selected from the group consisting of

4-(6-hydroxy-3-methylquinolin-2-yl)benzoic acid;

2-(4-(lH-tetrazol-5-yl)phenyl)-3-methylquinolin-6-ol;

4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-(lH-tetrazol-5-yl)phenyl)quinolin-6-ol;

1 -(6-hydroxyquinolin-2-yl)piperidine-4-carboxylic acid;

( lr,4r)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

(ls,4s)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

3- chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2-fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4-(2H-tetrazol-5-yl)phenyl)-4-chloroquinolin-6-ol;

4-(6-hydroxyquinolin-2-yl)phenyl)- 1 ,2,4-oxadiazol-5(2H)-one;

3-fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;

6-hydroxyquinolin-2-yl)thiophene-2-carboxylic acid;

6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylic acid;

3- fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4- chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

3-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

2- fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(4H)-one;

3- fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(4H)-one;

4- chloro-6-hydroxyquinolin-2-yl)benzoic acid;

2-chloro-4-(2H-tetrazol-5-yl)phenyl)quinolin-6-ol;

4-(6-hydroxyquinolin-2-yl)phenyl)-l,3,4-oxadiazol-2(3H)-one;

dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

6-hydroxyquinolin-2-yl)-3-methylbenzoic acid; 4-(3-chloro-6-hydroxyquinolin-2-yl)-3-fluorobenzoic acid;

3- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-thiadiazol-5(2H)-one;

4- (6-hydroxyquinolin-2-yl)-3-(trifluoromethyl)benzoic acid;

4- (6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)benzoic acid;

2- (4-carboxyphenyl)-6-hydroxyquinoline 1 -oxide;

5- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,3,4-thiadiazol-2(3H)-one;

5-(4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-3(2H)-one;

(lr,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

(ls,4s)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

3- chloro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

2- (5-(2H-tetrazol-5-yl)thiophen-2-yl)quinolin-6-ol;

5-(4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-thiadiazol-3(2H)-one;

3- fluoro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

1- (6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylic acid;

4- (5-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

(lr,4r)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylic acid; (ls,4s)-4-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)cyclohexanecarboxylic acid; 4-(5-bromo-6-hydroxyquinolin-2-yl)benzoic acid;

3- bromo-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4- (4-(dimethylamino)-6-hydroxyquinolin-2-yl)benzoic acid;

4-(4-fluoro-6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;

3- cyano-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-carboxy-2-chlorophenyl)-6-hydroxyquinoline 1 -oxide;

4- (3-cyano-6-hydroxyquinolin-2-yl)benzoic acid;

4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(8-fluoro-6-hydroxyquinolin-2-yl)benzoic acid; and

3- fluoro-4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid.

22. The method of Claim 21, wherein the GSNOR inhibitor comprises 3-chloro-4-(6- hydroxyquinolin-2- yl)benzoic acid .

23. The method of Claim 13, wherein the increase in CFTR protein at the plasma membrane is at least 20% over the treatment with corrector and potentiator alone at 3 hours post treatment.

24. A method of increasing the amount of CFTR protein at the plasma membrane for a prolonged period of time in a patient with cystic fibrosis, comprising administering a therapeutically effective amount of a GSNOR inhibitor.

25. The method of Claim 24 wherein the GSNOR inhibitor is administered in combination with a CFTR corrector.

26. The method of claim 25 wherein the CFTR corrector is selected from VX-809 and VX- 661.

27. The method of Claim 24 wherein the GSNOR inhibitor is administered in combination with a CFTR corrector and a CFTR potentiator.

28. The method of Claim 27, wherein the GSNOR inhibitor is administered in combination with the corrector VX-809 and the potentiator VX-770.

29. The method of Claim 27 wherein the GSNOR inhibitor is administered in combination with the corrector VX-661 and the potentiator VX-770.

30. The method of Claim 24 wherein the GSNOR inhibitor comprises compounds of Formula 1:

Formula 1 wherein m is selected from the group consisting of 0, 1, 2, or 3;

R₂b and R_2c are independently selected from the group consisting of hydrogen, halogen, C1-C3 alkyl, fluorinated C1-C3 alkyl, cyano, C1-C3 alkoxy, and N(CH3)₂;

X is selected from the group consisting of

n is selected from the group consisting of 0, 1, and 2;

R3 is independently selected from the group consisting of halogen, C1-C3 alkyl, fluorinated C1-C3 alkyl, cyano, C1-C3 alkoxy, and NR₄R₄' where R₄ and R₄' are independently selected from the group consisting of C1-C3 alkyl, or R₄ when taken together with R₄' form a ring with 3 to 6 members; and selected from the group consisting

Ύ O-NH ^U

31. The method of claim 30 wherein

32. The method of claim 30 wherein m is selected from the group consisting of 0 and 1; R₂b and R_2c are independently selected from the group consisting of hydrogen, chloro, fluoro, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂; n is selected from the group consisting of 0 and 1; and R₃ is independently selected from the group consisting of fluoro, chloro, bromo, methyl, trifluoromethyl, cyano, methoxy, and N(CH₃)₂.

33. The method of claim 32 wherein X is

34. The method of claim 33 wherein A is COOH.

35. The method of claim 30 wherein the GSNOR inhibitor is selected from the group consisting of

4-(6-hydroxy-3-methylquinolin-2-yl)benzoic acid;

2-(4-(lH-tetrazol-5-yl)phenyl)-3-methylquinolin-6-ol;

4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-(lH-tetrazol-5-yl)phenyl)quinolin-6-ol;

1 -(6-hydroxyquinolin-2-yl)piperidine-4-carboxylic acid;

( lr,4r)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

(ls,4s)-4-(6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

3- chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2-fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-(2H-tetrazol-5-yl)phenyl)-4-chloroquinolin-6-ol;

3- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(2H)-one; 3- fluoro-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4- (6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;

5- (6-hydroxyquinolin-2-yl)thiophene-2-carboxylic acid;

4-(6-hydroxyquinolin-2-yl)cyclohex-3-enecarboxylic acid;

4-(3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(4-chloro-3-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(3-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

3-(2-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(4H)-one;

3- (3-fluoro-4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-5(4H)-one;

4- (4-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

2- (2-chloro-4-(2H-tetrazol-5-yl)phenyl)quinolin-6-ol;

5- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,3,4-oxadiazol-2(3H)-one;

3- (dimethylamino)-4-(6-hydroxyquinolin-2-yl)benzoic acid;

4- (4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(6-hydroxyquinolin-2-yl)-3-methylbenzoic acid;

4-(3-chloro-6-hydroxyquinolin-2-yl)-3-fluorobenzoic acid;

3- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-thiadiazol-5(2H)-one;

4- (6-hydroxyquinolin-2-yl)-3-(trifluoromethyl)benzoic acid;

4- (6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)benzoic acid;

2- (4-carboxyphenyl)-6-hydroxyquinoline 1 -oxide;

5- (4-(6-hydroxyquinolin-2-yl)phenyl)-l,3,4-thiadiazol-2(3H)-one;

5-(4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-oxadiazol-3(2H)-one;

(lr,4r)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

(ls,4s)-4-(3-chloro-6-hydroxyquinolin-2-yl)cyclohexanecarboxylic acid;

3- chloro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

2- (5-(2H-tetrazol-5-yl)thiophen-2-yl)quinolin-6-ol;

5-(4-(6-hydroxyquinolin-2-yl)phenyl)-l,2,4-thiadiazol-3(2H)-one;

3- fluoro-4-(4-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

l-(6-hydroxy-3-(trifluoromethyl)quinolin-2-yl)piperidine-4-carboxylic acid;

4- (5-chloro-6-hydroxyquinolin-2-yl)benzoic acid;

3-bromo-4-(6-hydroxyquinolin-2-yl)benzoic acid; 4-(4-(dimethylamino)-6-hydroxyquinolin-2-yl)benzoic acid;

4-(4-fluoro-6-hydroxyquinolin-2-yl)-3-methoxybenzoic acid;

3- cyano-4-(6-hydroxyquinolin-2-yl)benzoic acid;

2- (4-carboxy-2-chlorophenyl)-6-hydroxyquinoline 1 -oxide;

4- (3-cyano-6-hydroxyquinolin-2-yl)benzoic acid;

4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid;

4-(8-fluoro-6-hydroxyquinolin-2-yl)benzoic acid; and

3- fluoro-4-(5-fluoro-6-hydroxyquinolin-2-yl)benzoic acid.

36. The method of Claim 35, wherein the GSNOR inhibitor comprises 3-chloro-4-(6- hydroxyquinolin-2- yl)benzoic acid .

37. The method of Claim 24 wherein the increase in the amount of CFTR protein at the plasma membrane is at least 10% at 3 hours.