WO2017062611A1 - Methods for the treatment of cystic fibrosis - Google Patents

Abstract

The present invention provides methods of treating or lessening the severity of cystic fibrosis in a patient, comprising the step of administering to said patient an effective amount of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (N91115, Cavosonstat).

Description

METHODS FOR THE TREATMENT OF CYSTIC FIBROSIS

RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Serial No. 62/238,478, filed October 7, 2015, entitled "Methods for the Treatment of Cystic Fibrosis" which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides methods of treating or lessening the severity of cystic fibrosis in a patient, comprising the step of administering to said patient an effective amount of 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (N91115, Cavosonstat).

BACKGROUND

[0003] 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. [0004] 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.

[0005] GSNO has been identified as a positive modulator of CFTR. GSNOR is the primary catabolizing enzyme of GSNO. Inhibition of GSNOR restores GSNO levels which improve F508del-CFTR function via nitrosation of chaperone proteins, prevention of CFTR proteosomal degradation, promotion of CFTR maturation, and maintainence of epithelial tight junctions.

[0006] 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)), as well as host defense (de Jesus-Berrios et al., Curr. Biol, 13: 1963-1968 (2003)). Collectively, data suggest GSNO as a primary physiological ligand for the GSNOR, which catabolizes GSNO and consequently reduces available GSNO and SNO's (Liu et al., (2001)), (Liu et al., Cell, 116(4), 617-628 (2004)), and (Que et al., Science, 308, (5728): 1618-1621 (2005)). Currently there is no curative treatment for CF; thus, there is a significant need for novel methods for treating or lessening the severity of cystic fibrosis in a patient and appropriate dose regimens for such treatments. The present invention satisfies these needs.

SUMMARY

[0007] The present invention relates to methods for treating patients with CF, 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 secretions, 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. The ultimate goal of CFTR modulator therapy is to maximize and maintain CFTR function, thereby restoring chloride transport. 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.

[0008] Preservation of GSNO, through inhibition of its primary catabolizing enzyme, 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 GSNO in pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis may occur via up-regulation of GSNOR enzyme activity. These 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.

[0009] The present invention provides methods for treating or lessening the severity of CF, comprising the step of administering to a patient in need an effective amount of 3-chloro- 4-(6-hydroxyquinolin-2-yl)benzoic acid (N91115). In one embodiment, N91115 is administered as a single agent CFTR modulator (monotherapy). In another embodiment, N91115 may be administered as a monotherapy in combination with one or more palliative care agents. In another embodiment, N91115 may be administered in combination with one or more secondary active agents. Administration of N91115 can occur concurrently with, prior to, or subsequent to, one or more secondary active agents and/or one or more palliative care agents. The invention encompasses pharmaceutically acceptable salts, stereoisomers, prodrugs, metabolites, and N-oxides of the described compound. Also encompassed by the invention are pharmaceutical compositions comprising N91115 and at least one

pharmaceutically acceptable carrier.

[0010] Also encompassed by the invention are pharmaceutical compositions comprising a) N91115 or a pharmaceutically acceptable salt thereof:

in an amount of about 200 mg to 1600 mg, and

b) a pharmaceutically acceptable carrier.

[0011] In another embodiment, the invention provides a therapeutic regimen for the treatment of CF comprising administering to a patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg to 1600 mg.

[0012] Also provided by this invention is a therapeutic regimen for the treatment of CF comprising administering to a patient N91115 or a pharmaceutically acceptable salt thereof: in an amount of about 200 mg to 800 mg, wherein the amount is administered twice per day.

[0013] Also provided by this invention is a therapeutic regimen for the treatment of CF patients comprising administering to a patient N91115 or a pharmaceutically acceptable salt thereof:

in an amount of about 400 mg to 1600 mg,

wherein the amount is administered daily.

[0014] The present invention also provides a method of treating or lessening the severity of cystic fibrosis in a patient by administering to the patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg or higher. In another embodiment, the method provides administering to the CF patient N91115 in an amount of about 200 mg or higher, administered twice per day. In another embodiment, the method provides

administering to the CF patient N91115 in an amount of about 400 mg or higher,

administered daily.

[0015] This invention also provides a method for treating a patient with cystic fibrosis by decreasing sweat chloride levels in a patient by administering to said patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg or higher. In another embodiment, the method includes administering N91115 in an amount of about 200 mg or higher, twice per day. In another embodiment, the method includes administering N91115 in an amount of about 400 mg or higher, once per day.

[0016] In other embodiments, this invention provides a method for treating a patient with cystic fibrosis by decreasing sweat chloride levels in a patient by administering to said patient N91115, wherein the sweat chloride is decreased by at least about 4mmol/L.

[0017] In other embodiments, this invention provides a method for administering N91115 to a cystic fibrosis patient, in an amount such that the sweat chloride is decreased by at least about 4 mmol/L.

[0018] The compositions of the present invention can be prepared in any suitable pharmaceutically acceptable dosage form.

[0019] 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.

[0020] 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 DRAWINGS

[0021] Figure 1: Demographic and Baseline Characteristics of the Patient Population dosed with 50 mg,100 mg, and 200 mg of N91115.

[0022] Figure 2: Adverse Event Summary for Patients dosed with 50 mg, 100 mg, and 200 mg of N91115.

[0023] Figure 3: S. Aureus Sputum Culture: Fold Change Shown (Day28 / Day 21).

[0024] Figure 4: P. Aeruginosa Sputum Culture: Fold Change Shown (Day28 / Day 21).

[0025] Figure 5A: Percent Predicted FEV1 (ppFEVl) is Shown as a Mean (95% CI)

Absolute Change from Baseline for Each Treatment Arm.

[0026] Figure 5B: Summary of Absolute and Relative Changes in ppFEVi.

[0027] Figure 6A: Selected Exploratory Endpoints are Summarized as Day 28 Change from Baseline.

[0028] Figure 6B: Waterfall Plots Depicting Change from Baseline in Sweat Chloride for Each Subject on Days 7, 14, 21, and 28.

[0029] Figure 6C: Within Group Mean Change (95% CI) from Baseline in Sweat Chloride at Day 28.

DETAILED DESCRIPTION [0030] A. Overview of the Invention

[0031] 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.

[0032] CF is a lethal genetic disease affecting approximately 70,000 people worldwide. 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 estimated 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. 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 CF 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.

[0033] Current therapies to treat CF lung disease include CFTR modulators that target the defective CFTR protein, and mucolytics, antibiotics, anti-inflammatory agents, anti-infectives and nutritional agents that target the downstream disease consequences that are secondary to the loss of CFTR function. Since the median predicted survival age is currently about 41 years, there is a large medical need for more efficacious therapies that address the underlying defect of CF and that would be used throughout the patient's life.

[0034] 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 cA P 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. Depending on the molecular consequence of the mutation and disease severity, CFTR stabilizers, such as N91115, amplifiers, activators, potentiators, and correctors may be coadministered to maximize clinical efficacy or therapeutic window, if needed.

[0035] There are many (>1000) different gene mutations for CF. Mutations affecting the CFTR gene cause a large variety of defects including altered CFTR channel gating (class III mutations such as G551D and G1349D) or impaired CFTR protein maturation (class II mutations such as F508del). Therefore, compounds increasing CFTR-dependent chloride transport are potentially useful as drugs to treat CF patients. In particular, pharmacological activators of CFTR, called potentiators, are useful to overcome the gating defect caused by class III CF mutations. Conversely, other compounds, called correctors, may help the F508del-CFTR protein to escape the endoplasmic reticulum and reach the plasma membrane. Potentiators are also useful for F508del. Indeed, this mutation causes also a gating defect, although less severe than that of classical class III mutations.

[0036] The most common mutation, F508del-CFTR (class II), results from a 3 base pair deletion that leads to the deletion of phenylalanine at position 508 of the full-length protein. The resulting F508del-CFTR protein is unstable and susceptible to rapid degradation in the 26S proteosome, with little if any F508del-CFTR at the plasma membrane. The F508del mutation is found in approximately 86% of all CF patients in the United States and Europe. In the F508del mutation, the deletion of an amino acid results in misfolded CFTR that is unstable and is targeted for degradation, which is facilitated by chaperone proteins. As a result, not enough CFTR reaches, or "traffics" to, the cell surface. F508del is therefore referred to as a trafficking mutation. In the United States, approximately 47% of CF patients are homozygous and have two copies of this mutation, and approximately 39% are heterozygous and have one copy.

[0037] In the lungs of CF patients, the lack of transport of chloride 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 F508del-CFTR that reaches the plasma membrane, or otherwise improving its function, offers the potential to improve the hydration of the airway surface fluid and reverse part of the underlying pathophysiology.

[0038] Inhibitors of S-nitrosoglutathione reductase (GSNOR), the primary catabolizing enzyme of S-nitrosoglutathione (GSNO), provide a novel therapeutic strategy in cystic fibrosis (CF). GSNO has been identified as a potential modulator of CFTR; 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, CFTR stabilization, increased cell surface CFTR, and increased CFTR function. Other effects demonstrated by GSNOR inhibitors and relevant to cystic fibrosis include potent bronchodilatory and anti-inflammatory effects in animal models of inflammatory lung and bowel disease. [0039] Increasing the amount of F508del-CFTR that reaches the plasma membrane, or otherwise improving its function, 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., 2012), prevention of CFTR proteosomal degradation, promotion of CFTR maturation, and maintenance of epithelial tight junctions.

[0040] 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, 2012).

[0041] The anti-inflammatory properties of GSNOR inhibition have been demonstrated in several in vitro and in vivo models (for example, WO2012/048181). 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 NFKB by nitrosation of NFKB regulatory proteins. GSNOR inhibition, therefore, offers a novel mechanism for targeting inflammatory pathways in CF.

[0042] The combination of improved F508del-CFTR and anti-inflammatory effects arising from GSNOR inhibition leads to clinical improvement in CF patients, which may be preceded by measurable changes in FEVi, sweat chloride, NPD, intestinal current measurements and inflammatory biomarkers in serum and airway secretions, sputum and/or bronchoalveolar lavage fluid (BALF). Other measurements of clinical improvement include the frequency of infective pulmonary exacerbations of CF, patient reported outcomes, and weight gain.

[0043] The present invention relates to the treatment of CF patients utilizing specific doses and dosage regimens for N91115. In some embodiments, the CF patients have at least one F508del-CFTR mutation. In some embodiments, the CF patients are homozygous for F508del-CFTR. In other embodiments the CF patients are heterozygous for F508del-CFTR. In still other embodiments, the CF patients have other mutations. N91115 (3-chloro-4-(6- hydroxyquinolin-2-yl)benzoic acid) is a highly potent, selective and reversible inhibitor of GSNOR (WO2012/048181).

[0044] N91115 was assessed for safety, pharmacokinetics and exploratory markers of activity in a randomized, controlled clinical study in CF patients homozygous for F508del- CFTR. The primary objective was to assess the safety of three different doses (50 mg, 100 mg, or 200 mg) of oral N91115 administered twice daily over 28 days in CF patients who were homozygous for the F508del-CFTR mutation. Secondary and exploratory objectives included the assessment of pharmacokinetics and the exploration of potential

pharmacodynamic biomarkers of GSNOR inhibition. N91115 was safe and well tolerated with no dose limiting toxicities and no significant safety findings across a comprehensive set of safety parameters (Example 1).

[0045] No statistically significant changes in spirometry parameters were observed during the study (Example 2). Compared to placebo, however, the percent predicted FEVi (ppFEVi) was numerically higher at the end of treatment with the 200 mg twice daily dose when mean and median relative and absolute changes were examined (Figures 5A and 5B).

[0046] Sweat chloride was assessed in patients as an exploratory marker of N91115's effect on CFTR function. A trend toward improvement in sweat chloride after 28 Days was observed in the 200 mg dose group (mean change compared with placebo of -5.2 mmol/L, 95% CI -11.7, 1.4; p = 0.11). A statistically significant within group change from baseline was observed after 28 Days in the 200 mg dose group (-4.1 mmol/L, 95% CI -6.5, -0.5; p = 0.032). The data suggest that 200 mg is the threshold dose for sweat chloride effects (Example 3). Furthermore, the magnitude of the effect on sweat chloride with N91115 was similar to that seen with combined correctors and potentiators in the F508del-CFTR homozygous population.

[0047] Accordingly, one embodiment of this invention provides pharmaceutical compositions comprising

a) N91115 or a pharmaceutically acceptable salt thereof: in an amount of about 200 mg to 1600 mg, and

b) a pharmaceutically acceptable carrier.

[0048] In a further embodiment, the pharmaceutical compositions comprise

a) N91115 or a pharmaceutically acceptable salt thereof:

in an amount of about 200 mg to 1200 mg,

in an amount of about 200 mg to 800 mg;

in an amount of about 200 mg to 400 mg;

in an amount of about 200 mg;

in an amount of about 300 mg; or

in an amount of about 400 mg; and

b) a pharmaceutically acceptable carrier.

[0049] N91115 is known to exist in different solid forms (PCT/US2016/050974). In one embodiment, Form A of N91115 is used in the pharmaceutical compositions of the invention. Form A is a free-form crystalline hemi-hydrate of N91115, with characteristic peaks in its X- ray powder diffraction pattern at 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, with all values + 0.2 degrees 2-theta.

[0050] In one embodiment, the pharmaceutical composition is administered twice per day (e.g., BID; Q12H). Alternatively, the pharmaceutical composition is administered once per day. Alternatively, the pharmaceutical composition is administered three-times per day (e.g., TID, Q8H).

[0051] In one embodiment, the pharmaceutical compositions of the invention are dosed orally.

[0052] In one embodiment, the pharmaceutical compositions of the invention include N91115 as a single agent CFTR modulator (i.e. N91115 administered without a secondary active agent).

[0053] In another embodiment, the invention provides a therapeutic regimen for the treatment of CF patients comprising administering to said patient N91115, or a

pharmaceutically acceptable salt thereof, in an amount of about 200 mg to 1600 mg.

[0054] Also provided by this invention is a therapeutic regimen for the treatment of CF patients comprising administering N91115 or a pharmaceutically acceptable salt thereof: in an amount of about 200 mg to 800 mg,

wherein the amount is administered twice per day.

The therapeutic regimens according to this invention are intended to include the

administration of N91115 in one or more dosage forms. [0055] In a further embodiment, the therapeutic regimen for the treatment of CF patients comprises administering N91115 or a pharmaceutically acceptable salt thereof:

in an amount of about 200 mg to 800 mg,

in an amount of about 200 mg to 600 mg,

in an amount of about 200 mg to 400 mg;

in an amount of about 200 mg;

in an amount of about 300 mg; or

in an amount of about 400 mg;

wherein the amount is administered twice per day.

[0056] Also provided by this invention is a therapeutic regimen for the treatment of CF patients comprising administering N91115 or a pharmaceutically acceptable salt thereof: in an amount of about 400 mg to 1600 mg,

wherein the amount is administered daily.

The therapeutic regimens according to this invention are intended to include the

administration of N91115 in one or more dosage forms.

[0057] In a further embodiment, the therapeutic regimen for the treatment of CF patients comprises administering N91115 or a pharmaceutically acceptable salt thereof:

in an amount of about 400 mg to 1600 mg,

in an amount of about 400 mg to 1200 mg,

in an amount of about 400 mg to 800 mg;

in an amount of about 400 mg;

in an amount of about 600 mg; or

in an amount of about 800 mg;

wherein the amount is administered daily.

[0058] Another embodiment of this invention provides a method of treating or lessening the severity of cystic fibrosis in a patient by administering to the patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg or higher. In another embodiment, the method provides administering to the CF patient N91115 in an amount of about 200 mg or higher, administered twice per day. In another embodiment, the method provides administering to the CF patient N91115 in an amount of about 400 mg or higher, administered daily.

[0059] In certain embodiments, the invention provides a method of treating or lessening the severity of cystic fibrosis in a patient by administering N91115 in an amount of about 200 mg to 800 mg, administered twice per day. In other embodiments, the dose of N91115 is about 200 mg to 600 mg, administered twice per day. In other embodiments, the dose of N91115 is about 200 mg to 400 mg, administered twice per day. In other embodiments, the dose of N91115 is about 200 mg, administered twice per day. In other embodiments, the dose of N91115 is about 300 mg administered twice per day. In other embodiments, the dose of N91115 is about 400 mg administered twice per day.

[0060] Another embodiment of this invention provides a method of treating or lessening the severity of cystic fibrosis in a patient by administering to the patient N91115, or a pharmaceutically acceptable salt thereof in an amount of about 400 mg or higher,

administered once per day. In certain embodiments, the dose of N91115 is in an amount of about 400 mg to 1600 mg, administered once per day. In other embodiments, the dose of N91115 is about 400 mg to 1200 mg, administered once per day. In other embodiments, the dose of N91115 is about 400 mg to 800 mg, administered once per day. In other

embodiments, the dose of N91115 is about 400 mg, administered once per day. In other embodiments, the dose of N91115 is about 800 mg administered once per day. In other embodiments, the dose of N91115 is about 600 mg administered once per day.

[0061] In some embodiments, the amount of N91115 is administered twice per day (e.g., BID; Q12H). Alternatively, the amount of N91115 is administered once per day.

Alternatively, the amount of N91115 is administered three-times per day (e.g., TID, Q8H). N91115 may be administered with or without food.

[0062] As it would be recognized, it is advantageous to have flexible dosing schedules. Accordingly, in another embodiment of this invention, the administration is twice daily, but not every 12 hours, optionally with meals.

[0063] N91115 has demonstrated improvement of sweat chloride levels when tested in CF patients at the 200 mg dose administered twice daily (Example 3). More patients in the 200 mg Q12H dose group had decreases in sweat chloride from their pretreatment values than in the other dose groups (placebo, 50 mg Q12H and 100 mg Q12H) (Figure 6A, Figure 6B, and Figure 6C). The data show that 200 mg is a threshold dose for the sweat chloride signal with placebo differences for the mean change of -5.2 mmol/L (95% CI -11.7, 1.4; p = 0.11) at 28 days. A statistically significant within group change from baseline was observed after 28 Days in the 200 mg dose group (-4.1 mmol/L, 95% CI -6.5, -0.5; p = 0.032).

[0064] Accordingly, this invention provides a method for treating a patient with cystic fibrosis by decreasing sweat chloride levels in a patient by administering to said patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg or higher. In another embodiment, the method includes administering N91115 in an amount of about 200 mg or higher, twice per day. In another embodiment, the method includes administering N91115 in an amount of about 400 mg or higher, once per day.

[0065] In another embodiment, this invention provides a method for treating a patient with cystic fibrosis by decreasing sweat chloride in a patient by administering to said patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of a) about 200 mg to 800 mg, twice per day, b) about 200 mg to 600 mg, twice per day, c) about 200 mg to 400 mg, twice per day, d) about 200 mg, twice per day, e) about 300 mg, twice per day, or f) about 400 mg, twice per day,

[0066] In another embodiment, this invention provides a method for treating a patient with cystic fibrosis by decreasing sweat chloride in a patient by administering to said patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of a) about 400 mg to 1600 mg, once per day, b) about 400 mg to 1200 mg, once per day, c) about 400 mg to 800 mg, once per day, d) about 400 mg, once per day, e) about 600 mg, once per day, or f) about 800 mg, once per day.

[0067] In other embodiments, this invention provides a method for treating a patient with cystic fibrosis by decreasing sweat chloride levels in a patient by administering to said patient N91115, wherein the sweat chloride is decreased by at least about 4mmol/L.

[0068] In other embodiments, this invention provides a method for administering N91115 to a cystic fibrosis patient, in an amount such that the sweat chloride is decreased by at least about 4 mmol/L.

[0069] In another embodiment, the invention provides a dosing regimen comprising administering N91115 or a pharmaceutically acceptable salt thereof to a cystic fibrosis patient in an amount such that the sweat chloride is decreased by at least about 4 mmol/L.

[0070] In one embodiment, the cystic fibrosis patients of the method have at least one copy of the F508del-CFTR mutation. In other embodiments, the cystic fibrosis patients of the method are homozygous for the F508del-CFTR mutation. In other embodiments, the cystic fibrosis patient of the method are heterozygous for the F508del-CFTR mutation. In other embodiments, the cystic fibrosis patients of the method have other CF mutations.

[0071] In another embodiment, the invention provides pharmaceutical compositions that are useful in treating or lessening the severity of cystic fibrosis in a patient by administering to said patient an effective amount of the GSNOR inhibitor N91115. The GSNOR inhibitor of the pharmaceutical composition can be administered as a single agent CFTR modulator, which can be administered concurrently with, prior to, or subsequent to other palliative care agents. Alternatively, the GSNOR inhibitor of the pharmaceutical composition can be administered concurrently with, prior to, or subsequent to, one or more secondary active agents, including other CFTR modulators, and/or with other palliative care agents.

[0072] B. Definitions

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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. [0079] As used herein, a "secondary active agent" is a compound or therapy that modulates CFTR function. 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.

[0080] As used herein, a "CFTR corrector" is a compound that promotes maturation and delivery of CFTR proteins to the apical surface.

[0081] As used herein, a "CFTR potentiator" is a compound that activates apical CFTR by increasing the open time of the channel.

[0082] As used herein, "gene therapy" is any therapy directed toward the genetic defect in CF.

[0083] As used herein a "CFTR amplifier" is any compound that increases CFTR activity.

[0084] As used herein, "monotherapy" is treatment with a GSNOR inhibitor as the sole modulator of CFTR, without a secondary active agent (i.e. without an agent that modulates CFTR function).

[0085] 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.

[0086] "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.

[0087] 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.

[0088] The phrase "therapeutically effective amount" as it relates to the secondary active agent of the present invention or the palliative care agent of the present invention shall mean the dosage that provides the specific pharmacological response for which the agent is administered in a significant number of subjects in need of such treatment.

[0089] C. Pharmaceutical Compositions

[0090] Pharmaceutical compositions of the invention include N91115 and at least one pharmaceutically acceptable carrier. In one embodiment, the N91115 pharmaceutical composition can be administered as a monotherapy. In another embodiment, the

pharmaceutical composition of the invention includes N91115 in combination with one or more secondary active agents. In another embodiment, the pharmaceutical composition of the invention includes N91115 in combination with one or more palliative care agents.

[0091] 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 or palliative care agents.

[0092] 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.

[0093] The compounds of the pharmaceutical compositions of the present invention 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).

[0094] In one embodiment, the secondary active agent(s) of the pharmaceutical combination is selected from a compound or therapy that modulates CFTR function. In one embodiment, the secondary active agent(s) is selected from CFTR correctors and/or CFTR potentiators. In one embodiment, the secondary active agent is selected from one or more CFTR amplifiers.

[0095] In one embodiment, N91115 may be used with any single or combination of palliative agents including mucolytic agents, bronchodilators, antibiotics, anti-infective agents, an anti-inflammatory agents, a nutritional agents, or other palliative agents known to manage CF.

[0096] 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.

[0097] Preferably, the pharmaceutically acceptable dosage form is administered orally. N91115 has been formulated and delivered orally via capsule (US 62/216,771,

PCT/US2016/051009).

[0098] It has been shown that N91115 can exist in several solid forms

(PCT/US2016/050974). In one embodiment, the pharmaceutical compositions of the invention are prepared with Form A of N91115. Form A is a free-form crystalline hemi- hydrate of N91115, with further characteristics described in PCT/US2016/050974.

[0099] 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. [00100] 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.

[00101] 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.

[00102] 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.

[00103] 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.

[00104] Pharmaceutical compositions according to the invention 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.

[00105] D. Kits Comprising the Compositions of the Invention

[00106] 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.

[00107] E. Methods of Treatment

[00108] The invention encompasses methods of preventing progression or treating cystic fibrosis through use of one or more of the disclosed pharmaceutical compositions. The methods include administering a therapeutically effective amount of N91115 as a

monotherapy in the treatment of cystic fibrosis. The monotherapy can be administered with one or more palliative agents.

[00109] The methods also comprise administering a therapeutically effective amount of N91115 in combination with one or more secondary agent(s) to a patient in need. N91115 can be administered concurrently with, prior to, or subsequent to, one or more secondary active agents.

[00110] In one embodiment, the method is a method of treating or lessening the severity of cystic fibrosis in a patient, comprising the step of administering to said patient an effective amount of the pharmaceutical composition described herein.

[00111] The compound N91115 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.

[00112] 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.

[00113] The patient can be a human patient with any disease causing mutation of CF, whether homozygous or heterozygous. As used herein, the terms patient and subject may be used interchangeably.

[00114] 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 progression of the symptoms or

complications, alleviate the symptoms or complications, or eliminate the disease, condition, or disorder. More specifically, "treating" includes reducing sweat chloride levels, 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.

[00115] F. Uses

[00116] 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.

[00117] The present invention provides 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.

[00118] 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 may positively modulate CFTR.

GSNOR inhibitors are 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) 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.

[00119] 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. EXAMPLES

[00120] 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 U.S. patents and applications, are specifically incorporated by reference.

[00121] Example 1: Safety

[00122] N91115 was examined as a single CFTR modulator in a randomized double-blind, placebo-controlled, parallel group, multicenter study in which patients were assigned to one of three doses of N91115 or placebo. Patients remained on their background regimen of palliative care agents. N91115 was dosed twice daily for 28 days and withdrawn during a two week follow up phase. N91115 was administered orally as 50 mg capsules in a sufficient amount for each of the three doses (US 62/216,771, PCT/US2016/051009, US 62/216,765, and PCT/US2016/050974).

[00123] The capsules were prepared with the components and amounts listed below comprising 50 mg of N91 1 15. The capsule was prepared using a direct blend formulation process, followed by encapsulation into empty hypromellose capsules using automated equipment, in Table 1 below, grades/brands were Lactose ono hydrate: NF, EP, JP: Mod. Spray Dry Fast Flo; Dibasic Calcium Phosphate: USP, EP; Pregelatinized Starch: NF, EP (1500 Partially Pregelatinized Maize Starch 2001 ); Magnesium Stearate: NF, EP, HyQual VG; Talc, USP, BP,EPJP; Colloidal Silicon Dioxide : NF, EP, Aerosil 200.

Table 1, Capsule formulation.

[00124] A total of 51 patients homozygous for F508del-CFTR were enrolled among the 4 treatment groups. Patients were enrolled with equal randomization to placebo or N91115 at doses of 50 mg, 100 mg, or 200 mg administered Q12H for 28 days. Demographic and baseline characteristics are shown in Figure 1. Safety assessments were based on clinical evaluations, laboratory tests, adverse events, spirometry, and 12-lead ECGs. Additional safety oversight was provided by an independent Data Monitoring Committee (IDMC).

Secondary endpoints included pharmacokinetic parameters of N91115 and its primary metabolite. Exploratory markers of effect included sweat chloride, systemic biomarkers of inflammation, body weight, patient reported outcomes using quality of life questionnaires.

[00125] As determined by the IDMC, no dose-limiting toxicities were observed during the study. The frequency of adverse events was similar among the treatment groups (Figure 2). There were 3 treatment emergent serious adverse events (infective pulmonary exacerbations of CF or PEs), 1 in the placebo group, and 1 each in the N91115 100 and 200 mg dose groups. The PE in the patient on placebo resulted in study drug discontinuation. No PEs, serious or non-serious, occurred in the 200 mg dose group during the treatment phase, however, 3 patients in that dose group experienced a PE (1 serious, 2 non-serious) during the follow-up phase.

[00126] Results: There were no adverse effects on safety parameters including 12-lead ECGs, laboratory chemistry and hematology parameters (including liver function tests: white blood cell counts and absolute neutrophil counts), sputum microbiology, or spirometry. There were no significant effects on these safety parameters measured at Baseline and predose on Day 1, 7, 14, 21, and 28. Sputum cultures in patients who could spontaneously produce sputum were done at Baseline and on Day 28 (Figures 3 and 4).

[00127] Sputum for microbiology testing was collected predose on Days 1 and 28 and was evaluated for potential treatment related effects. Sputum samples were obtained from expectorated sputum in those patients who were able to produce an adequate quantity. Ability to produce sputum was not a requirement for this study. More patients on N91115 had decreases in their S. Aureas and P. Aeruginosa levels at the 200 mg dose than on placebo. S. Aureus and Pseudomonas Aeruginosa quantitative sputum culture results are shown grouped by treatment arm for each patient; day 28 fold change from baseline are reported (Figure 3, S. Aureus Sputum Culture) and (Figure 4, P. Aeruginosa Sputum Culture).

[00128] Example 2: Spirometry

[00129] For the study detailed in Example 1, spirometry was performed at screening; predose on Days 1, 7, 14, 21, and 28; and on Day 42. Spirometry was performed according to the current ATS/ERS Task Force guidelines (Miller, 2005). The spirometer used to assess lung function was calibrated prior to procedures by study staff on all relevant study days. Subjects were allowed to continue their usual dosing schedule for long-acting and short- acting β- agonists and anticholinergics. In order to standardize spirometry for patients on short-acting β- agonists, all spirometry done at screening, predose on each dosing day when in the clinic and on Day 42 was performed 10-15 minutes after receiving 2 puffs of inhaled albuterol.

[00130] Results: Figure 5A shows Percent Predicted FEVi (ppFEVi) as a mean (95% CI) absolute change from baseline for each treatment arm. Figure 5B shows a summary of absolute and relative changes in ppFEVi. No statistically significant changes in spirometry parameters were observed during the study. Compared to placebo, however, the percent predicted FEVi (ppFEVi) was numerically higher at the end of treatment with the 200 mg twice daily dose when mean and median relative and absolute changes were examined (Figures 5A and 5B).

[00131] Example 3: Sweat Chloride

[00132] Sweat chloride was examined in CF patients homozygous for F508del-CFTR as a marker of CFTR modulation (trial description in Example 1). Methods: A study qualifying sweat chloride test was done during the screening period for any patient who did not have a previously documented sweat test > 60 mEq/L to confirm the diagnosis of CF. Baseline sweat chloride was then assessed within 48 hours prior to dosing on Day 1. Follow-up sweat chloride tests were done within 2 hours postdose on Days 7, 14, 21 and Day 28, and at the follow-up visit on Day 42. Sweat chloride collection in this study was performed at the site according to the Cystic Fibrosis Foundation's Therapeutic Development Network's (TDN) SOPs. The Macroduct^® collection system (Wescor, Logan UT) was used. Pretreatment or "Baseline" and post-treatment values and change from baseline at each time point were summarized.

[00133] Results: Sweat testing was performed at Baseline; Days 7, 14, 21, and 28; and the follow up visit (Day 42). Baseline and follow-up sweat chloride data were available for 47 patients. Data is summarized in Figure 6A, which shows sweat chloride change from baseline and response versus placebo (among other exploratory endpoints). As illustrated in the waterfall plots of Figure 6B, more patients in the 200 mg Q12H dose group had decreases in sweat chloride from Baseline than in the other dose groups (Figure 6B). The mean changes from Baseline on Day 28 are summarized in Figure 6C. The data show that 200 mg is a threshold dose for the sweat chloride signal with placebo differences for the mean change of - 5.2 mmol/L (95% CI -11.7, 1.4; p = 0.11) at 28 days. A statistically significant within group change from baseline was observed after 28 Days in the 200 mg dose group (-4.1 mmol/L, 95% CI -6.5, -0.5; p = 0.032).

Claims

Claims

1. A method for treating or lessening the severity of cystic fibrosis in a patient by

administering to the patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg or greater.

2. The method according to claim 1, wherein N91115, or a pharmaceutically acceptable salt thereof, is in an amount of about 200 mg.

3. The method according to claim 1, wherein N91115, or a pharmaceutically acceptable salt thereof, is in an amount of about 400 mg.

4. The method according to any one of claim 1, 2 or 3 wherein N91115 is administered twice per day.

5. The method according to any one of claim 1, 2, 3 or 4 wherein N91115 is

administered as a monotherapy.

6. A method for treating a patient with cystic fibrosis by decreasing sweat chloride levels in a patient by administering to said patient N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200 mg or higher.

7. The method of claim 6 wherein N91115 is administered twice a day.

8. The method of claim 7 wherein N91115 is administered in an amount of a) about 200 mg to 800 mg,

b) about 200 mg to 600 mg,

c) about 200 mg to 400 mg,

d) about 200 mg,

e) about 300 mg, or

f) about 400 mg.

9. The method of claim 6 wherein N91115 is administered once a day.

10. The method of claim 9 wherein N91115 is administered in an amount of a) about 400 mg to 1600 mg,

b) about 400 mg to 1200 mg,

c) about 400 mg to 800 mg,

d) about 400 mg,

e) about 600 mg, or

f) about 800 mg.

11. A method for treating a patient with cystic fibrosis by decreasing sweat chloride

levels in a patient by administering to said patient N91115, wherein the sweat chloride is decreased by at least about 4mmol/L.

12. A therapeutic regimen for the treatment of CF patients, comprising administering N91115, or a pharmaceutically acceptable salt thereof, in an amount of about 200mg to 1600mg.

13. The therapeutic regimen according to claim 12, wherein N91115 or a

pharmaceutically acceptable salt thereof, is:

in an amount of about 200 mg to 800 mg,

wherein the amount is administered twice per day.

14. The therapeutic regimen according to claim 13, wherein N91115 or a

pharmaceutically acceptable salt thereof, is:

in an amount of about 200 mg to 600 mg;

in an amount of about 200 mg to 400 mg;

in an amount of about 200 mg;

in an amount of about 300 mg; or

in an amount of about 400 mg;

wherein the amount is administered twice per day.

15. The therapeutic regimen according to claim 12, wherein N91115 or a

pharmaceutically acceptable salt thereof, is: in an amount of about 400 mg to 1600 mg,

wherein the amount is administered daily.

16. The therapeutic regimen according to claim 15, wherein the N91115, or a

pharmaceutically acceptable salt thereof, is:

in an amount of about 400 mg to 1600 mg;

in an amount of about 400 mg to 1200 mg;

in an amount of about 400 mg to 800 mg;

in an amount of about 400 mg;

in an amount of about 600 mg; or

in an amount of about 800 mg;

wherein the amount is administered once per day.

17. The therapeutic regimen according to claim 12, 13, 14, 15, 16, wherein N91115 is administered as a monotherapy.