Solid forms of n-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide

[0001] The present invention relates to solid forms, for example the crystalline forms of N- (4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- ( trifluoromethyl) -1,4-dihydroquinoline-3-carboxamide, which is a modulator of transmembrane conductivity regulator ("CFTR"). The invention also relates to pharmaceutical compositions containing the crystalline forms of N- (4- (7-azabicyclo [2.2.1] heptan-7yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (difluoromethyl) -1,4-dihydroquinoline -3-carboxamide, and methods related thereto.

BACKGROUND OF THE INVENTION [0002] Transporters having an ATP binding cassette are a family of membrane transporter proteins that regulate the transport of many different pharmacological agents, potentially toxic drugs and xenobiotics, as well as anions. They are homologous membrane proteins that bind and use cellular adenosine triphosphate (ATP) for their specific activities. Some of these transporters have been discovered as multi-drug resistance proteins (such as MDR1-P glycoprotein or multi-drug resistance protein, MRP1) that protect malignant tumor cells from chemotherapeutic agents. So far, 48 such transporters have been identified and grouped into 7 families depending on their similarity and function.

[0003] One member of the transporter family having an ATP binding cassette often associated with disease is the cAMP / ATP regulated anion channel, CFTR. CFTR is expressed in a wide variety of cell types, including lymphatic and secretory epithelial cells, where it regulates the flow of anions across the membrane, as well as the activity of other ion channels and proteins. In epithelial cells, normally CFTR functioning is critical to maintaining electrolyte transport in the body, including respiratory and digestive tissue. CFTR consists of about 1480 amino acids encoding a protein composed of tandem repeats of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are connected by a large, polar, regulatory (R) -domain with multiple phosphorylation sites that regulates channel activity and its movement in the cell.

[0004] The CFTR coding gene has been identified and sequenced (see, Gregory, RJ et al. (1990) Nature 347: 382-386; Rich, DP et al. (1990) Nature 347: 358-362), Riordan, JR et al. . (1989) Science 245: 1056-1073). A defect in this gene causes mutations in CFTR causing cystic fibrosis ("CF"), the most common lethal genetic disease in humans. About one in every 2,500 children in the United States is affected by cystic fibrosis. In the general US population, up to 10 million people carry a single copy of the defective gene with no apparent disease effects. In contrast, individuals with two copies of the CF associated gene suffer from the debilitating and lethal effects of CF, including chronic lung disease.

[0005] In patients with cystic fibrosis, mutations in CFTR expressed endogenously in respiratory epithelia lead to a reduced peak anion secretion resulting in an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to increased mucus accumulation in the lungs and associated microbial infections that eventually cause CF patients to die. Additionally, in addition to respiratory disease, patients with CF usually have gastrointestinal problems and pancreatic failure, which, if left untreated, leads to death. In addition, most men with cystic fibrosis are infertile and fertility is reduced in women with cystic fibrosis. Unlike the severe effects of two copies of the CF-related gene, individuals with a single copy of the CF-associated gene show increased resistance to cholera and dehydration due to diarrhea - which probably explains the relatively high frequency of the CF gene in the population.

[0006] CFTR gene sequence analysis on CF chromosomes revealed many disease-causing mutations (Cutting, GR et al (1990) Nature 346: 366-369; Dean, M. et al (1990) Cell 61: 863: 870; and Kerern, BS. Et al (1989) Science 245: 1073-1080; Kerem, BS et al (1990) Proc. Natl. Acad. Sci. USA 87: 8447-8451). To date, more than 1,000 disease-causing mutations have been identified in the CF gene (<a href="http://www.genet.sickkids.on.ca/cftr/">http://www.genet.sickkids.on.ca/cftr/</a>). The most common mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence and is often called ΔF508-CFTR. This mutation occurs in about 70 percent of cases of cystic fibrosis and is associated with severe disease.

[0007] Deletion of residue 508 in ΔF508-CFTR prevents the correct folding of the resulting protein. This causes the inability of the mutated protein to exit the ER and move to the plasma membrane. As a result, the number of channels present in the membrane is much lower than that observed in cells expressing wild-type CFTR. In addition to the movement disorder, this mutation causes channel gating disorder. In total, the reduced number of channels in the membrane and its abnormal gating lead to reduced anion transport across the epithelium, resulting in abnormal ion and fluid transport. (Quinton, PM (1990), FASEB J. 4: 27092727). However, studies have shown that the reduced number of ΔF508-CFTR in the membrane is functional, but less than wild-type CFTR. (Dolmans et al. (1991), Nature Lond. 354: 526-528; Denning et al., Supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to ΔF508-CFTR, R117H-CFTR and G551D-CFTR, other disease-causing CFTR mutations that cause disruption of movement, synthesis and / or gating of the channel can be increased or decreased to alter anion secretion and modify disease progression and / or severity.

[0008] Although CFTR transports many molecules in addition to anions, it is obvious that this role (transport of anions, chloride and bicarbonate) is one element in the important mechanism of ion and water transport across the epithelium. Other elements include epithelial Na channel⁺, ENaC, co-transporter Na⁺/ 2Cl^-/ K⁺, Na pump⁺-K⁺-ATPazowa and K channels⁺ laterol basal membranes, which are responsible for the uptake of chloride into the cell.

[0009] These elements work together to achieve directional transport across the epithelium by their selective expression and location in the cell. Chloride absorption occurs through the coordinated activity of ENaC and CFTR present on the apical membrane and the Na pump⁺-K⁺-ATPasic and C1 channels expressed on the laterolateral surface. Secondary active transport of chloride from the lumen side of the duct leads to an intracellular accumulation of chloride, which can then passively leave the cell through Cl ion channels^-, causing vector transport. Arrangement of the co-transporter Na⁺/ 2Cl^-K⁺, Na pumps⁺-K⁺-ATPazowa and K channels⁺ laterol basal membrane on the laterol basal surface and CFTR on the lumen side of the duct coordinates the secretion of chloride by CFTR on the lumen side of the duct. Since it is likely that water is never actively transported by itself, its flow through the epithelium depends on small osmotic gradients through the epithelium produced by the high flow of sodium and chloride ions.

[0010] The abnormal bicarbonate transport resulting from the CFTR mutation is believed to cause defects in some secretory functions. See, e.g., "Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis," Paul M. Quinton, Lancet 2008; 372: 415-417.

[0011] Mutations in CFTR, which are associated with moderate CFTR dysfunction, are also observed in patients with conditions that have some CF disease symptoms, but they do not meet the CF diagnostic criteria. These include congenital bilateral vas deferens, idiopathic chronic pancreatitis, chronic bronchitis and chronic rhinitis and sinusitis. Other diseases in which the mutated CFTR is considered a risk factor, including modifying genes or environmental factors, include primary sclerosing cholangitis, allergic bronchopulmonary fungal fungal disease, and asthma.

[0012] Cigarette smoke, hypoxia and environmental factors that trigger hypoxia signal transmission have also been shown to interfere with CFTRs and may contribute to some form of respiratory disease such as chronic bronchitis. Diseases that may result from CFTR malfunction but do not meet the CF diagnostic criteria are characterized as CFTR-related diseases.

[0013] In addition to cystic fibrosis, modulation of CFTR activity may be beneficial in other diseases that are not directly caused by mutations in CFTR, such as secretory diseases and diseases associated with the folding of other CFTR-mediated proteins. CFTR regulates chloride and bicarbonate flow through epithelials from many cells by controlling fluid flow, protein dissolution, mucus viscosity and enzymatic activity. CFTRs can cause blockage of the airways or ducts in many organs, including the liver and pancreas. Potentiators are compounds that increase the gating activity of CFTR present in the cell membrane. A candidate for treatment with tonic substances can be any disease that includes thickening of the mucus, fluid regulation disorder, mucus clearance disorder, or blockage of the ducts leading to inflammation and tissue destruction.

[0014] These include, but are not limited to, chronic obstructive pulmonary disease (COPD), asthma, tobacco-induced COPD, chronic bronchitis, rhinitis and sinusitis, constipation, dry eye disease and Sjogren's syndrome, gastroesophageal disease reflux, gallstones, prolapse of the rectum and inflammatory bowel disease. COPD is characterized by a limited airflow that progresses and is not fully reversible. The restriction of airflow is caused by excessive mucus secretion, emphysema and bronchiolitis. Mutant or wild-type CFTR activators offer the possibility of treating excessive mucus secretion and disturbed mucociliary clearance, which often occurs in COPD. In particular, increasing the anion secretion by CFTR may facilitate fluid transport to liquids on the surface of the airways to hydrate mucus and optimize the viscosity of periciliary fluid. This would lead to increased mucociliary clearance and reduction of COPD related symptoms. In addition, by preventing ongoing infection and inflammation through improved airway cleansing, CFTR modulators can counteract or slow down the destruction of airway parenchyma that characterizes emphysema, and reduce or reverse the increase in the number and size of mucus secreting cells that underlies mucus hypersecretion. respiratory diseases. Dry eye disease is characterized by a decrease in the production of watery tear fluid and abnormal lipid, protein and mucin profiles in the tear film. There are many causes of dry eye, some of which include age, Lasik eye surgery, arthritis, medications, chemical / thermal burns, allergies, and diseases such as cystic fibrosis and Sjogren's syndrome. An increase in anion secretion by CFTR would affect fluid transport from corneal endothelial cells and from secretory glands surrounding the eye as the corneal hydration increases. This would help alleviate the symptoms associated with dry eye disease. Sjogren's syndrome is an autoimmune disease in which the immune system attacks the glands that produce moisture in the body, including the eye, mouth, skin, respiratory tissue, liver, vagina and intestine. Symptoms include dry eye, mouth and vagina, as well as lung disease. This disease is also associated with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis and polymyositis / dermatomyositis. It is thought that this disease, whose treatment options are limited, causes abnormal protein movement. Modulators of CFTR activity can hydrate the various organs affected and can help alleviate the associated symptoms. Individuals with cystic fibrosis suffer from recurrent episodes of intestinal obstruction and a higher incidence of prolapse of the rectum, gallstones, gastroesophageal reflux disease, gastrointestinal malignancies and inflammatory bowel disease, which indicates that CFTR function may play an important role in preventing such diseases.

[0015] As discussed above, deletion of residue 508 in ΔF508-CFTR is believed to prevent normal folding of the resulting protein, resulting in the inability of the mutated protein to exit the ER and move to the plasma membrane. As a result, an insufficient number of mature proteins is present on the plasma membrane and chloride transport in epithelial tissues is significantly reduced. In fact, it has been shown that the cellular phenomenon of abnormal CFTR processing in ER by ER machinery underlies not only CF disease, but a wide range of other isolated or hereditary diseases. Two ways in which ERs may malfunction are either loss of ER protein conjugation leading to their degradation, or ER accumulation of these damaged / incorrectly folded proteins [Aridor M, et al., Nature Med., 5 (7), p. . 745-751 (1999); Shastry, BS, et al., Neurochem. International, 43, pp. 1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, pp. 211-222 (2002); Morello, JP et al., TIPS, 21, pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198 (1999)]. Diseases associated with the first class of ER malfunction are cystic fibrosis (due to improperly folded ΔF508-CFTR, as discussed above), hereditary emphysema (induced by a1-antitrypsin; non Piz variants), hereditary hemochromatosis, clotting-fibrinolysis deficiencies such as protein C deficiency, hereditary Type 1 angioedema, lipid disorders such as familial hypercholesterolemia, type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as intrinsic storage disease pseudo-Hurler, mucopolysaccharidosis (caused by lysosomal processing enzymes, Sandhof / Tay-Sachs disease (caused by β-hexosaminidase), Crigler-Najiar syndrome type II (induced by UDPglucuronyl-sialyl transferase), poliendocrinopathy / hyperinsulinemia, diabetes mellitus (induced by insulin receptor), Laron's dwarfism (induced by growth hormone receptor), myeloperoxidase deficiency, primary hypoparathyroidism (prepro-parathoma induced) due to tyrosinase). Diseases associated with the second class of ER malfunction are type 1 CDG glycanosis, hereditary emphysema (induced by a1-Antitrypsin (PiZ Variant)), congenital hyperthyroidism, congenital bone fragility (induced by Type I, II, IV procollagen), hereditary hypofibrinogenemia (caused by fibrinogen) ), ACT deficiency (induced by α1-antichymotrypsin), diabetes insipidus (DI), pituitary DI (induced by vasopressin / V2 receptor), renal DI (induced by aquaporin II), Charcot-Mari-Tooth syndrome (caused by peripheral myelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease (caused by eAPP and presenilins), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick disease, various polyglutamine neurological disorders, such as Huntington's disease, type I cerebellar ataxia, cerebrospinal muscular atrophy, atrophy of the dentate nucleus, red nucleus, pale knob and hypothalamic nucleus and myotonic dystrophy, as well as spongiform encephalopathies such as hereditary Creutzfeldt-Jakob disease (caused by prion protein treatment defect), Fabry disease (caused by lysosomal α-galactosidase A), Straussler-Scheinker treatment (caused by defect Prp), infertility, pancreatitis, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, chloride channel disorders, congenital myotonia (Thomson forms and

Becker syndrome), type III Bartter syndrome, Dent's disease, hyperplexia, epilepsy, lysosomal storage diseases, Angelman syndrome, primary ciliary dyskinesia (PCD), PCD with reverse visceral (also known as Cartagener's syndrome), PCD without reverse visceral and ciliary aplasia and liver disease.

[0016] Other diseases in which the mutation in CFTR is involved include male infertility due to congenital bilateral vas deficient (CBAVD), benign lung disease, idiopathic pancreatitis and allergic bronchopulmonary fungal fungal disease (ABPA). See, "CFIR-opathies: disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations," Peader G. Noone and Michael R. Knowles, Respir. Res. 2001, 2: 328-332 (which is incorporated herein by reference).

[0017] In addition to increasing CFTR activity, reducing anion secretion by CFTR modulators may be beneficial for treating secretory diarrhea in which epithelial water transport is significantly increased as a result of the transport of activated chloride secreting substances. This mechanism involves an increase in cAMP concentration and CFTR stimulation.

[0018] Although there are many causes of diarrhea, the main consequences of diarrheal diseases resulting from excessive chlorine transport are common to all of them and include dehydration, acidosis, impaired growth and death. Acute and chronic diarrhea is a major medical problem in many regions of the world. Diarrhea is both a significant factor in malnutrition and a leading cause of death (5,000,000 deaths / year) in children under five.

[0019] Secretory diarrhea is also a serious condition in patients with acquired immune deficiency syndrome (AIDS) and chronic inflammatory bowel disease (IBD). Sixteen million people traveling from industrialized countries to developing countries develop diarrhea each year, with the severity and number of cases of diarrhea changing depending on the country and region of travel.

[0020] Accordingly, there is a need to develop potent and selective CFTR enhancers in the form of mutant and wild-type human CFTR. These mutated forms of CFTR include, but are not limited to, ΔF508del, G551D, R117H, 2789 + 5G&gt; A.

[0021] There is also a need for modulators of CFTR activity and compositions thereof that can be used to modulate CFTR activity in a mammalian cell membrane.

[0022] There is a need for methods of treating diseases caused by CFTR mutation using such modulators of CFTR activity.

[0023] There is a need for methods to modulate CFTR activity in an ex vivo mammalian cell membrane.

[0024] In addition, there is a need for stable solid forms of this compound that can easily be used in pharmaceutical compositions suitable for use as therapeutic agents.

SUMMARY OF THE INVENTION [0025] The present invention relates to the solid forms of N- (4- (7-azabicyclo [2.2.1] heptan-7yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1,4 -dihydroquinoline-3-carboxamide (hereinafter "Compound 1") with the following structure:

<img file="PL2358680T3_D0001.tif" />

[0026] Compound 1 and pharmaceutically acceptable compositions thereof are useful for treating or reducing the severity of various diseases, disorders or conditions including, but not limited to, cystic fibrosis, pancreatitis, sinusitis, hereditary emphysema, hereditary hemochromatosis, clotting-fibrinolysis deficiencies, such as protein C deficiency, hereditary type 1 angioedema, lipid processing disorders such as familial hypercholesterolemia, type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as intracellular / pseudo-Hurler inclusion disease, mucopolysaccharidosis, Sandhof / Tay-Sachs disease, Crigler-Najiar type II syndrome, poliendocrynopathy / hyperinsulinemia, diabetes mellitus, hypoplasia, larvaemia type 1 CDG glycanose, hereditary emphysema, congenital hyperthyroidism, congenital bone fragility, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (DI), pituitary DI, renal DI, Charcot-Mari-Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick disease, various polyglutamine neurological disorders such as Huntington's disease, cerebellum cerebellar ataxia type I, bulbous spinal muscular atrophy, atrophy of the dentate nucleus, red nucleus, pale knob and hypothalamic nucleus and myotonic dystrophy, as well as spongiform encephalopathies such as hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye disease, pancreatic failure, osteoporosis, osteopenia, Gorham's syndrome, disorders chloride, congenital myotonia (Thomson and Becker forms), Bartter syndrome type III, Dent's disease, hyperplexia, epilepsy, lysosomal storage diseases, Angelman's syndrome, primary ciliary dyskinesia (PCD), PCD with reverse viscera (also known as Kartagener syndrome), PCD without reverse viscera and cilia aplasia, and Sjogren's disease.

[0027] In one aspect, Compound 1 is a substantially crystalline, pure free form of Form A.

[0028] The methods described herein can be used to prepare compositions of the present invention containing Form A. The amounts and characteristics of the ingredients used in the methods are described below.

BRIEF DESCRIPTION OF THE DRAWINGS [0 029] Figure 1 is an X-ray powder diffraction pattern of Form A.

[0030] Figure 2 is a conformational image of Form A based on single crystal X-ray analysis.

[0031] Figure 3 shows the FTIR spectrum of Form A.

DETAILED DESCRIPTION OF THE INVENTION [0032] Definitions [0033] As used herein, the following definitions apply, unless otherwise indicated.

[0034] As used herein, the term "ABC transporter" means an ABC transporter protein or fragment thereof comprising at least one binding domain, wherein the protein or fragment thereof is present / present in vivo or in vitro. As used herein, the term "binding domain" means a domain on an ABC transporter that can bind a modulator. See, e.g., Hwang, TC et al., J. Gen. Physiol. (1998): 111 (3), 477-90. [0035] As used herein, the term "CFTR" means transmembrane conductivity regulator responsible for cystic fibrosis or its mutant having regulatory activity, including, but not limited to, ΔF508 CFTR, R117H CFTR and G551D CFTR (see CFTR mutations, e.g. <a href="http://www.genet.sickkids.on.ca/cftr/">http://www.genet.sickkids.on.ca/cftr/</a>).

[0036] As used herein, the term "modulating" means increasing or decreasing in a measurable amount.

[0037] As used herein, the term "normal CFTR" or "normal CFTR function" means wild type CFTR without any disturbance due to environmental factors such as smoking, environmental pollution or anything that causes inflammation in the lung.

[0038] As used herein, the term "lowered CFTR" or "lowered CFTR" means a lower level of CFTR than normal or a lower CFTR performance than normal.

[0039] As used herein, the term "crystalline" refers to compounds or compositions in which structural units are ordered in fixed geometric patterns or networks, such that crystalline solids exhibit rigid long-range ordering. The structural units that make up the crystal structure can be atoms, molecules or ions. Crystalline solids have a specific melting point.

[0040] As used herein, the term "substantially crystalline" means a solid material that is ordered in established geometric patterns or networks that exhibit rigid long-range ordering. For example, substantially crystalline materials exhibit more than about 85% crystallinity (e.g., more than about 90% crystallinity or more than about 95% crystallinity). It should also be noted that "substantially crystalline" includes the "crystalline" descriptor as defined in the previous paragraph.

In one aspect, the invention relates to the form N- (4- (7-azabicyclo [2.2.1] heptan-7yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1,4- dihydroquinoline-3-carboxamide characterized as Form A.

[0042] In some embodiments, Form A has one or more peaks: from about 7.7 to about 8.1 degrees, for example about 7.9 degrees; from about 11.7 to about 12.1 degrees, for example about 11.9 degrees; from about 14.2 to about 14.6 degrees, for example about 14.4 degrees; and about 15.6 to about 16.0 degrees, for example about 15.8 degrees; on X-ray powder diffraction pattern obtained using Cu K alpha radiation.

[0043] In some embodiments, Form A has one or more peaks: from about 7.8 to about 8.0 degrees, for example about 7.9 degrees; from about 11.8 to about 12.0 degrees, for example about 11.9 degrees; from about 14.3 to about 14.5 degrees, for example about 14.4 degrees; and about 15.7 to about 15.9 degrees, for example about 15.8 degrees; on X-ray powder diffraction pattern obtained using Cu K alpha radiation.

[0044] In other embodiments, Form A has one or more peaks: from about 7.7 to about 8.1 degrees, for example about 7.9 degrees; from about 21.6 to about 22.0 degrees, for example about 21.8 degrees; and about 23.6 to about 24.0 degrees, for example about 23.8 degrees; on X-ray powder diffraction pattern obtained using Cu K alpha radiation.

[0045] In yet other embodiments, Form A has one or more peaks from about: 7.8 to about 8.0 degrees, for example about 7.9 degrees; from about 21.7 to about 21.9 degrees, for example about 21.8 degrees; and about 23.7 to about 23.9 degrees, for example about 23.8 degrees; on X-ray powder diffraction pattern obtained using Cu K alpha radiation.

[0046] In some embodiments, Form A has one or more of the following peaks measured in degrees on an X-ray powder diffraction pattern: a peak from about 7.7 to about 8.1 degrees (e.g., about 7.9 degrees); a peak from about 9.1 to about 9.5 degrees, (e.g., about 9.3 degrees); a peak from about 11.7 to about 12.1 degrees (e.g., about 11.9 degrees); a peak from about 14.2 to about 14.6 degrees (e.g., about 14.4 degrees); a peak from about 14.9 to about 15.3 degrees (e.g. about 15.1 degrees); peak from about 15.6 to about

16.0 degrees (e.g., about 15.8 degrees); a peak from about 16.8 to about 17.2 degrees (e.g., about 17.0 degrees); a peak from about 17.5 to about 17.9 degrees (e.g., about 17.7 degrees); a peak from about 19.1 to about 19.5 degrees (e.g., about 19.3 degrees); a peak from about 19.9 to about 20.3 degrees (e.g., about 20.1 degrees); a peak from about 21.2 to about 21.6 degrees (e.g., about 21.4 degrees); a peak from about 21.6 to about 22.0 degrees (e.g., about 21.8 degrees); peak from around

23.2 to about 23.6 degrees (e.g., about 23.4 degrees); a peak from about 23.6 to about 24.0 degrees, (e.g., about 23.8 degrees); a peak from about 25.4 to about 25.8 degrees (e.g., about 25.6 degrees); a peak from about 26.6 to about 27.0 degrees, (e.g., about 26.8 degrees); a peak from about 29.2 to about 29.6 degrees (e.g., about 29.4 degrees); a peak from about 29.5 to about 29.9 degrees (e.g., about 29.7 degrees); a peak from about 29.9 to about 30.3 degrees (e.g. about 30.1 degrees); and a peak from about 31.0 to about 31.4 degrees (e.g., about 31.2 degrees).

[0047] In some embodiments, Form A has one or more of the following peaks measured in degrees on an X-ray powder diffraction pattern: a peak from about 7.8 to about 8.0 degrees (e.g., about 7.9 degrees); a peak from about 9.2 to about 9.4 degrees (e.g., about 9.3 degrees); a peak from about 11.8 to about 12.0 degrees (e.g., about 11.9 degrees); a peak from about 14.3 to about 14.5 degrees (e.g., about 14.4 degrees); a peak from about 15.0 to about 15.2 degrees (e.g. about 15.1 degrees); a peak from about 15.7 to about 15.9 degrees (e.g., about 15.8 degrees); a peak from about 16.9 to about 17.1 degrees (e.g., about 17.0 degrees); a peak from about 17.6 to about 17.8 degrees (e.g., about 17.7 degrees); a peak from about 19.2 to about 19.4 degrees (e.g., about 19.3 degrees); a peak from about 20.0 to about 20.2 degrees (e.g., about 20.1 degrees); a peak from about 21.3 to about 21.5 degrees (e.g., about 21.4 degrees); a peak from about 21.7 to about 21.9 degrees (e.g. about 21.8 degrees); peak from around

23.3 to about 23.5 degrees (e.g., about 23.4 degrees); a peak from about 23.7 to about 23.9 degrees (e.g., about 23.8 degrees); a peak from about 25.5 to about 25.7 degrees, (e.g., about 25.6 degrees): a peak from about 26.7 to about 26.9 degrees, (e.g., about 26.8 degrees); peak from around

29.3 to about 29.5 degrees (e.g., about 29.4 degrees); a peak from about 29.6 to about 29.8 degrees (e.g., about 29.7 degrees); a peak from about 30.0 to about 30.2 degrees (e.g., about 30.1 degrees); and a peak from about 31.1 to about 31.3 degrees, (e.g., about 31.2 degrees).

[0048] In some embodiments, Form A is characterized by a diffraction pattern as shown in Figure 1.

[0049] In one aspect, the invention relates to a pharmaceutical composition comprising Form A and a pharmaceutically acceptable excipient or carrier.

[0050] In one aspect, the present invention provides Form A for use in a method of treating a CFTR mediated disease in a human.

[0051] In some embodiments, the method comprises administering an additional therapeutic agent.

[0052] In some embodiments, the disease is selected from cystic fibrosis, pancreatitis, sinusitis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies such as protein C deficiency, hereditary Type 1 angioedema, lipid disorders such as familial hypercholesterolemia , Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as intracellular inclusion / pseudo-Hurler disease, mucopolysaccharidosis, Sandhof / Tay-Sachs disease, Crigler-Najiar syndrome type II, poliendocrinopathy / hyperinsulinemia, diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG type 1 glycanosis, hereditary emphysema, congenital emphysema hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (DI), pituitary DI, renal DI, Charcot-Mari-Tooth syndrome, Perlizaeus-Merzbacher diseases, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, various polyglutamine neurological disorders such as Huntington's disease, cerebellum cerebellar ataxia type I, cerebrospinal atrophy atrophy of the toothed nucleus, red nucleus, pale knob and hypothalamic nuclei and myotonic dystrophy, as well as spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye disease, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, chloride channel disorders, congenital myotonia (Thomson and Becker forms), Bartter's type syndrome III, Denta diseases, hyperplexia, epilepsy, lysosomal storage diseases, Angelman syndrome, primary ciliary dyskinesia (PCD), PCD with reverse viscera (also known as Kartagener syndrome), PCD without reverse viscera and cilia aplasia, and Sjogren's disease.

[0053] In one embodiment, the present invention provides an effective amount of Form A for use in a method of treatment of human cystic fibrosis.

[0054] In one aspect, the present invention relates to a pharmaceutical packet or kit comprising Form A and a pharmaceutically acceptable carrier.

[0055] In one aspect, the invention relates to the crystalline form N- (4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1 , 4-dihydroquinoline-3-carboxamide with a rhombohedral crystal system, the R-3 space group, and with the following unit cell dimensions: a = 19.1670 (4) A, b = 19.1670 (4) A, c = 33, 6572 (12) A, α = 90 °, β = 90 ° and γ = 120 °.

[0056] In one embodiment, the present invention provides the crystalline form N- (4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) - 1,4-dihydroquinoline-3-carboxamide with unit cell dimensions: a = 19.1670 (4) A, b = 19.1670 (4) A, c = 33.6572 (12) A.

[0057] Uses, preparations and administration [0058] Pharmaceutically acceptable compositions [0059] In one aspect of the present invention there are provided pharmaceutically acceptable compositions, which compositions comprise Form A described herein, and optionally contain a pharmaceutically acceptable carrier, excipient or vehicle. In some embodiments, the compositions further comprise one or more additional therapeutic agents.

[0060] As described above, the pharmaceutically acceptable compositions of the present invention further comprise a pharmaceutically acceptable carrier, excipient or vehicle which, as used herein, includes any and all of solvents, diluents or other liquid vehicles, the excipients facilitate forming dispersions or suspensions, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, glidants and the like that match the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used to formulate pharmaceutically acceptable compositions and known techniques for their preparation. Except where any of the usual carrier media is incompatible with the compounds of the invention, for example by causing any undesirable biological activity or otherwise adversely interacting with any of the other components of the pharmaceutically acceptable composition, their use is considered to be covered by the present invention. Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid or potassium sorbate, mixtures of partial saturated glycerides of vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; Sesame oil; oil; corn oil and soybean oil; glycols; such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, non-stick agents, coating agents, sweeteners, flavors and flavors, preservatives and antioxidants may also be present in the composition, according to the decision of the person preparing the recipe.

[0061] Uses of compounds and pharmaceutically acceptable compositions [0062] In yet another aspect, the present invention provides Form A of compound 1 for use in a method of treating or reducing the severity of a condition, disease or disorder in which a CFTR mutation is involved. In some embodiments, the present invention provides a method of treating a condition, disease or disorder in which CFTR activity is deficient.

[0063] In some embodiments, the present invention provides Form A of Compound 1 for use in a method of treatment of diseases associated with reduced CFTR activity due to mutations in the CFTR encoding gene or environmental factors (e.g., cigarette smoke). These diseases include cystic fibrosis, chronic bronchitis, recurrent bronchitis, acute bronchitis, male infertility due to congenital bilateral vas deference (CBAVD), female infertility due to congenital uterine and vaginal (CAUV), chronic idiopathic idiopathic pancreatitis pancreatitis, acute idiopathic pancreatitis, chronic rhinitis and sinusitis, primary sclerosing cholangitis, allergic bronchopulmonary fungal mycosis, diabetes mellitus, dry eye, constipation, allergic bronchopulmonary fungal mycosis (ABPA), bone diseases (e.g. osteoporosis) and asthma.

[0064] In some embodiments, the present invention provides Form A of Compound 1 for use in a method of treatment of diseases associated with the proper functioning of CFTR. These diseases include chronic obstructive pulmonary disease (COPD), chronic bronchitis, recurrent bronchitis, acute bronchitis, rhinitis and sinusitis, constipation, pancreatitis, including chronic pancreatitis, recurrent pancreatitis and acute pancreatitis. pancreatic insufficiency, male infertility caused by congenital bilateral vas deferens (CBAVD), mild lung disease, idiopathic pancreatitis, liver disease, hereditary emphysema, gallstones, gastroesophageal reflux disease, gastrointestinal malignancy, inflammatory bowel disease, constipation, diabetes, arthritis, osteoporosis and osteopenia.

[0065] In certain embodiments, the present invention provides Form A of Compound 1 for use in a method of treating diseases associated with the proper functioning of CFTR including hereditary hemochromatosis, coagulation-fibrinolysis deficiencies such as protein C deficiency, Type 1 hereditary angioedema, lipid processing disorders such like familial hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as intracellular inclusion / pseudo-Hurler disease, mucopolysaccharidosis, Sandhof / Tay-Sachs disease, Crigler-Najiar type II syndrome, poliendocrinopathy / hyperinsulinemia, diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, CD malaroma hyperthyroidism, congenital bone fragility, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (DI), pituitary DI, renal DI, syndrome

Charcot-Mari-Tooth, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, various polyglutamine neurological disorders such as Huntington's disease, cerebrospinal ataxia And, bullet-spinal muscular atrophy, atrophy of the dentate nucleus, red nucleus, pale knob and hypothalamic nucleus and myotonic dystrophy, as well as spongiform encephalopathies such as hereditary Creutzfeldt-Jakob disease (caused by a defect in prion protein processing), Fabry disease, Straussler-Scheinker syndrome, Gorham syndrome, chloride channel disorders, congenital myotonia (Thomson and Becker forms), Bartter type III syndrome , Denta's disease, hyperplexia, epilepsy, lysosomal storage disease, Angelman's syndrome, primary ciliary dyskinesia (PCD), PCD with reverse bowel orientation (also known as Kartagener's syndrome), PCD without reverse bowel and cilia aplasia or Sjogren's disease.

[0066] According to an alternative preferred embodiment, the present invention provides a composition comprising Form A for use in a method of treating cystic fibrosis.

[0067] According to the invention, the "effective amount" of Form A or a pharmaceutically acceptable composition thereof is an amount effective to treat or reduce the severity of one or more of the diseases, disorders or conditions listed above.

[0068] Form A or a pharmaceutically acceptable composition thereof can be administered using any amount or route of administration effective to treat or reduce the severity of one or more of the diseases, disorders or conditions listed above. [0069] In certain embodiments, Form A or a pharmaceutically acceptable composition thereof is useful for treating or reducing the severity of cystic fibrosis in patients having residual CFTR activity in the apical membrane of the respiratory or non-respiratory epithelium. The presence of residual CFTR activity on the epithelial surface can easily be detected using methods known in the art, e.g. standard electrophysiological, biochemical or histochemical techniques. Such methods identify CFTR activity using in vivo or ex vivo electrophysiological techniques to measure Cl concentrations^- in sweat or saliva or ex vivo biochemical or histochemical techniques for monitoring cell surface density. Using such methods, residual CFTR activity can easily be detected in heterozygous or homozygous patients for a wide variety of mutations, including homozygous or heterozygous patients for the most common mutation, ΔF508. [0070] In another embodiment, Form A or a pharmaceutically acceptable composition described herein is useful for treating or reducing the severity of cystic fibrosis in patients with residual CFTR activity induced or increased by pharmacological methods or gene therapy. Such methods increase the amount of CFTR present on the cell surface, thereby inducing a previously absent CFTR activity in the patient or increasing the existing level of residual CFTR activity in the patient.

[0071] In one embodiment, Form A or a pharmaceutically acceptable composition described herein is useful for treating or reducing the severity of cystic fibrosis in patients with certain genotypes having residual CFTR activity, e.g., class III mutations (impaired regulation or gating), class IV (changed conductivity) or class V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV, and V cystic fibrosis Tansmembrane Conductance Regulator Defects and Opportunities of Therapy; Current Opinion in Pulmonary Medicine 6: 521 - 529. 2000). Other patient genotypes that show residual CFTR activity include patients who are homozygous for one of these classes or heterozygous for any of the other mutation classes, including class I mutations, class II mutations, or an unclassified mutation.

[0072] In one embodiment, Form A or a pharmaceutically acceptable composition described herein is useful for treating or reducing the severity of cystic fibrosis in patients with certain clinical phenotypes, e.g., with a moderate to mild clinical phenotype, which usually correlates with the level of residual CFTR activity in apical epithelial membrane. Such phenotypes include patients exhibiting pancreatic insufficiency or patients diagnosed with idiopathic pancreatitis or congenital bilateral vas deferens or mild lung disease.

[0073] The exact amount required will vary from individual to individual depending on the species, age and general health of the individual, severity of the infection, specific agent, method of administration, etc. Compounds of the invention are preferably formulated in a dosage unit form for easy administration and uniformity dose. As used herein, the expression "dosage unit form" refers to a physically discrete unit of agent appropriate for the patient being treated. However, it should be understood that the decision regarding the total daily use of the compounds and compositions of the present invention will be made by the attending physician in the field of sound medical judgment. For any particular patient or organism, the specific level of effective dose will depend on many factors including the disorder being treated and the severity of the disorder, the activity of the particular agent employed; the specific composition used; the age, body weight, general health, sex and diet of the patient; the time of administration, route of notification and the rate of secretion of the particular compound employed; duration of treatment; drugs used in combination or concurrently with the specific compound employed, and other factors well known in the medical science. As used herein, the term "patient" means an animal, preferably a mammal and most preferably a human. [0074] The pharmaceutically acceptable compositions of the present invention can be administered to humans or animals orally, rectally, parenterally, into the subarachnoid reservoir, vaginally, intraperitoneally, topically (in the form of powders, ointments, drops or a patch), buccal, in the form of a nasal or oral spray, etc. depending on the severity of the infection being treated. In certain embodiments, the compounds of the invention may be administered orally or parenterally at doses of about 0.01 mg / kg to about 50 mg / kg, and preferably from about 0.5 mg / kg to about 25 mg / kg of body weight of the subject per day one or more times a day to obtain the desired therapeutic effect.

[0075] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the field of the invention such as, for example, water or other solvents, solubilizing agents, and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular cottonseed, peanut oils, corn oils, (embryo, olive, castor and sesame), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and esters of fatty acids and sorbitan and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting, emulsifying and suspending agents, sweetening, flavoring and flavoring agents.

[0076] Injectable preparations, for example, sterile injectable aqueous or oily suspensions may be formulated in accordance with what is known in the art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable substrates and solvents that may be used include water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile, fixed oils are typically used as the solvent or suspending medium. For this purpose, any mild non-volatile oil containing synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0077] Injectable preparations can be sterilized, for example by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0078] In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound after subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. Then the rate of absorption of the compound depends on its dissolution rate, which, in turn, may depend on the size of the crystals and the crystal form. Alternatively, delayed absorption of a parenterally administered compound is achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot preparations are prepared by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide copolymer. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microcapsules that are compatible with body tissues.

[0079] Compositions for rectal or vaginal administration are preferably suppositories that can be easily prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at ambient temperature but liquid at body temperature and thus dissolve in the rectum or vaginal cavity and release the active compound.

[0080] Solid dosage forms for oral administration include capsules, tablets, pills, powders and capsules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or diluents such as starches, lactose, sucrose, glucose, mannitol and acid silicon, b) binders such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) dissolution retarding agents such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbing agents, such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. [0081] Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols, etc. Solid dosage forms in the form of tablets, dragees, capsules , pills and granules can be prepared with coatings and coatings, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may additionally contain opacifying agents and may also be in a composition that releases the active ingredient (s) only, or preferentially, in a certain part of the gastrointestinal tract, possibly in a delayed manner. Examples of coating compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols etc.

[0082] The active compounds may also be in the form of microcapsules with one or more excipients as indicated above. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and coatings such as enteric coatings, controlled release coatings and other coatings well known in the art of pharmaceutical formulation. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose or starch. Such dosage forms may also contain, as is usually used, additional substances other than inert diluents, for example tabletting glidants or other tabletting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may additionally contain opacifying agents and may also be in a composition that releases the active ingredient (s) only, or preferentially, in a certain part of the gastrointestinal tract, possibly in a delayed manner. Examples of coating compositions that can be used include polymeric substances and waxes.

[0083] Dosage forms for topical or transdermal administration of a compound of the present invention include ointments, pastes, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers as may be required. The ophthalmic preparation, ear drops and eye drops are also intended to be included within the scope of the invention. Furthermore, it is intended that the present invention relates to the use of transdermal patches that have the additional advantage of providing controlled administration of a compound to an organism. Such dosage forms are prepared by dissolving or spreading the compound in a suitable vehicle. Absorption enhancers can also be used to increase the flow of the compound through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0084] It will also be appreciated that Form A or a pharmaceutically acceptable composition described herein can be used in combination therapies, i.e. Form A or a pharmaceutically acceptable composition described herein can be administered simultaneously with, before or after one or more desired therapeutic agents or medical procedures. The specific combination therapies (therapeutic agents or procedures) to be used in the combined mode will also take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies used may also achieve the desired effect for the same disorder (for example, the inventive compound may be administered concurrently with another agent used to treat the same disorder) or different effects may be obtained (e.g., controlling any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease or condition are known to be "appropriate for the disease or condition being treated."

[0085] In one embodiment, the additional agent is selected from a mucolytic agent, bronchodilator, antibiotic, anti-infective agent, anti-inflammatory agent, CFTR modulator other than the compound of the present invention or a nutrient.

[0086] In one embodiment, the additional agent is an antibiotic. Exemplary antibiotics useful herein include tobramycin, including tobramycin inhalation powder (TIP), azithromycin, aztreonam, including aztreonam aerosol, amikacin, including its liposome preparations, ciprofloxacin, including its preparations suitable for administration by inhalation , including aerosol preparations, combinations of two antibiotics, e.g. fosfomycin and tobramycin.

[0087] In another embodiment, the additional agent is a mucolytic. Exemplary mucolytics useful herein include Pulmozyme®.

[0088] In another embodiment, the additional agent is a bronchodilator. Exemplary bronchodilators include albuterol, metaprotenerol sulfate, pirbuterol acetate, salmeterol or tetrabulin sulfate.

[0089] In another embodiment, the additional agent is effective in reconstituting liquids on the surface of the lung airways. Such measures improve the movement of salt into and out of the cells, which allows better hydration of mucus in the lung airways, and thus its easier purification. Examples of such agents include hypertonic saline, denuphosol, ([[(3S, 5R) -5- (4-amino-2-oxopyrimidin-1-yl) -3-hydroxyoxolan-2-yl] methoxyhydroxyphosphoryl] - [[(2R, 3S, 4R, 5R) -5- (2,4-dioxopyrimidin-1-yl) -3,4-dihydroxyoxolan-2-yl] methoxyhydroxyphosphoryl] oxyhydroxyphosphoryl] hydrogen phosphate) tetrasodium or bronchitol (mannitol for inhalation).

[0090] In another embodiment, the additional agent is an anti-inflammatory agent, i.e., an agent that can reduce inflammation in the lungs. Exemplary of such useful agents include ibuprofen, docosahexaenoic acid (DHA), sildenafil, inhalation glutathione, pioglitazone, hydroxychloroquine or simavastatin.

[0091] In another embodiment, the additional agent reduces the activity of the epithelial sodium channel blocker (ENaC) either directly by blocking the channel or indirectly by modulating proteases that lead to increased ENaC activity (e.g., serine proteases, channel activating proteases). Examples of such agents include kamostat (trypsin-like protease inhibitor), QAU145, 552-02, GS-9411, INO-4995, Aerolytic and amiloride. Additional agents that reduce the activity of the epithelial sodium channel blocker (ENaC) can be found, for example, in PCT Publication No. WO2009 / 074575, the content of which is fully incorporated herein.

[0092] Among others, the diseases described herein, combinations of CFTR modulators, such as Form A, and agents that lower ENaC activity, can be used to treat Liddle syndrome, inflammation or allergy involving cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive lung disease, asthma, respiratory tract infections, lung cancer, xerostomy and keratoconjunctivitis sire, respiratory tract infections (acute and chronic; viral and bacterial) and lung cancer.

[0093] Combinations of CFTR modulators, such as Form A, and ENaC-lowering agents are also useful in the treatment of diseases involving blockage of the epithelial sodium channel, which also includes diseases other than respiratory diseases that are associated with abnormal fluid regulation by epithelium , possibly involving abnormal physiology of protective surface liquids, e.g. xerostomy (dry mouth) or keratoconjunctivitis sire (dry eye). In addition, blockade of the sodium channel in the kidney can be used to improve diuresis, and thus to cause a hypotensive effect.

[0094] Asthma includes both congenital (non-allergic) and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchial asthma, exercise asthma, occupational asthma and asthma caused by bacterial infection. Asthma treatment should also be understood to include treatment of individuals, e.g. younger than 4 or 5 years of age, showing signs of wheezing, and diagnosed or diagnosed as "wheezing children", an established category of patients with high medical relevance and currently often identified as being in early or early asthma. (For convenience, this particular asthmatic condition is called "wheezing syndrome.") Prophylactic efficacy in the treatment of asthma will be demonstrated by a reduced incidence or severity of an attack of symptoms, e.g. an acute asthmatic attack or bronchoconstriction attack, improved lung function or bronchial hyperreactivity. . This may be further demonstrated by the reduced requirement for other symptomatic therapy, i.e. therapy for or intended to limit or stop a symptomatic attack when it occurs, e.g. anti-inflammatory (e.g. corticosteroid) or bronchodilator. The prophylactic benefit in asthma may, in particular, be seen in patients prone to "morning decrease in peak expiratory flow". "Morning decreases in peak expiratory flow" are a recognized asthmatic syndrome, common among a significant percentage of asthmatics, and characterized by an asthma attack, e.g. between hours around 4-6 am, i.e. at any time point significantly distant from any previously administered symptomatic asthma therapy.

[0095] Chronic obstructive pulmonary disease includes chronic bronchitis or dyspnoea associated therewith, emphysema, as well as exacerbation of airway hyperresponsiveness as a consequence of using other drugs, in particular other inhaled drugs. In some embodiments, combinations of CFTR modulators, such as Form A, and agents that reduce ENaC activity are useful for treating bronchitis of any type or origin, e.g. with acute, peanut-induced, catarrhal, crustal, chronic or tuberculous bronchitis.

[0096] In another embodiment, the additional agent is a CFTR modulator other than Form A of Compound 1, i.e., an agent that exerts a modulation effect on CFTR activity. For example, such agents include ataluren ("PTC124®"; 3- [5- (2-fluoro-phenyl) -1,2,4-oxadiazol-3-yl] -benzoic acid), synapultide, lanolutide, depelestat (human recombinant neutrophil elastase inhibitor ), kobiproston (7 - {(2R, 4aR, 5R, 7aR) -2 - [(3S) -1,1-difluoro3-methylpentyl] -2-hydroxy-6-oxooctahydrocyclopenta [b] pyran-5-yl} acid heptanoic acid) or (3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid. In another embodiment, the additional agent is (3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid.

[0097] In another embodiment, the additional agent is a nutrient. Examples of such agents include pancrelipase (pancreatic enzyme replacement), including Pancrease®, Pancreacarb®, Ultrase® or Creon®, Liprotomase® (formerly Trizytek®), Aquadeks® or inhalation glutathione. In one embodiment, the additional nutrient is pancrelipase.

[0098] In one embodiment, the additional agent is a CFTR modulator other than the compound of the present invention.

[0099] The amount of additional therapeutic agent present in the compositions of the present invention will not be higher than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably, the amount of additional therapeutic agent in the compositions disclosed herein will range from about 50% to 100% of the amount normally present in a composition comprising that therapeutic agent as the only active agent.

[0100] Form A or a pharmaceutically acceptable composition thereof described herein can be incorporated into the coating composition of an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Accordingly, the present invention, in another aspect, includes an implantable coating composition comprising a compound of the present invention generally described above and in the classes and subclasses herein, and a carrier suitable for coating the implantable device. In yet another aspect, the present invention includes an implantation device coated with a composition comprising a compound of the present invention generally described above and in the classes and subclasses set forth herein, and a carrier suitable for coating said implantation device. Suitable coatings and general manufacture of coated injection devices are described in US Patent Nos. 6099562; 5886026; and 5304121. Coatings are usually biocompatible polymeric materials such as hydrogel polymer, polydimethylsiloxane, polycaprolactone, polyethylene glycol, poly (lactic acid), ethylene / vinyl acetate and mixtures thereof. The coatings may optionally be further coated with a suitable top coat of fluorosilicon, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to provide controlled release characteristics to the composition.

[0101] Another aspect of the invention relates to modulation of CFTR activity in a biological sample or in a patient (e.g., in vitro or in vivo), the method comprising administering to or contacting the biological sample with Form A or a pharmaceutically acceptable composition thereof. As used herein, the term "biological sample" includes, but is not limited to, cell cultures or their extracts; biopsy materials obtained from a mammal or their extracts; and blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof.

[0102] CFTR modulation in a biological sample is useful for a variety of purposes that are known to the person skilled in the art. Examples of such purposes include, but are not limited to, CFTR in biological and pathological phenomena; and comparative assessment of new CFTR modulators.

[0103] In yet another embodiment, there is provided a method of modulating anion channel activity in vitro or in vivo, comprising the step of contacting said channel with Form A or a pharmaceutically acceptable composition thereof described herein. In preferred embodiments, the anion channel is a chloride channel or a bicarbonate channel. In other preferred embodiments, the anion channel is a chloride channel.

[0104] According to an alternative embodiment, the present invention provides a method of increasing the number of functional CFTRs in a cell membrane comprising the step of contacting said cell with Form A or a pharmaceutically acceptable composition thereof described herein.

[0105] According to another preferred embodiment, CFTR activity is measured by measuring the transmembrane potential voltage. Methods of measuring transmembrane potential voltage in a biological sample may include any of the methods known in the art, such as an optical membrane potential test or other electrophysiological methods.

[0106] The optical membrane potential test uses voltage sensitive FRET sensors described by Gonzalez and Tsien (See, Gonzalez, JE and RY Tsien (1995) "Voltage sensing by fluorescence resonance energy transfer in single cells." Biophys J 69 (4 ): 1272-80, and Gonzalez, JE and RY Tsien (1997); "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer" Chem Biol 4 (4): 269-77) in combination with devices for measuring fluorescence changes, such as the Voltage / Ion Probe Reader (VIPR) (See, Gonzalez, JE, K. Oades, et al. (1999) "Cell-based assays and instrumentation for screening ion-channel targets" Drug Discov Today 4 (9): 431-439).

[0107] These voltage-sensitive tests are based on a change in resonance fluorescence excitation energy transfer (FRET) between a film-soluble, voltage-sensitive dye, DiSBAC2 (3), and a fluorescent phospholipid, CC2-DMPE, which is attached to the outer layer plasma membrane and acts as a FRET donor. Changes in membrane potential (Vm) cause the redistribution of negatively charged DiSBAC2 (3) across the plasma membrane and the amount of energy transferred from CC2-DMPE changes accordingly. Changes in fluorescence emissions can be monitored using_™ VIPR II device, which is an integrated liquid dispenser and fluorescence detector designed to conduct cell-based studies in 96- or 384-well microtiter plates.

[0108] In another aspect, the present invention provides a kit for use in measuring the activity of CFTR or a fragment thereof in an in vitro or in vivo biological sample comprising (i) a composition comprising Form A or any of the above forms; and (ii) instructions for a) contacting the composition with the biological sample, and b) measuring the activity of this CFTR or fragment thereof. In one embodiment, the kit further includes instructions for a) contacting the additional composition with the biological sample; b) measuring the activity of this CFTR or a fragment thereof in the presence of this additional compound, and c) comparing the CFTR activity in the presence of the additional compound with the density of CFTR in the presence of Form A described herein. In preferred embodiments, the kit is used to measure the density of CFTR.

[0109] For a more detailed understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are provided for the purpose of illustrating the invention and are not to be construed as limiting the invention to any extent.

EXAMPLES [0110] Methods and materials [0111] XRPD (X-ray powder diffraction) [0112] X-ray powder diffraction (XRPD) data was recorded at room temperature using a Rigaku / MSC MiniFlex Desktop X-ray powder diffractometer (Rigaku, The Woodlands, TX). X-rays were generated using a Cu lamp operating at 30 kV and 15 mA with a Ke suppression filter. The divergence gap was variable with the dispersion and receiving slots set at 4.2 degrees and 0.3 mm gap respectively. The fixed time (FT) scanning mode was used, with a 0.02 degree step width and a 2.0 second counting time. The X-ray powder diffractometer was calibrated using a standard of 75% sodalite (Na3Al4Si4O12Cl) and 25% silicon (Rigaku, catalog number 2100 / ALS). A six-sample table with zero background sample holders (SH-LBSL511-RNDB) was used. The powder sample was placed in the intended area and flattened with a glass plate.

[0113] FTIR Spectroscopy (Fourier Transform Infrared) [0114] FTIR spectra were recorded with a Thermo Scientific spectrometer, Nicolet 6700 FT-IR with an intelligent orbital sample compartment, diamond window, using Omnic software, 7.4. The sample powder was placed directly on the diamond crystal and pressure was applied to match the surface of the sample to the surface of the diamond crystal. The background spectrum was recorded and then the sample spectrum was collected. Collection settings were as follows:

Detector: DTGS KBr;

Beam splitter: KBr;

Source: IR;

Scan range: 4000 - 400 cm^-1;

Strengthening: 8.0;

Optical speed: 0.6329 cm / s;

Aperture: 100;

Number of scans: 32; and Resolution: 4 cm^-1.

[0115] Example 1: Preparation of 4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxylic acid (7).

<img file="PL2358680T3_D0002.tif" />

Diethyl malonate 3 (276 g, 1.3 mol) and toluene (100 ml) were combined under a nitrogen atmosphere in a three-neck, 1 L round bottom flask equipped with a Dean-Stark cooler. The solution was heated to 140 ° C with stirring and this temperature was maintained for 4 h. The reaction mixture was cooled to 70 ° C and hexane (600 ml) was slowly added. The resulting suspension was stirred allowing it to warm to room temperature. The solid was filtered off, washed with 10% ethyl acetate in hexane (2 x 400 mL) then dried under vacuum to give a white solid (350 g, 94% yield) as the desired condensation product, 2 - ((2-chloro-5- diethyl (trifluoromethyl) phenylamino) methylene) malonate 4. ¹H NMR (400 MHz, DMSO-d6) δ11,28 (d, J = 13.0 Hz, 1H), 8.63 (d, J = 13.0 Hz, 1H), 8.10 (s, 1H) , 7.80 (d, J = 8.3 Hz, 1H), 7.50 (dd, J = 1.5, 8.4 Hz, 1H), 4.24 (q, J = 7.1 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 1.27 (m, 6H).

[0117] Preparation of ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxylate (5). Dowtherm® (200 ml, 8 ml / g) was placed in a three-necked 1-flask, which was degassed at 200 ° C for 1 h. The solvent was heated to 260 ° C and 2 - ((2-chloro) was added in portions over 10 min. -5- (trifluoromethyl) phenylamino) methylene) diethyl malonate (25 g, 0.07 mol). The resulting mixture was stirred at 260 ° C for 6.5 hours (h) and the ethanol obtained as a byproduct was removed by distillation. The mixture was allowed to cool slowly to 80 ° C. Hexane (150 ml) was added slowly over 30 minutes (min), followed by an additional 200 ml hexane. The mixture was stirred until room temperature was reached. The solid was filtered off, washed with hexane (3 χ 150 mL) then dried in vacuo to give ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxylate as a light brown solid ( 13.9 g, 65% yield).¹H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 8.39 (s, 1H), 8.06 (d, J = 8.3 Hz, 1H), 7.81 (d , J = 8.4 Hz, 1H), 4.24 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).

[0118] Preparation of ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate (6). Ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxylate 5 (100 g, 0.3 mol), ethanol (1250 ml, 12.5) was placed in a 5-necked flask. ml / g) and triethylamine (220 ml, 1.6 mol). 10 g of 10% Pd / C (50% moisture) was then added to the reactor at 5 ° C. The reaction mixture was stirred vigorously under a hydrogen atmosphere for 20 h at 5 ° C, after which the reaction mixture was concentrated to a volume of about 150 ml. The product, ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate, as a suspension with Pd / C, was used directly in the following step.

[0119] Preparation of 4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxylic acid (7). Ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate 6 (58 g, 0.2 mol, crude reaction suspension containing Pd / C) was suspended in NaOH (814 ml, 5 M, 4 , 1 mol) in a 1 L reflux flask and heated at 80 ° C for 18 h and then heated at 100 ° C for 5 h. The reaction mixture was filtered hot through a pad of Celite to remove Pd / C and Celite was washed 1 N NaOH. The filtrate was acidified to about pH 1 to give a thick, white precipitate. The precipitate was filtered off, then washed with water and cold acetonitrile. The solid was then dried in vacuo to give 4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxylic acid 7 as a white solid (48 g, 92% yield).¹H NMR (400.0 MHz, DMSO-d6) δ 15.26 (s, 1H), 13.66 (s, 1H), 8.98 (s, 1H), 8.13 (dd, J = 1, 6.8.8 Hz, 1H), 8.06 - 7.99 (m, 2H).

[0120] Example 2: Preparation of 4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) aniline

<img file="PL2358680T3_D0003.tif" />

[0121] Preparation of 7- [4-nitro-3- (trifluoromethyl) phenyl] -7-azabicyclo [2.2.1] heptane (20). To a flask containing 7-azabicyclo [2.2.1] heptane 9 hydrochloride (4.6 g, 34.43 mmol, obtained under nitrogen, a solution of 4-fluoro-1-nitro-2- (trifluoromethyl) benzene 8 (6.0 was added) g, 28.69 mmol) and triethylamine (8.7 g, 12.00 mL, 86.07 mmol) in acetonitrile (50 mL) The reaction flask was heated at 80 ° C under nitrogen for 16 h. The reaction mixture was allowed to cool and then partitioned between water and dichloromethane. The organic layer was washed with 1 M HCl, dried over Na2SO4, filtered and concentrated to dryness. Purification by silica gel chromatography (0-10% ethyl acetate in a mixture of hexanes) gave 7- [4-nitro-3- (trifluoromethyl) phenyl] -7-azabicyclo [2.2.1] heptane 10 (7.2 g, 88% yield) as a yellow solid.¹H NMR (400.0 MHz, DMSO-d6) δ 8.03 (d, J = 9.1 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.25 (dd , J = 2.6, 9.1 Hz, 1H), 4.59 (s, 2H), 1.69 - 1.67 (m, 4H), 1.50 (d, J = 7.0 Hz, 4H).

[0122] Preparation of 4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) aniline (11). From the flask containing 7- [4-nitro-3- (trifluoromethyl) phenyl] -7-azabicyclo [2.2.1] heptane 10 (7.07 g, 24.70 mmol) and 10% Pd / C (0 71 g, 6.64 mmol), gas was removed and then purged with nitrogen. Ethanol (22 ml) was added and the reaction flask connected to a hydrogen balloon. After vigorous stirring for 12 h, the reaction mixture was purged with nitrogen and Pd / C was removed by filtration. The filtrate was concentrated under reduced pressure to give a dark oil and the residue was purified by silica gel chromatography (0-15% ethyl acetate in hexanes) to give 4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) aniline 11 as a purple solid (5.76 g, 91% yield). ¹H NMR (400.0 MHz, DMSO-d6) δ 6.95 (dd, J = 2.3, 8.8 Hz, 1H), 6.79 (d, J = 2.6 Hz, 1H), 6 , 72 (d, J = 8.8 Hz, 1H), 4.89 (s, 2H), 4.09 (s, 2H), 1.61 - 1.59 (m, 4H) and 1.35 ( d, J = 6.8 Hz, 4H).

[0123] Example 3: Preparation of W (4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1,4-dihydroquinoline -3-carboxamide (compound 1).

<img file="PL2358680T3_D0004.tif" />

[0124] For a solution of 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylic acid 7 (9.1 g, 35.39 mmol) and 4- (7-azabicyclo [2.2.1] heptane-7 -yl) -2- (trifluoromethyl) aniline 11 (9.2 g, 35.74 mmol) in 2-methyltetrahydrofuran (91.00 mL) was added at room temperature cyclic propylphosphonic acid anhydride (50% solution in ethyl acetate, 52, 68 ml, 88.48 mmol) and pyridine (5.6 g, 5.73 ml, 70.78 mmol). The reaction flask was heated at 65 ° C for 10 h under nitrogen. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and quenched with saturated Na2CO3 solution (50 mL). The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with water, dried over Na2SO4, filtered and concentrated to give a light brown solid. The crude solid product was suspended in ethyl acetate / diethyl ether (2: 1), collected by vacuum filtration and washed twice more with ethyl acetate / diethyl ether (2: 1) to give the product as a light yellow crystalline powder. The powder was dissolved in warm ethyl acetate and adsorbed on Celite. Purification by silica gel chromatography (0-50% ethyl acetate in dichloromethane) gave N- (4- (7-azabicyclo [2.2.1] heptan-7-yl) -2- (trifluoromethyl) phenyl) -4- oxo-5- (trifluoromethyl) -1,4-dihydroquinoline-3-carboxamide (compound 1) as a white crystalline solid (Form A) (13.5 g, 76% yield). LC / MS m / z 496.0 [M + H]⁺, retention time 1.48 min (RP-C18, 10-99% CH3CN / 0.05% TFA within 3 min). ¹H NMR (400.0 MHz, DMSO-d6) δ 13.08 (s, 1H), 12.16 (s, 1H), 8.88 (s, 1H), 8.04 (dd, J = 2, 1, 7.4 Hz, 1H), 7.95 - 7.88 (m, 3H), 7.22 (dd, 2.5, 8.9 Hz, 1H), 7.16 (d, J = 2 , 5 Hz, 1H),

4.33 (s, 2H), 1.67 (d, J = 6.9 Hz, 4H), 1.44 (d, J = 6.9 Hz, 4H).

[0125] The powder diffraction pattern of Form A is shown in Figure 1.

[0126] Table 1 gives representative XRPD peaks of Form A.

Table 1. XRPD peaks of Form A

<td>2θ (degrees)</td><td>Intensity (%)</td>

<td> 7,90</td><td> 100,0</td>

<td> 9,28</td><td> 10,8</td>

<td> 11,90</td><td> 12,8</td>

<td> 14,38</td><td> 35,2</td>

<td> 15,08</td><td> 12,6</td>

<td> 15,80</td><td> 34,1</td>

<td> 16,96</td><td> 25,2</td>

<td> 17,66</td><td> 13,8</td>

<td> 19,28</td><td> 39,4</td>

<td> 20,06</td><td> 20,2</td>

<td> 21,36</td><td> 14,5</td>

<td> 21,80</td><td> 94,2</td>

<td> 23,40</td><td> 30,0</td>

<td> 23,80</td><td> 92,0</td>

<td> 25,64</td><td> 8,9</td>

<td> 26,82</td><td> 6,4</td>

<td> 29,36</td><td> 8,1</td>

<td> 29,72</td><td> 18,1</td>

<td> 30,14</td><td> 14,2</td>

<td> 31,20</td><td> 9,9</td>

[0127] The conformational images of Form A based on a single crystal X-ray analysis are shown in Figure 2. Diffraction data was collected with a Bruker Apex II diffractometer equipped with a sealed lamp as a CuK-alpha source and an Apex II CCD detector. The structure was solved and improved using the SHELX program (Sheldrick, GM, Acta Cryst A64, pp. 112-122 (2008)). Based on intensities, statistics and symmetry, the structure was solved and corrected in a rhombohedral crystal system, the R-3 space group. The dimensions of the Form A unit cell were: a = 19.1670 (4) A, b = 19.1670 (4) A, c = 33.6572 (12) A, α = 90 °, β = 90 ° and γ = 120 °.

[0128] FTIR spectra of Form A are shown in Figure 3.

[0129] Table 2 below shows representative FTIR peaks of Form A.

Table 2. Form A FTIR peaks

<td>Position (cm^-1)</td><td>Intensity</td>

<td> 407,4</td><td> 46,07</td>

<td> 436,7</td><td> 72,55</td>

<td> 471,5</td><td> 61,17</td>

<td> 497,8</td><td> 63,61</td>

<td> 505,7</td><td> 60,34</td>

<td> 532,9</td><td> 61,14</td>

<td> 567,8</td><td> 54,31</td>

<td> 590,7</td><td> 55,23</td>

<td> 614,4</td><td> 64,01</td>

<td> 649,7</td><td> 50,74</td>

<td> 661,0</td><td> 49,82</td>

<td> 686,8</td><td> 51,43</td>

<td> 726,1</td><td> 53,80</td>

<td> 751,4</td><td> 35,60</td>

<td> 798,1</td><td> 48,21</td>

<td> 808,8</td><td> 48,47</td>

<td> 824,8</td><td> 42,25</td>

<td> 875,5</td><td> 52,89</td>

<td> 898,6</td><td> 71,77</td>

<td> 918,7</td><td> 68,93</td>

<td> 977,7</td><td> 42,31</td>

<td> 1008,1</td><td> 64,09</td>

<td> 1047,3</td><td> 35,70</td>

<td> 1072,5</td><td> 53,76</td>

<td> 1091,2</td><td> 43,79</td>

<td> 1113,4</td><td> 28,46</td>

<td> 1131,4</td><td> 30,00</td>

<td>Position (cm^-1)</td><td>Intensity</td>

<td> 1153,0</td><td> 34,61</td>

<td> 1168,3</td><td> 40,13</td>

<td> 1199,3</td><td> 74,26</td>

<td> 1221,8</td><td> 48,07</td>

<td> 1253,1</td><td> 47,84</td>

<td> 1277,6</td><td> 36,67</td>

<td> 1291,7</td><td> 48,07</td>

<td> 1310,8</td><td> 55,99</td>

<td> 1329,1</td><td> 63,21</td>

<td> 1352,8</td><td> 42,30</td>

<td> 1433,2</td><td> 42,45</td>

<td> 1463,0</td><td> 63,68</td>

<td> 1526,0</td><td> 35,86</td>

<td> 1574,0</td><td> 60,60</td>

<td> 1607,5</td><td> 60,30</td>

<td> 1662,6</td><td> 55,12</td>

<td> 1740,9</td><td> 86,74</td>

<td> 2870,0</td><td> 81,63</td>

<td> 2947,7</td><td> 75,12</td>

<td> 2963,8</td><td> 75,30</td>

<td> 3092,7</td><td> 84,58</td>

[0130] Tests for detecting and measuring the properties of ΔF508CFTR amplification compounds [0131] Optical methods for testing membrane potential testing properties of F508-CFTR modulating compounds [0132] Fluorescent voltage detection dyes are used in the test to measure changes in membrane potential using a fluorescent plate reader ( e.g. FLIPR III, Molecular Devices, Inc.) as a functional growth readout of ΔF508-CFTR in NIH 3T3 cells. The driving force for the response is the creation of a chloride ion gradient in combination with channel activation by a single step of adding liquid after previously treating the cells with compounds and then adding a voltage detecting dye.

[0133] Identification of enhancers [0134] To identify ΔF508-CFTR enhancers, a double addition HTS test format was developed. This HTS test uses fluorescent voltage sensing dyes to measure changes in membrane potential on FLIPR III as a measure of the gating (conductivity) of ΔF508 CFTR in temperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force of the answer is the Cl ion gradient^- in combination with channel activation with forskolin in a single liquid addition step using a fluorescence plate reader such as FLIPR III after treatment of cells with enhancing compounds (or DMSO medium control) followed by the addition of a redistributable dye.

[0135] Solutions

Washing solution # 1: (in mM) NaCl 160, KCl 4.5, CaCl2 2, MgCl2 1, HEPES 10, pH 7.4 with NaOH.

Chloride free purging solution: Chloride salts in purging solution # 1 are replaced with gluconate salts.

[0136] Cell culture [0137] For optical measurements of membrane potential, NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used. Cells are maintained at 37 ° C in 5% CO₂and 90% moisture in Dulbecco's modified Eagle medium supplemented with mM glutamine, 10% fetal bovine serum, 1 X NEAA, β-ME, 1 X pen / strep and ₂ mM HEPES in 175 cm culture bottles. For all optical tests, cells were plated at ~ 20,000 / well in 384-well matrigel-coated plates and cultured for 2 h at 37 ° C before culturing at 27 ° C for 24 h for the boost test. For the corrected tests, cells were cultured at 27 ° C or 37 ° C with or without compounds for 16-24 hours. Electrophysiological tests to study the modulation properties of ΔF508-CFTR by compounds.

[0138] Ussing chamber test [0139] Ussing chamber experiments were performed on polarized airway epithelial cells expressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulators identified in optical tests. Non-CF and CF airway epithelium was isolated from bronchial tissue, cultured as previously described (Galietta, LJV, Lantero, S., Gazzolo, A., Sacco, O., Romano, L., Rossi,

GA, and Zegarra-Moran, O. (1998) In Vitro Cell. Dev. Biol. 34, 478-481) and sown on_™ Costar® Snapwell filters^™ previously coated with NIH3T3 conditioned medium. After four days, the apical medium was removed and the cells were grown on an air-liquid interface for&gt; 14 days before use. This resulted in the formation of a single layer of completely differentiated cylindrical cells that produced cilia, which are characteristic of the respiratory epithelium. HBE non-CF was isolated from non-smokers who did not have any known lung disease. CFHBE was isolated from patients homozygous for ΔF508-CFTR.

[0140] HBE grown on Costar® Snapwell ™ cell culture inserts was embedded in a Ussing chamber (Physiologic Instruments, Inc., San Diego, CA) and transepithelial resistance and short-circuit current were measured in the presence of side-basal to a peak Cl gradient^- (Isc) using a voltage-clamp system (Department of Bioengineering, University of Iowa, IA). Briefly, HBE was tested under voltage-clamp recording conditions (Vhold = 0 mV) at 37 ° C. The basolateral solution contained (in mM) 145 NaCl, 0.83 K2HPO4, 3.3 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 10 Glucose, 10 HEPES (pH adjusted to 7.35 NaOH) and the overhead solution contained (in mM) 145 NaGluconate, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, 10 HEPES (pH adjusted to 7.35 NaOH).

[0141] Identification of enhancing compounds [0142] A typical protocol used a Cl concentration gradient^- from the basolateral to the apical membrane. To establish this gradient, normal Ringer's fluid was used on the laterolateral membrane, while on the top of the NaCl membrane it was replaced with an equimolar solution of sodium gluconate (adjusted to pH 7.4 to NaOH) to produce a large Cl "concentration gradient through the epithelium. Forskolin (10 μΜ) and all test compounds were added to the apical side of the cell culture inserts. The performance of putative ΔF508-CFTR enhancers was compared with the known enhancer, genistein.

[0143] Patch clamp recording [0144] Total current Cl^- in ΔF508-NΊH3T3 cells were monitored using a perforated-patch recording configuration as previously described (Rae, J., Cooper, K., Gates, P. and Watsky, M. (1991) J. Neurosci. Methods 37, 15 -26). Voltage clamp recording was performed at 22 ° C using an Axopatch 200B patch clamp amplifier (Axon Instruments Inc., Foster City, CA). The pipette solution contained (in mM) 150 N-methyl-D-glucamine (NMDG) -Cl, 2 MgCl2, 2 CaCl2, 10 EGTA, 10 HEPES and 240 μg / ml amphotericin-B (pH adjusted to 7.35). The extracellular medium contained (in mM) 150 NMDG-Cl, 2 MgCl2, 2 CaCl2, 10 HEPES (pH adjusted to HCl 7.35). Pulse generation, data collection and analysis were performed on a computer equipped with a Digidata 1320 A / D interface in combination with Clampex 8 (Axon Instruments Inc.). To activate ΔF508-CFTR, 10 μΜ forskolin and 20 μΜ genistein were added to the bath and the voltage-voltage relationship was monitored every 30 s.

[0145] Identification of enhancers [0146] The ability of enhancers ΔF508-CFTR to increase the macroscopic current ΔF508-CFTR Cl^- (Ia_F508) in NIH3T3 cells stably expressing ΔF508-CFTR were also examined using peforated-patch registration techniques. Enhancers identified in optical tests induced a dose-dependent increase in IApsos with similar strength and performance observed in optical tests. In all cells tested, the reversal potential before and during use of the enhancer was about -30 mV, which is calculated EC1 (-28 mV).

[0147] Cell culture [0148] NIH3T3 mouse fibroblasts stably expressing ΔP508-CFTR are used for whole cell registration. Cells are maintained at 37 ° C in 5% CO₂ and humidity of 90% in Eagle's Dulbecco's modified medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen / strep and 25 mM HEPES in 175 cm²breeding bottles. For whole-cell registration, 2500-5000 cells were plated on poly-L-lysine-coated coverslips and cultured for 24-48 h at 27 ° C before use to test for enhancer activity; and incubated with or without a correction compound at 37 ° C to measure the activity of the correction compounds. [0149] Single channel recording [0150] wt-CFTR gating activity and temperature-corrected ΔF508-CFTR expressed in NIH3T3 cells were observed using recording on excised and inverted membrane fragments as previously described (Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, RG, Pavirani, A., Lecocq, JP., Lazdunski, Μ. (1991) Nature 354, 526-528) using an Axopatch 200B patch clamp amplifier (Axon Instruments Inc.). The pipette contained (in mM): 150 NMDG, 150 aspartic acid, and 5 CaCl₂, 2 MgCl₂ and HEPES (pH adjusted to 7.35 with Tris base). The bath contained (in mM): 150 NMDG-Cl, 2 MgCl2, 5 EGTA, 10 TES and 14 Tris bases (pH adjusted to 7.35 HCl). After excision, both wt- and ΔF508CFTR were activated by the addition of 1 mM Mg-ATP, 75 nM cAMP-dependent protein kinase catalytic unit (PKA; Promega Corp. Madison, WI) and 10 mM NaF to inhibit protein phosphatases, which counteracted power outage. The pipette potential was maintained at 80 mV. Channel activity was analyzed for membrane fragments containing &lt;2 active channels. The maximum number of simultaneous openings determined the number of active channels during the experiment. To determine the amplitude of the intensity of a single channel, the data recorded from 120 s activity of ΔF508-CFTR was filtered off-line at 100 Hz, and then used to construct amplitude histograms with all points that were matched with multigauss functions using the Bio-Patch Analysis software (Bio Logic Comp. France). The total microscopic current and opening probability (Po) were determined from 120 s of channel activity. Po was determined using the Bio-Patch software or the relationship Po = I / i (N), where I = average current, i = current amplitude for a single channel, and N = the number of active channels in the fragment.

[0151] Cell culture [0152] NIH3T3 mouse fibroblasts stably expressing ΔP508-CFTR are used for patch clamp registration using dissected membranes. Cells are maintained at 37 ° C 5 in 5% CO2 and 90% humidity in Dulbecco's modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, β-ME, 1 X pen / strep and 25 mM HEPES at 175 cm² breeding bottles. For single-channel recording, 2500-5000 cells were plated on poly-L-lysine-coated coverslips and grown for 24-48 h at 27 ° C before use.

[0153] Form A of Compound 1 is useful as a modulator of transporters having an ATP binding cassette. EC was measured₅₀ ^ m) Form A of Compound 1 is less than 2.0 μΜ. The effectiveness of Form A of Compound 1 was calculated to be between 100% and 25%. It should be noted that 100% efficacy means the maximum response obtained with 4-methyl 2- (5-phenyl-1H-pyrazol-3-yl) phenol.