HK1158650B - 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 - Google Patents

Description

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

RELATED APPLICATIONS

The present application claims priority benefits of U.S. provisional application serial No. US61/107,813, filed on 23/10/2008, entitled "solid form of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide," the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to solid state forms, e.g., crystalline forms, of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide, which are cystic fibrosis transmembrane conductance regulator ("CFTR") modulators. The invention also relates to pharmaceutical compositions comprising crystalline forms of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide and methods of using the same.

Background

ATP cassette transporters (ATP cassette transporters) are a family of membrane transporters that regulate the transport of a wide range of pharmacological agents, potentially toxic drugs and exogenous chemicals, as well as anions. They are homologous membrane proteins that bind and use cellular Adenosine Triphosphate (ATP) to achieve their specific activity. It has been found that some of these transporters are multi-drug resistant proteins (e.g., MDR1-P glycoprotein, or multi-drug resistant protein MRP1) that protect malignant cancer cells from chemotherapeutic agents. To date, 48 such transporters have been identified, classified into families 7 based on their sequence identity and function.

One member of the ATP cassette transporter family commonly associated with disease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressed in a variety of cell types, including absorptive and secretory epithelial cells, where it regulates anion flow through the membrane, and regulates the activity of other ion channels and proteins. In epithelial cells, the normal function of CFTR is critical to maintain electrolyte transport throughout the body, including respiratory and digestive tissues. CFTR is composed of approximately 1480 amino acids that encode a protein composed of tandem repeats of transmembrane domains, each containing 6 transmembrane helices and a1 nucleotide binding domain. The 2 transmembrane domains are linked by a large, polar, regulatory (R) -domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (see Gregory, R.J. et al (1990) Nature 347: 382-Strata 386; Rich, D.P. et al (1990) Nature 347: 358-Strata 362), Riordan, J.R. et al (1989) Science 245: 1066-1073). Defects in this gene cause mutations in CFTR, leading to cystic fibrosis ("CF"), the most common fatal genetic disease of the human body. In the united states, cystic fibrosis affects about one in 2,500 infants. In the general U.S. population, up to 1 million people carry a single copy of such a defective gene, but without significant adverse effects. In contrast, individuals with 2 copies of genes associated with CF can develop debilitating and fatal effects of CF, including chronic lung disease.

In patients with cystic fibrosis, mutations in the endogenously expressed CFTR in the respiratory epithelium result in reduced anion secretion from the apical membrane, imbalanceing ion and fluid transport. The resulting decrease in anion transport leads to an increase in lung mucus accumulation in CF patients with microbial infections that ultimately lead to death. In addition to respiratory disease, CF patients typically have gastrointestinal problems and pancreatic insufficiency, which if left untreated, can lead to death. Furthermore, most men with cystic fibrosis are unable to give birth, while women with cystic fibrosis have reduced fertility. In contrast to the serious consequences of 2 copies of the CF associated gene, individuals carrying a single copy of the CF associated gene showed increased resistance to cholera and diarrhea-probably accounting for the relatively high frequency of the CF gene in this population.

Sequence analysis of the CFTR gene of the CF chromosome revealed numerous disease-causing mutations (Cutting, G.R. et al (1990) Nature 346: 366-. To date, more than 1000 disease-causing mutations in the CF gene have been identified (http:// www.genet.sickkids.on.ca/cftr /). The most common mutation is a deletion of phenylalanine at position 508 of the amino acid sequence of CFTR, commonly referred to as Δ F508-CFTR. This mutation occurs in about 70% of cystic fibrosis cases and is associated with severe disease.

Deletion of residue 508 in Δ F508-CFTR prevents the nascent protein from folding correctly. This results in the mutant protein not being able to exit the ER and reach the plasma membrane. As a result, the number of channels present in the membrane is much smaller than that observed in cells expressing wild-type CFTR. In addition to impaired trafficking, this mutation leads to defective channel gating. This adds up to the reduced number of channels in the membrane and the defect in gating leading to reduced anion transport through the epithelium, leading to defective ion and liquid transport (Quinton, P.M, (1990), FASEB J.4: 2709-. However, studies have shown that a reduced amount of af 508-CFTR in the membrane is functional, although less than wild-type CFTR. (Dolmans et al (1991), Nature Lond.354: 526-. In addition to Δ F508-CFTR, R117H-CFTR and G551D-CFTR, other disease-causing CFTR mutations that result in defects in trafficking, synthesis and/or channel gating can be up-or down-regulated to alter anion secretion and alter disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions, it is clear that this role (transport of anions, chloride and bicarbonate) represents one of the important mechanisms of transport of ions and water across epithelial cells. Other elements include epithelial Na + channelsENaC, Na +/2Cl-/K + cotransporter, Na⁺-K⁺ATP-ase Pump and basolateral Membrane K⁺Channels, which are responsible for the uptake of chloride ions into the cell.

These elements work together to achieve targeted transport across epithelial cells through their selective expression and localization in cells. Through ENaC and CFTR present on the apical membrane with Na expressed on the basolateral surface of the cell⁺-K⁺The coordinating activity between the ATPase pump and the Cl channel, the absorption of chloride ions takes place. The secondary active transport of chloride ions from the luminal side leads to intracellular chloride ion accumulation, which can then passively pass through Cl^-The ion channel leaves the cell, resulting in vector transport. Na on the outer surface of the substrate⁺/2Cl^-/K⁺Cotransporter, Na⁺-K⁺ATP-ase Pump and basolateral Membrane K⁺The arrangement of CFTR on the channel and luminal side coordinates the secretion of chloride ions by CFTR on the luminal side. Since water may never actively transport itself, it flows across the epithelium relying on a slight trans-epithelial osmotic gradient created by the bulk flux of sodium and chloride ions.

It is postulated that defects in bicarbonate transport due to mutations in CFTR result in defects in some secretory functions. See, for example, "cytotoxic fibrosis: an imperired bicarbonate session and brucoviscidosis, "Paul M.Quinton, Lancet 2008; 372: 415-417.

Mutations in CFTR associated with moderate CFTR dysfunction are also evident in patients with diseases that share some disease manifestations with CF but do not meet the diagnostic criteria for CF. They include congenital bilateral vasectomy, idiopathic chronic pancreatitis, chronic bronchitis, and chronic rhinosinusitis (rhinosinusitis). Other diseases in which mutant CFTR is considered to be a risk factor along with modifier genes or environmental factors include primary sclerosing cholangitis, allergic bronchopulmonary aspergillosis, and asthma.

Smoking, hypoxia and environmental factors that induce hypoxic signals have also been shown to impair CFTR function and may cause some forms of respiratory disease, such as chronic bronchitis. Diseases that may be due to defective CFTR function but do not meet the diagnostic criteria for CF are characterized as CFTR-associated diseases.

In addition to cystic fibrosis, modulation of CFTR activity is beneficial in other diseases that are not directly a result of CFTR mutation, such as CFTR-mediated secretory diseases and other protein folding diseases. CFTR regulates chloride and bicarbonate flow through many cellular epithelia to control fluid movement, protein solubilization, mucus viscosity, and enzyme activity. Defects in CFTR can lead to blockage of the airways or ducts in many organs, including the liver and pancreas. Enhancers are compounds that enhance the gating activity of CFTR present in the cell membrane. Any disease involving mucus thickening, impaired fluid regulation, impaired mucus clearance, or blocked conduits leading to inflammation and tissue destruction may be candidates for enhancers.

These diseases include, but are not limited to, Chronic Obstructive Pulmonary Disease (COPD), asthma, smoking-induced COPD, chronic bronchitis, rhinosinusitis, constipation, dry eye, sjogren's syndrome, gastroesophageal reflux disease, gallstones, rectal prolapse, and inflammatory bowel disease. COPD is characterized by progressive and non-fully reversible airflow limitation. Airflow limitation is due to mucus hypersecretion, emphysema, and bronchiolitis. Activators of mutant or wild-type CFTR provide a potential treatment for mucus hypersecretion and impaired mucociliary clearance common in COPD. Specifically, increasing the secretion of anions across the CFTR promotes fluid transport into airway surface liquids to hydrate mucus and optimize periciliary fluid viscosity. This results in enhanced mucociliary clearance and a reduction in symptoms associated with COPD. In addition, by preventing the resulting progressive infection and inflammation (due to improved airway clearance), CFTR modulators can prevent or slow down the destruction of airway parenchyma and reduce or reverse the increase in the number and size of mucus secreting cells that underlie mucus hypersecretion in airway disease characterized by emphysema. Dry eye disease is characterized by reduced tear production and abnormal tear film fluid, protein and mucin properties. 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. Increased anion secretion by CFTR can increase fluid transport from the corneal endothelial cells and secretory glands surrounding the eye to enhance corneal hydration. This helps to alleviate symptoms associated with dry eye. Sjogren's syndrome is an autoimmune disease in which the immune system attacks the systemic fluid-producing glands, including the eye, mouth, skin, respiratory tissues, liver, vagina and digestive tract. Symptoms include dry eyes, mouth and vagina, and lung disease. The disease is also associated with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, and polymyositis/dermatomyositis. Protein trafficking defects are thought to cause the disease, thereby limiting treatment options. Modulators of CFTR activity may hydrate the various organs affected by the disease and help alleviate the associated symptoms. Individuals with cystic fibrosis have repeated episodes of ileus and high incidences of rectal prolapse, gallstones, gastroesophageal reflux disease, GI malignancies, and inflammatory bowel disease, suggesting that CFTR function may play an important role in preventing such diseases.

As described above, it is believed that the deletion of residue 508 in Δ F508-CFTR prevents the nascent protein from folding correctly, resulting in the inability of the mutant protein to exit the ER and be transported to the plasma membrane. As a result, insufficient amounts of mature protein are present in the plasma membrane and chloride transport within epithelial tissues is significantly reduced. In fact, this cellular phenomenon of ER deficiency, which processes CFTR via the ER mechanism, has been shown to be the underlying basis not only for CF disease, but also for a wide range of other independent and genetic diseases. Two pathways leading to a failure of the ER mechanism are degradation by loss of coupling to ER export of proteins (coupling), or ER accumulation by these defective/misfolded proteins [ Aridor M et al, Nature med., 5(7), pp745-751 (1999); shasty, B.S. et al, neurochem.International, 43, pp 1-7 (2003); rutishauser, J, et al, Swiss Med Wkly, 132, pp 211-; morello, JP et al, TIPS, 21, pp.466-469 (2000); bross P. et al, HumanMut., 14, pp.186-198(1999) ]. The diseases associated with ER dysfunction of the first category are cystic fibrosis (caused by misfolded AF 508-CFTR as described above), hereditary emphysema (caused by a 1-antitrypsin; non-Piz variants), hereditary hemochromatosis, coagulation-fibrinolysis defects (coagulolysis-defibrinogenetics), such as protein C deficiency, hereditary angioedema type 1, lipid processing defects, such as familial hypercholesterolemia, chylomicronemia type 1, betalipoproteinemia, lysosomal storage diseases, such as I-cell disease/pseudohurler disease (pseudoudo-Hurler), mucopolysaccharidosis (caused by lysosomal processing enzymes), Sandhof/Tay-Sachs (caused by beta-hexosaminidase), Crigler-Najjar type II (caused by UDP-glucose-transferase-type II), hereditary pulmonary emphysema-fibrinolysis disorders (stroke-diabetes-type I-cell disease), mucopolysaccharidosis (caused by lysosome processing enzymes), and Alzheimer's disease (sandhoff-Sachs), and Creutzfeld-Najjar (caused by beta-hexosaminidase) ) Multiple endocrine adenopathy/hyperinsulinemia, diabetes (caused by insulin receptors), Laron dwarfism (Laron dwarfism) (caused by growth hormone receptors), myeloperoxidase deficiency, primary hypoparathyroidism (caused by pro-parathyroid prohormone), melanoma (caused by tyrosinase). Diseases associated with the latter class of ER dysfunction are Glycanossis CDG type 1 (hypoglycoprotein syndrome), hereditary emphysema (caused by alpha 1-antitrypsin (PiZ variant)), congenital hyperthyroidism, osteogenesis imperfecta (caused by procollagen types I, II, IV), hereditary hypofibrinogenemia (caused by fibrinogen), ACT deficiency (caused by alpha 1-antichymotrypsin), Diabetes Insipidus (DI), neurogenic (neuropygian) DI (caused by vasopressin hormone/V2-receptor), renal DI (caused by aquaporin-II), Charcot-Marie-Tooth syndrome (caused by peripheral myelin protein 22), Pai-Mei disease, neurodegenerative diseases, such as Alzheimer's disease (caused by beta APP and senescent proteins), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine (polyglutamine) neurological disorders, such as Huntington's disease, spinocerebellar ataxia type I (spinocerebellar ataxia), spinobulbar muscular atrophy, dentatorubral pallidoluysical hypothalamic atrophy, and myotonic dystrophy, and spongiform encephalopathies, such as Creutzfeldt-Jakob disease (caused by a defect in prion protein processing), Fabry's disease (caused by lysosomal α -galactosidase A), Straussler-Scheinker syndrome (caused by a defect in Prp processing), infertility, pancreatitis, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, Gorham's channel pathology (chloridizelopaphia), congenital myotonic muscle spasticity (Thomson and Becker types), Debarter's syndrome type III, Dehybride's disease, seismoexia syndrome (perk syndrome), Epilepsy, startle syndrome, lysosomal storage disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with side-to-side transposition (also known as catagen syndrome), PCD without side-to-side transposition and ciliary dysplasia (ciriary atlas) and liver disease.

Other diseases associated with mutations in CFTR include male infertility due to Congenital Bilateral Absence of Vas Deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis, and allergic bronchopulmonary aspergillosis (ABPA). See "CFTR-opathies: discrete phenotypes associated with cyclic synthesis regulator genes, "peer g.noone and Michael r.knowles, respir.res.2001, 2: 328-332 (incorporated herein by reference).

In addition to upregulating CFTR activity, the reduction of anion secretion by CFTR modulators may be beneficial for the treatment of secretory diarrhoea, in which epithelial water transport is significantly increased due to secretagogue activated chloride transport. The mechanisms include elevation of cAMP and stimulation of CFTR.

Although there are many causes of diarrhea, the major consequences of diarrheal disease caused by excessive chloride ion transport are common, including dehydration, acidosis, impaired development and death. Acute and chronic diarrhea represent a major medical problem in many areas of the world. Diarrhea is an important factor in malnutrition and is the leading cause of death in children under 5 years of age (5,000,000 deaths per year).

Secretory diarrhea is also a dangerous condition in patients with acquired immunodeficiency syndrome (AIDS) and chronic Inflammatory Bowel Disease (IBD). Each year 1600 million travelers from developed to developing countries develop diarrhea, the severity and number of cases of diarrhea varying with the country and region traveled.

Thus, there is a need for potent and selective CFTR enhancers for human CFTR wild-type and mutant forms. These mutant CFTRs include, but are not limited to, Δ F508del, G551D, R117H, 2789+5G- > A.

There is also a need for modulators of CFTR activity and compositions thereof, which can be used to modulate CFTR activity in mammalian cell membranes.

There is a need for methods of treating diseases caused by mutations in CFTR using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivo cell membrane of a mammal.

Furthermore, there is a need for stable solid forms of said compounds which are easy to use in pharmaceutical compositions suitable for use as therapeutic agents.

Summary of The Invention

The present invention relates to solid forms of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (hereinafter "compound 1") having the following structure:

compound 1 and pharmaceutically acceptable compositions thereof are useful in the treatment or lessening the severity of a variety of diseases, disorders, or conditions, including, but not limited to, cystic fibrosis, pancreatitis, sinusitis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, e.g., protein C deficiency, hereditary angioedema type 1, lipid processing deficiencies, e.g., familial hypercholesterolemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohulerosis, mucopolysaccharidoses, Sanhoff disease/Tay-Sachs disease, Creutzfeldt-Jakob-Naphur syndrome type II, polyendocrinopathy/hyperinsulinemia, diabetes, Larren dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, Glycanosis CDG type 1, Hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, charcot-marie syndrome, pelizaeus-merzbacher disease, neurodegenerative diseases, such as alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders, such as huntington's disease, spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubral pallidoluysian atrophy and myotonic dystrophy, and spongiform encephalopathies, such as hereditary creutzfeldt-jakob disease, fabry disease, Straussler-Scheinker syndrome, COPD, xerophthalmia, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, chloride tunnel disease, osteoporosis, alzheimer's disease, parkinson's, Congenital myotonia (Thomson and Becker types), barter syndrome type III, Dent's disease, startle syndrome, epilepsy, startle syndrome, lysosomal storage disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with left and right transposition (also known as catagory syndrome), PCD without left and right transposition and ciliary dysplasia and sjogren's disease.

In one aspect, compound 1 is form a (form a) in substantially crystalline neat free form.

The processes described herein can be used to prepare the compositions of the present invention comprising form a. The amounts and characteristics of the ingredients used in the process are as described herein.

Brief Description of Drawings

Figure 1 is an X-ray powder diffraction pattern of form a.

Figure 2 is a conformational diagram of form a based on single X-ray crystal analysis.

Figure 3 provides FTIR spectra of form a.

Detailed Description

Definition of

The following definitions as used herein shall apply unless otherwise stated.

The term "ABC-transporter" as used herein refers to an ABC-transporter or a fragment thereof comprising at least one binding domain, wherein the protein or fragment thereof is present in vivo or in vitro. The term "binding domain" as used herein refers to a domain on an ABC-transporter that can bind to a modulator. See, e.g., Hwang, t.c. et al, j.gen.physiol. (1998): 111(3),477-90.

The term "CFTR" as used herein means a cystic fibrosis transmembrane conductance regulator or mutant with regulator activity thereof, including but not limited to Δ F508CFTR, R117H CFTR and G551D CFTR (see, for example, http:// www.genet.sickkids.on.ca/CFTR/, for CFTR mutations).

The term "modulate" as used herein means to increase or decrease in a measurable amount.

The term "normal CFTR" or "normal CFTR function" as used herein refers to a CFTR similar to the wild type without any damage due to environmental factors such as smoking, pollution or any condition that produces inflammation in the lungs.

The term "reduced CFTR" or "reduced CFTR function" as used herein refers to less than normal CFTR or less than normal CFTR function.

As used herein, "crystalline" means a compound or composition in which the structural units are arranged in a fixed geometric pattern or lattice such that the crystalline solid has a rigid, large range of order. The structural units constituting the crystal structure may be atoms, molecules or ions. Crystalline solids exhibit a defined melting point.

The term "substantially crystalline" as used herein means a solid substance arranged in a fixed geometric pattern or lattice having a rigid, large range of order. For example, a substantially crystalline material has a crystallinity greater than about 85% (e.g., greater than about 90% crystallinity or greater than about 95% crystallinity). It should also be noted that the term "substantially crystalline" includes the subject word "crystalline" as defined in the preceding paragraph.

In one aspect, the invention features a form of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide characterized in that it is crystalline form a.

In some embodiments, form a is characterized by one or more of the following peaks in an X-ray powder diffraction obtained using Cu ka radiation: about 7.7 to about 8.1 degrees, such as about 7.9 degrees; about 11.7 to about 12.1 degrees, such as about 11.9 degrees; about 14.2 to about 14.6 degrees, such as about 14.4 degrees; and a peak at about 15.6 to about 16.0 degrees, e.g., about 15.8 degrees.

In some embodiments, form a is characterized by one or more of the following peaks in an X-ray powder diffraction obtained using Cu ka radiation: about 7.8 to about 8.0 degrees, such as about 7.9 degrees; from about 11.8 to about 12.0 degrees, such as about 11.9 degrees; about 14.3 to about 14.5 degrees, such as about 14.4 degrees; and about 15.7 to about 15.9 degrees, such as about 15.8 degrees.

In other embodiments, form a is characterized by one or more of the following peaks in an X-ray powder diffraction obtained using Cu ka radiation: about 7.7 to about 8.1 degrees, such as about 7.9 degrees; about 21.6 to about 22.0 degrees, such as about 21.8 degrees; and about 23.6 to about 24.0 degrees, such as about 23.8 degrees.

In other embodiments, form a is characterized by one or more of the following peaks in an X-ray powder diffraction obtained using Cu ka radiation: about 7.8 to about 8.0 degrees, such as about 7.9 degrees; about 21.7 to about 21.9 degrees, such as about 21.8 degrees; and about 23.7 to about 23.9 degrees, such as about 23.8 degrees.

In some embodiments, form a is characterized by one or more of the following peaks measured in degrees in an X-ray powder diffraction pattern: a peak at about 7.7 to about 8.1 degrees (e.g., about 7.9 degrees); a peak at about 9.1 to about 9.5 degrees (e.g., about 9.3 degrees); a peak at about 11.7 to about 12.1 degrees (e.g., about 11.9 degrees); a peak at about 14.2 to about 14.6 degrees (e.g., about 14.4 degrees); a peak at about 14.9 to about 15.3 degrees (e.g., about 15.1 degrees); a peak at about 15.6 to about 16.0 degrees (e.g., about 15.8 degrees); a peak at about 16.8 to about 17.2 degrees (e.g., about 17.0 degrees); a peak at about 17.5 to about 17.9 degrees (e.g., about 17.7 degrees); a peak at about 19.1 to about 19.5 degrees (e.g., about 19.3 degrees); a peak at about 19.9 to about 20.3 degrees (e.g., about 20.1 degrees); a peak at about 21.2 to about 21.6 degrees (e.g., about 21.4 degrees); a peak at about 21.6 to about 22.0 degrees (e.g., about 21.8 degrees); a peak at about 23.2 to about 23.6 degrees (e.g., about 23.4 degrees); a peak at about 23.6 to about 24.0 degrees (e.g., about 23.8 degrees); a peak at about 25.4 to about 25.8 degrees (e.g., about 25.6 degrees); a peak at about 26.6 to about 27.0 degrees (e.g., about 26.8 degrees); a peak at about 29.2 to about 29.6 degrees (e.g., about 29.4 degrees); a peak at about 29.5 to about 29.9 degrees (e.g., about 29.7 degrees); a peak at about 29.9 to about 30.3 degrees (e.g., about 30.1 degrees); and a peak at about 31.0 to about 31.4 degrees (e.g., about 31.2 degrees).

In some embodiments, form a is characterized by one or more of the following peaks measured in degrees in an X-ray powder diffraction pattern: a peak at about 7.8 to about 8.0 degrees (e.g., about 7.9 degrees); a peak at about 9.2 to about 9.4 degrees (e.g., about 9.3 degrees); a peak at about 11.8 to about 12.0 degrees (e.g., about 11.9 degrees); a peak at about 14.3 to about 14.5 degrees (e.g., about 14.4 degrees); a peak at about 15.0 to about 15.2 degrees (e.g., about 15.1 degrees); a peak at about 15.7 to about 15.9 degrees (e.g., about 15.8 degrees); a peak at about 16.9 to about 17.1 degrees (e.g., about 17.0 degrees); a peak at about 17.6 to about 17.8 degrees (e.g., about 17.7 degrees); a peak at about 19.2 to about 19.4 degrees (e.g., about 19.3 degrees); a peak at about 20.0 to about 20.2 degrees (e.g., about 20.1 degrees); a peak at about 21.3 to about 21.5 degrees (e.g., about 21.4 degrees); a peak at about 21.7 to about 21.9 degrees (e.g., about 21.8 degrees); a peak at about 23.3 to about 23.5 degrees (e.g., about 23.4 degrees); a peak at about 23.7 to about 23.9 degrees (e.g., about 23.8 degrees); a peak at about 25.5 to about 25.7 degrees (e.g., about 25.6 degrees); a peak at about 26.7 to about 26.9 degrees (e.g., about 26.8 degrees); a peak at about 29.3 to about 29.5 degrees (e.g., about 29.4 degrees); a peak at about 29.6 to about 29.8 degrees (e.g., about 29.7 degrees); a peak at about 30.0 to about 30.2 degrees (e.g., about 30.1 degrees); and a peak at about 31.1 to about 31.3 degrees (e.g., about 31.2 degrees).

In some embodiments, form a is characterized by a diffraction pattern as provided in figure 1.

In one aspect, the invention features a pharmaceutical composition comprising form a and a pharmaceutically acceptable adjuvant or carrier.

In one aspect, the invention features a method of treating a CFTR mediated disease in a human, comprising administering to the human an effective amount of form a.

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

In some embodiments, the disease is selected from cystic fibrosis, pancreatitis, sinusitis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis defects, e.g., protein C defect, hereditary angioedema type 1, lipid processing defects, e.g., familial hypercholesterolemia, chylomicronemia type 1, abeta-lipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohuler disease, mucopolysaccharidosis, sandhoff/tay-sachs disease, creutzfeldt-jakob syndrome type II, polyendocrinopathy/hyperinsulinemia, diabetes, larron dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, Glycanosis CDG type 1, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, and multiple sclerosis, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, Charcot-Marie-Turkey syndrome, Peyer-Meyer's disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders, such as Huntington's disease, spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and myotonic dystrophy, as well as spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, xerophthalmia, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, chloride tunnel disease, congenital myotonia (Thomson and Becker types), Batt syndrome type III, Dent's disease, panic syndrome, shock syndrome, Epilepsy, startle syndrome, lysosomal storage disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with side-to-side transposition (also known as catagory syndrome), PCD without side-to-side transposition and ciliary dysplasia, and sjogren's disease.

In one embodiment, the present invention provides a method of treating cystic fibrosis in a human comprising administering to the human an effective amount of form a.

In one aspect, the invention features a pharmaceutical pack or kit comprising form a and a pharmaceutically acceptable carrier.

In one aspect, the invention features N- (4- (7-azabicyclo [2.2.1]]A crystalline form of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide having a trigonal crystal system (trigonal crystal system), an R-3 space group and the following unit cell dimensions:α is 90 °, β is 90 °, and γ is 120 °.

In one embodiment, the invention provides N- (4- (7-azabicyclo [2.2.1] s]A crystalline form of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide having the following unit cell dimensions:

use, formulation and administration

Pharmaceutically acceptable compositions

In one aspect of the invention pharmaceutically acceptable compositions are provided, wherein these compositions comprise form a as described herein and optionally comprise a pharmaceutically acceptable carrier, adjuvant or excipient. In some embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

As noted above, the pharmaceutically acceptable compositions of the present invention also comprise a pharmaceutically acceptable carrier, adjuvant or excipient, which, as used herein, includes any and all solvents, diluents or other liquid carriers, dispersing or suspending agents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 16 th edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980) describe various carriers used in formulating pharmaceutically acceptable compositions and known techniques for preparing them. In addition to any conventional carrier media which is incompatible with the compounds of the present invention, e.g., by causing any undesirable biological effect or by interfering in a deleterious manner with any other component of the pharmaceutically acceptable composition, the use of any conventional carrier media is also contemplated as falling within the scope of the present invention. Some examples of substances that can be used 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, partial glyceride mixtures of saturated 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, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, lanolin, 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 powder; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive 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; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

Use of compounds and pharmaceutically acceptable compositions

In another aspect, the invention provides a method of treating or lessening the severity of a condition, disease or disorder associated with a mutation in CFTR. In some embodiments, the present invention provides a method of treating a condition, disease, or disorder associated with a deficiency in CFTR activity, comprising administering to a subject, preferably a mammal, in need thereof a composition comprising compound 1 form a.

In some embodiments, the invention provides a method of treating a disease associated with reduced CFTR function due to a mutation in a gene encoding CFTR or environmental factors (e.g., smoking). These include cystic fibrosis, chronic bronchitis, recurrent bronchitis, acute bronchitis, male infertility due to congenital bilateral vasectomy (CBAVD), female infertility due to congenital uterine and vaginal deficits (CAUV), Idiopathic Chronic Pancreatitis (ICP), idiopathic recurrent pancreatitis, idiopathic acute pancreatitis, chronic rhinosinusitis, primary sclerosing cholangitis, allergic bronchopulmonary aspergillosis, diabetes, dry eye, constipation, allergic bronchopulmonary aspergillosis (ABPA), bone diseases (e.g., osteoporosis), and asthma.

In some embodiments, the invention provides a method of treating a disease associated with normal CFTR function. These diseases include Chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis, constipation, pancreatitis, including chronic pancreatitis, recurrent pancreatitis and acute pancreatitis, pancreatic insufficiency, male infertility due to congenital bilateral absence of the vas deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis, liver disease, hereditary emphysema, gallstones, gastroesophageal reflux disease, gastrointestinal malignancies, inflammatory bowel disease, constipation, diabetes, arthritis, osteoporosis, and osteopenia.

In some embodiments, the invention provides a method of treating a disease associated with normal CFTR function, including hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, e.g., protein C deficiency, hereditary angioedema type 1, lipid processing deficiencies, e.g., familial hypercholesterolemia, chylomicronemia type 1, abeta-lipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohulerian disease, mucopolysaccharidoses, sandhoff/tay-sachs disease, cre-na syndrome type II, polyendocrinopathy/hyperinsulinemia, diabetes, larron dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, Glycanosis CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, diabetes, dwarfism, achlordosis, diabetes mellitus type 1, and the like, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, Charcot-Marie-Tourette syndrome, Pepper-Meyer's disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders, such as Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and myotonic dystrophy, and spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease (caused by defects in prion protein processing), Fabry disease, Straussler-Scheinker syndrome, Gorham syndrome, chloride channel disorders, myotonia congenital myotonia (Thomson and Becker types), Batt syndrome type III, Dent's disease, shock syndrome, epilepsy, shock syndrome, lysosomal disease, depot disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with side-to-side transposition (also known as catagory syndrome), PCD without side-to-side transposition and ciliary dysplasia, or sjogren's disease, comprising the step of administering to said mammal an effective amount of a composition comprising crystalline form a described herein.

According to an alternative preferred embodiment, the present invention provides a method of treating cystic fibrosis comprising the step of administering to said mammal a composition comprising the step of administering to said mammal an effective amount of a composition comprising form a as described herein.

According to the present invention, an "effective amount" of form a or a pharmaceutically acceptable composition is an amount effective to treat or reduce the severity of one or more diseases, disorders or conditions as described above.

Form a or a pharmaceutically acceptable composition thereof may be administered using any amount and any route of administration effective to treat or reduce the severity of one or more diseases, disorders, or conditions as described above.

In some embodiments, form a or a pharmaceutically acceptable composition thereof is suitable for treating or lessening the severity of cystic fibrosis in a patient who exhibits residual CFTR activity in the apical membrane of the respiratory epithelium or non-respiratory epithelium. The presence of residual CFTR activity on the surface of epithelial cells can be readily detected using methods known in the art, such as standard electrophysiological, biochemical or histochemical techniques. These methods use in vivo or ex vivo electrophysiological techniques, sweat or saliva Cl^-Concentration measurements or ex vivo biochemical or histochemical techniques to monitor cell surface density to identify CFTR activity. Using these methods, residual can be readily detected in patients who are heterozygous or homozygous for a variety of different mutants, including patients who are heterozygous or homozygous for the most common mutant, Δ F508CFTR activity of (a).

In another embodiment, form a or a pharmaceutically acceptable composition thereof described herein is suitable for treating or lessening the severity of cystic fibrosis in a patient having residual CFTR activity induced or enhanced with pharmacological methods or gene therapy. These methods increase the amount of CFTR present on the cell surface, thereby inducing a hitherto absent CFTR activity in the patient or increasing the existing level of residual CFTR activity in the patient.

In another embodiment, form A or a pharmaceutically acceptable composition thereof described herein is suitable for use in or for lessening the severity of cystic fibrosis in a patient having a genotype indicative of residual CFTR activity, e.g., a class III mutation (impaired regulation or gating), a class IV mutation (altered Conductance), or a class V mutation (reduced synthesis) (Lee R.Choo-Kang, Pamela L., Zeitinin, Type I, II, III, IV, and V cysteine fibrous nucleic acid construct Regulator Defects and Opportunities of therapy, Current Opinion in Pulmonary Medicine 6: 521-. Other patient genotypes that exhibit residual CFTR activity include patients that are homozygous for one of these types or heterozygous for any other type of mutation, including a class I mutation, a class II mutation, or a mutation without classification.

In one embodiment, form a or a pharmaceutically acceptable composition thereof described herein is suitable for treating or lessening the severity of cystic fibrosis in a patient having certain clinical phenotypes, e.g., mild to moderate clinical phenotypes typically associated with an amount of residual CFTR activity in the apical membrane of epithelial cells. These phenotypes include patients exhibiting pancreatic insufficiency or diagnosed with idiopathic pancreatitis and congenital bilateral absence of the vas deferens or mild lung disease.

The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the infection, the particular drug, its mode of administration, and the like. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to physically discrete units of a drug suitable for the patient to be treated. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend upon a variety of factors including the disease to be treated and the severity of the disease; the activity of the particular compound used; the specific composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed, and other factors well known in the medical arts. The term "patient" as used herein refers to an animal, preferably a mammal, most preferably a human.

The pharmaceutically acceptable compositions of the present invention may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powders, ointments, drops, or patches), bucally, and as an oral or nasal spray, depending on the severity of the infection to be treated. In some embodiments, the compounds of the present invention may be administered orally or parenterally at dosage levels of from about 0.01mg/kg to about 50mg/kg, preferably from about 0.5mg/kg to about 25mg/kg, of the subject's body weight once or more times daily to achieve the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers 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, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents, for example, sterile injectable aqueous or oleaginous suspensions. 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 excipients or solvents that may be used are water, ringer's solution, u.s.p. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (e.g., oleic acid) may be used in the preparation of injectables.

Injectable formulations 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 just prior to use.

In order to prolong the effect of the compounds of the invention, it is often desirable to slow the absorption of the compounds injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of the compound will then depend on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delaying absorption of a parenterally administered compound form may be achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot dosage forms (depot forms) are prepared by forming microencapsule matrices of the compounds in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer used, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injections can also be prepared by entrapping the compound in liposomes or microemulsions which are compatible with body tissues.

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these 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 extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite, 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 forms may also comprise buffering agents.

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 and the like. Solid dosage forms of tablets, dragees (dragees), capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents, and may also be of a composition: they release one or more active ingredients only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may 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 and the like.

The active compound may also be present in microencapsulated form with one or more of the above-mentioned excipients. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulating art. In these solid dosage forms, the active compound may be mixed with at least one inert diluent, for example sucrose, lactose or starch. These dosage forms may also, for example, contain, as is common practice, other substances than inert diluents, e.g., tableting lubricants and other tableting 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 optionally contain opacifying agents, and may also be of a composition: they release one or more active ingredients only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymers and waxes.

Dosage forms for topical or transdermal administration of the compounds of the invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers which may be required. Ophthalmic formulations, ear drops and eye drops are also encompassed within the scope of the present invention. Furthermore, the present invention encompasses the use of transdermal patches, which have the additional advantage of controllably delivering the compound to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be used to increase the flux 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.

It will also be appreciated that form a or a pharmaceutically acceptable composition thereof described herein may be used in combination therapy, that is, form a or a pharmaceutically acceptable composition thereof described herein may be administered simultaneously, prior to, or after one or more additional desired treatments or medical procedures. The particular combination of treatments (treatments or manipulations) used in a combination treatment regimen will take into account the compatibility of the desired therapeutic effect to be achieved with the desired treatment and/or manipulation. It will also be appreciated that the treatments used may achieve the desired effect on the same disease (e.g. the compounds of the invention may be administered simultaneously with another drug used to treat the same disease), or they may achieve different effects (e.g. control of any adverse effects). As used herein, an additional therapeutic agent that treats or prevents a particular disease or condition by normal administration is referred to as "appropriate for the disease or condition being treated.

In one embodiment, the additional active agent is selected from the group consisting of mucolytics (mucolytics), bronchodilators, antibiotics, anti-infective agents, anti-inflammatory agents, CFTR modulators or nutritional agents other than the compounds of the invention.

In one embodiment, the additional active agent is an antibiotic. Typical antibiotics for use herein include tobramycin, including Tobramycin Inhalation Powder (TIP), azithromycin, aztreonam, aerosol forms including aztreonam, amikacin, including liposomal formulations thereof, ciprofloxacin, including formulations thereof suitable for administration by inhalation, levofloxacin, including aerosol formulations thereof, and combinations of two antibiotics, such as fosfomycin and tobramycin.

In another embodiment, the additional active agent is a mucolytic (mucolytics). Exemplary mucolytics for use herein include

In another embodiment, the additional active agent is a bronchodilator. Typical bronchodilators include salbutamol, metaproterol sulfate, pirbuterol acetate, salmeterol or tetrabulin sulfate.

In another embodiment, the additional active agent is effective to restore lung airway surface liquid. Such agents improve the movement of salts inside and outside the cell, making the mucus in the lung airways more easily hydrated and thus more easily cleared. Typical such agents include hypertonic physiological saline, denufosol sodium ([ [ (3S, 5R) -5- (4-amino-2-oxopyrimidin-1-yl) -3-hydroxyoxocyclopenta-2-yl ] methoxy-hydroxyphosphoryl ] [ [ (2R, 3S, 4R, 5R) -5- (2, 4-dioxopyrimidin-1-yl) -3, 4-dihydroxyoxocyclopenta-2-yl ] methoxy-hydroxyphosphoryl ] oxy-hydroxyphosphoryl ] hydrogen phosphate) or bronchitol (an inhalation formulation of mannitol).

In another embodiment, the additional active agent is an anti-inflammatory agent, i.e. an active agent that can reduce inflammation in the lungs. Typical such active agents for use herein include ibuprofen, docosahexaenoic acid (DHA), sildenafil, inhaled glutathione, pioglitazone, hydroxychloroquine or simvastatin (simvastatin).

In another embodiment, the additional active agent reduces the activity of an epithelial sodium channel blocker (ENaC) (either directly by blocking the channel or indirectly by modulating a protease (e.g., serine protease, channel activating protease) that results in an increase in ENaC activity). Typical such agents include camostat (a trypsin-like protease inhibitor), QAU145, 552-02, GS-9411, INO-4995, Aerolytic and amiloride. Additional active agents that reduce the activity of epithelial sodium channel blockers (enacs) can be found, for example, in PCT publication No. WO2009/074575, the entire contents of which are incorporated herein by reference in their entirety.

In other diseases described herein, a CFTR modulator, e.g., form A, in combination with an agent that reduces ENaC activity is useful in treating Liddle syndrome, inflammatory or allergic conditions, including cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive pulmonary disease, asthma, respiratory infections, lung cancer, xerostomia and keratoconjunctivitis sicca (keratojunctional sis), respiratory infections (acute and chronic; viral and bacterial), and lung cancer.

The combination of a CFTR modulator, such as form a, with an agent that reduces ENaC activity is also useful in the treatment of diseases that block epithelial sodium channel mediated, including diseases other than respiratory diseases, which are associated with dysregulation of fluid through the epithelium, possibly involving physiological abnormalities of the protective surface liquid on its surface, such as xerostomia (dry mouth) or keratoconjunctivitis sicca (dry eye). Furthermore, blocking the epithelial sodium channel in the kidney may be used to promote diuresis and thereby induce a hypotensive effect.

Asthma includes intrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchial asthma, exercise-induced asthma, occupational asthma and asthma induced following bacterial infection. Treatment of asthma is also to be understood as including treatment of subjects below 4 or 5 years of age exhibiting symptoms of wheezing and diagnosed or diagnosable as "wheezy infants", established patient categories of major medical concern and patients currently identified as typically having initial or early wheezing (for convenience this particular asthmatic condition is referred to as "wheezy infant syndrome"). Prophylactic efficacy in the treatment of asthma is evidenced by a reduction in the frequency or severity of symptomatic attack, e.g., of acute asthma or bronchoconstrictor attack, improvement in lung function, or improvement in airway hyperreactivity. It may further be evidenced by a reduced need for other palliative therapies, i.e., therapies used or expected to limit or interrupt the onset of symptoms at the time of onset, such as anti-inflammatory (e.g., corticosteroids) or bronchodilation. In particular, prophylactic benefit in asthma is evident in subjects prone to "morning dipping". "morning dipping" is a recognized asthma syndrome, common to a large percentage of asthmatic patients and characterized by asthma attacks, e.g., a few hours between about 4-6am, i.e., at a time generally substantially distant from any previously administered palliative asthma therapy.

Chronic obstructive pulmonary diseases include chronic bronchitis or dyspnea associated therewith, emphysema, and exacerbation of airway hyperreactivity following other drug therapies, particularly other inhaled drug therapies. In some embodiments, a CFTR modulator, e.g., crystalline form a, in combination with an agent that reduces ENaC activity is used to treat bronchitis, of whatever type or origin, including, e.g., acute bronchitis, arachidic bronchitis, catarrhal bronchitis, croupus bronchitis, chronic bronchitis, or tuberculous bronchitis.

In another embodiment, the additional agent is a CFTR modulator that is not compound 1 form a, i.e., an agent that has the effect of modulating CFTR activity. Typical such agents include astaluren (r) ((r))3- [5- (2-fluorophenyl) -1, 2, 4-oxadiazol-3-yl]Benzoic acid), sinapultide, lanokift, dilactat (human recombinant neutrophil elastase inhibitor), coprostane (7- { (2R, 4aR, 5R, 7aR) -2- [ (3S) -1, 1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta [ b]Pyran-5-yl } 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 active agent is (3- (6- (1- (2, 2-difluorobenzo [ d ]))][1，3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid.

In another embodiment, the additional active agent is a nutritional agent. Typical such agents include pancreatic lipase (pancreatic enzyme substitute), includingOr(prior one))、 Or glutathione inhalation. In one embodiment, the other nutritional agent is pancrelipase.

In one embodiment, the additional agent is a CFTR modulator that is not a compound of the invention.

The amount of the other therapeutic agent in the composition of the present invention will not exceed the amount normally administered in a composition containing the therapeutic agent as the only active ingredient. Preferably, the amount of the other therapeutic agent in the presently disclosed compositions will be about 50% to 100% of the content in a typical composition containing the therapeutic agent as the only therapeutically active agent.

Form a or a pharmaceutically acceptable composition thereof described herein may also be incorporated into compositions that coat implantable medical devices, such as prostheses, prosthetic valves, vascular grafts, stents and catheters. Thus, the present invention, in another aspect, includes a composition for coating an implantable device comprising a compound of the present invention as generally described above and in the classes and subclasses herein, and a carrier suitable for coating said implantable device. In another aspect, the present invention includes an implantable device coated with a composition comprising a compound of the present invention as generally described above and in the classes and subclasses herein, and a carrier suitable for coating the implantable device. General preparation of suitable coatings and coated implantable devices is described in U.S. Pat. nos. 6,099,562, 5,886,026, and 5,304,121. The coating is typically a biocompatible polymeric material such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate and mixtures thereof. The coating may optionally be further covered with a surface layer of a suitable fluorosilicone, polysaccharide, polyethylene glycol, phospholipid, or combinations thereof to impart controlled release characteristics to the composition.

Another aspect of the invention relates to modulating CFTR activity (e.g., in vitro or in vivo) in a biological sample or patient, comprising administering to or contacting the patient form a or a pharmaceutically acceptable composition thereof described herein. The term "biological sample" as used herein includes, without limitation, cell cultures and extracts thereof; biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Modulation of CFTR in a biological sample can be used for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, the study of CFTR in biological and pathological phenomena; and comparative evaluation of novel CFTR modulators.

In another embodiment, there is provided a method of modulating the activity of an anion channel in vitro or in vivo comprising the step of contacting the channel with form a or a pharmaceutically acceptable composition thereof described herein. In a preferred embodiment, the anion channel is a chloride channel or a bicarbonate channel. In other preferred embodiments, the anion channel is a chloride channel.

According to an alternative embodiment, the present invention provides a method of increasing the number of functional CFTR in a cell membrane, said method comprising the step of contacting said cell with form a or a pharmaceutically acceptable composition thereof as described herein.

According to another preferred embodiment, the activity of CFTR is determined by measuring the transmembrane voltage potential. The means for measuring the voltage potential across the membrane in a biological sample may employ any method known in the art, such as optical membrane potentiometry or other electrophysiological methods.

Optical membrane potential assays employ voltage-sensitive FRET sensors as described by Gonzalez and Tsien (see Gonzalez, J.E., and R.Y.Tsien (1995) "Voltage sensing by fluorescence response energy transfer in single cells," Biophys J69 (4): 1272-80 and Gonzalez, J.E.and R.Y.Tsien (1997), "Improved indicators of fluorescence response energy transfer" Chem Biol 4 (4): 269-77) in combination with an instrument for measuring fluorescence change, such as a Voltage/ion Probe Reader (PR) (see Gonzalez, J.E., K.Oadades et al (1999) "Cell analysis and discharge analysis" 439).

These voltage-sensitive assays are based on changes in Fluorescence Resonance Energy Transfer (FRET) between the membrane-soluble, voltage-sensitive dye DiSBAC2(3) and the fluorescent phospholipid CC2-DMPE, which is attached to the outer leaflet of the plasma membrane and serves as a FRET donor. Membrane potential (V)_m) Resulting in a negatively charged DiSBAC₂(3) Redistributed across the plasma membrane, the amount of energy transferred from the CC2-DMPE changed accordingly. The change in fluorescence emission can be made using VIPR^TMII monitoring, which is an integrated liquid processor and fluorescence detector, is designed for 96-or 384-well microtiter plate based screening.

In another aspect, the present invention provides a kit for determining the activity of CFTR or a fragment thereof in a biological sample in vitro or in vivo, said kit comprising: (i) a composition comprising form a or any of the above embodiments; and (ii) instructions for: a) contacting the composition with the biological sample; and b) determining the activity of said CFTR or fragment thereof. In one embodiment, the kit further comprises instructions for: a) contacting an additional composition with the biological sample; b) determining the activity of the CFTR or fragment thereof in the presence of the additional compound; and c) comparing the CFTR activity in the presence of the additional compound to the density of CFTR in the presence of form a described herein. In a preferred embodiment, the kit is used to determine the density of CFTR.

In order that the invention described herein may be more fully understood, the following examples are provided. It should be understood that these examples are for illustration only and are not to be construed as limiting the invention in any way.

Examples

Method and material

XRPD (X-ray powder diffraction)

X-ray powder diffraction (XRPD) data were recorded at room temperature using a Rigaku/MSC MiniFlex Desktop powder X-ray diffractometer (Rigaku, The Woodlands, TX). X-rays were generated using a Cu tube operating at 30kV and 15mA with a K β suppression filter. The divergence slit was variable due to scattering and the receiving slit was set at 4.2 degrees and 0.3mm slit, respectively. The scan pattern is a Fixed Time (FT) using 0.02 degree steps and a 2.0 second count time. Calibration of the powder X-ray diffractometer using reference standards: 75% sodalite (Na)₃Al₄Si₄O₁₂Cl) and 25% silicon (Rigaku, Cat # 2100/ALS). A6-sample station (SH-LBSI511-RNDB) with zero background sample racks was used. The powder sample was placed on the recessed area and pressed flat with a glass slide.

FTIR (Fourier transform Infrared) spectroscopy

FTIR spectra were collected from a Thermo Scientific, Nicolet 6700FT-IR spectrometer with intelligent rail sampling compartment, diamond window, application software Omnic, 7.4. The powder sample was placed directly on the diamond crystal and pressed to adhere the sample surface to the diamond crystal surface. A background spectrum is collected and then a sample spectrum is collected. The acquisition settings were as follows:

a detector: DTGS KBr;

spectroscope: KBr;

source: IR;

scanning range: 4000-400cm^-1；

Gain: 8.0;

optical speed: 0.6329 cm/sec;

pore diameter: 100, respectively;

scanning and numbering: 32, a first step of removing the first layer; and

resolution ratio: 4cm^-1。

Example 1: preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7).

2-chloro-5- (trifluoromethyl) aniline 2(200g, 1.023mol), diethyl 2- (ethoxymethylene) malonate 3(276g, 1.3mol) and toluene (100mL) were combined in a three-necked 1-L round bottom flask equipped with a Dean-Stark condenser under a nitrogen atmosphere. The solution was heated to 140 ℃ with stirring and the temperature was maintained for 4 h. The reaction mixture was cooled to 70 ℃ and hexane (600mL) was added slowly. The resulting slurry was stirred and allowed to warm to room temperature. The solid was collected by filtration, washed with 10% ethyl acetate in hexane (2x400mL) and then dried in vacuo to give a white solid (350g, 94% yield) as the desired condensation product diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate 4.¹H NMR(400MHz，DMSO-d₆)δ11.28(d，J＝13.0Hz，1H)，8.63(d，J＝13.0Hz，1H)，8.10(s，1H)，7.80(d，J＝8.3Hz，1H)，7.50(dd，J＝1.5，8.4Hz，1H)，4.24(q，J＝7.1Hz，2H)，4.17(q，J＝7.1Hz，2H)，1.27(m，6H)。

Preparation of ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (5). Adding into a 3-neck 1-L flask(200mL, 8mL/g) and degassed at 200 ℃ for 1 h. The solvent was heated to 260 ℃ and diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate 4(25g, 0.07mol) was added in portions over 10 min. The resulting mixture was stirred at 260 ℃ for 6.5 hours, and the resulting ethanol by-product was removed by distillation. The mixture was slowly cooled to 80 ℃. Hexane (150mL) was added slowly over 30 minutes (min) followed by a 200mL batch of hexane. The slurry was stirred until it reached room temperature. The solid was filtered, washed with hexane (3 × 150mL), then dried in vacuo to give 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid ethyl ester 5 as a tan solid (13.9g, 65% yield).¹H NMR(400MHz，DMSO-d₆)δ11.91(s，1H)，8.39(s，1H)，8.06(d，J＝8.3Hz，1H)，7.81(d，J＝8.4Hz，1H)，4.24(q，J＝7.1Hz，2H)，1.29(t，J＝7.1Hz，3H)。

Preparation of ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate (6). To a 3-neck 5-L flask were added 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid ethyl ester 5(100g, 0.3mol), ethanol (1250mL, 12.5mL/g), and triethylamine (220mL, 1.6 mol). Then 10g of 10% Pd/C (50% wet) was added to the flask at 5 ℃. The reaction was stirred vigorously at 5 ℃ under a nitrogen atmosphere for 20h, 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 6, was used directly in the next step as a slurry with Pd/C.

Preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7). In a 1-L flask with reflux condenser, ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate 6(58g, 0.2mol, crude reaction slurry containing Pd/C) was suspended in NaOH (814mL 5M, 4.1mol), heated at 80 ℃ for 18H, and then at 100 ℃ for an additional 5H. The reaction was filtered warm through the packed Celite to remove Pd/C and the Celite was rinsed with 1N NaOH. Acidifying the filtrateTo about pH 1, a thick white precipitate was obtained. The precipitate was filtered and then rinsed 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 (48g, 92% yield).¹H NMR(400.0MHz，DMSO-d₆)δ15.26(s，1H)，13.66(s，1H)，8.98(s，1H)，8.13(dd，J＝1.6，7.8Hz，1H)，8.06-7.99(m，2H)。

Example 2: preparation of 4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) aniline

7- [ 4-Nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]Preparation of heptane (20). To a compound containing 7-azabicyclo [2.2.1]A flask of heptane hydrochloride 9(4.6g, 34.43mmol, from nitrogen atmosphere) was charged with a solution of 4-fluoro-1-nitro-2- (trifluoromethyl) benzene 8(6.0g, 28.69mmol) and triethylamine (8.7g, 12.00mL, 86.07mmol) in acetonitrile (50 mL). The reaction flask was heated at 80 ℃ for 16h under a nitrogen atmosphere. The reaction mixture was cooled and then partitioned between water and dichloromethane. The organic layer was washed with 1M HCl and Na₂SO₄Drying, filtering and concentrating to dryness. Purification by silica gel chromatography (0-10% ethyl acetate in hexane) afforded 7- [ 4-nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]Heptane 10(7.2g, 88% yield) as a yellow solid.¹H NMR(400.0MHz，DMSO-d₆)δ8.03(d，J＝9.1Hz，1H)，7.31(d，J＝2.4Hz，1H)，7.25(dd，J＝2.6，9.1Hz，1H)，4.59(s，2H)，1.69-1.67(m，4H)，1.50(d，J＝7.0Hz，4H)。

4- (7-azabicyclo [2.2.1]]Preparation of hept-7-yl) -2- (trifluoromethyl) aniline (11). 7- [ 4-nitro-3- (trifluoromethyl) phenyl is added]-7-azabicyclo [2.2.1]A flask of heptane 10(7.07g, 24.70mmol) and 10% Pd/C (0.71g, 6.64mmol) was evacuated and then flushed with nitrogenAnd (4) qi. Ethanol (22mL) was added and the reaction flask was fitted with a hydrogen balloon. After 12h of vigorous stirring, the reaction mixture was purged with nitrogen and the 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 hexane) to give 4- (7-azabicyclo [2.2.1] 4]Hept-7-yl) -2- (trifluoromethyl) aniline 11 as a purple solid (5.76g, 91% yield).¹H NMR(400.0MHz，DMSO-d₆) δ 6.95(dd, J ═ 2.3, 8.8Hz, 1H), 6.79(d, J ═ 2.6Hz, 1H), 6.72(d, J ═ 8.8Hz, 1H), 4.89(s, 2H), 4.09(s, 2H), 1.61-1.59(m, 4H) and 1.35(d, J ═ 6.8Hz, 4H).

Example 3: preparation of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (Compound 1).

To 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylic acid 7(9.1g, 35.39mmol) and 4- (7-azabicyclo [2.2.1] at room temperature]To a solution of hept-7-yl) -2- (trifluoromethyl) aniline 11(9.2g, 35.74mmol) in 2-methyltetrahydrofuran (91.00mL) was added propylphosphonic acid cyclic anhydride (50% solution in ethyl acetate, 52.68mL, 88.48mmol) and pyridine (5.6g, 5.73mL, 70.78 mmol). The reaction flask was heated at 65 ℃ for 10h under a nitrogen atmosphere. After cooling to room temperature, the reaction was diluted with ethyl acetate and saturated Na₂CO₃The reaction was stopped by the 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 and Na₂SO₄Dried, filtered and concentrated to give a tan solid. The crude solid product was slurried in ethyl acetate/diethyl ether (2: 1), collected by vacuum filtration, and washed twice with ethyl acetate/diethyl ether (2: 1) to give the product as a pale 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) afforded N- (4- (7-azabicyclo [ 2.2.1)]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (compound 1) as a white crystalline solid (form a) (13.5g, 76% yield). LC/MS M/z 496.0[ M + H ]]⁺Retention time 1.48min (RP-C)₁₈，10-99％CH₃CN/0.05% TFA, within 3 min).¹H NMR(400.0MHz，DMSO-d₆)δ13.08(s，1H)，12.16(s，1H)，8.88(s，1H)，8.04(dd，J＝2.1，7.4Hz，1H)，7.95-7.88(m，3H)，7.22(dd，2.5，8.9Hz，1H)，7.16(d，J＝2.5Hz，1H)，4.33(s，2H)，1.67(d，J＝6.9Hz，4H)，1.44(d，J＝6.9Hz，4H)。

The powder diffraction pattern of the crystal form A is shown in figure 1.

Table 1 below provides representative XRPD peaks for form a.

TABLE 1 form A XRPD peaks

2 theta (degree)

Strength (%)

2 theta (degree)	Strength (%)
		7.90	100.0
9.28	10.8
		11.90	12.8
14.38	35.2
		15.08	12.6
15.80	34.1
		16.96	25.2
17.66	13.8
		19.28	39.4
20.06	20.2
		21.36	14.5
21.80	94.2
		23.40	30.0
23.80	92.0
		25.64	8.9
26.82	6.4
		29.36	8.1
29.72	18.1
		30.14	14.2
31.20	9.9

The conformational diagram of form a based on single crystal X-ray analysis is shown in figure 2. Diffraction data were acquired using a Bruker Apex II diffractometer equipped with a sealed-tube CuK- α source and Apex II CCD detector. The structure was analyzed and precisely defined using the SHELX program (Sheldrick, g.m., Acta cryst.a64, pp.112-122 (2008)). Structures in trigonal crystal systems (trigonal crystal systems) and R-3 space groups are analyzed and precisely defined based on intensity, statistics and symmetry. Form a has the following unit cell dimensions:α is 90 °, β is 90 ° and γ is 120 °.

The FTIR spectrum of form a is provided in figure 3.

Table 2 below provides representative FTIR peaks for form a.

TABLE 2 form A FTIR peaks

Position (cm)-1)	Strength of
		407.4	46.07
436.7	72.55
		471.5	61.17
497.8	63.61
		505.7	60.34
532.9	61.14
		567.8	54.31
590.7	55.23
		614.4	64.01
649.7	50.74
		661.0	49.82
686.8	51.43
		726.1	53.80
751.4	35.60
		798.1	48.21
808.8	48.47
		824.8	42.25
875.5	52.89
		898.6	71.77
918.7	68.93
		977.7	42.31
1008.1	64.09
		1047.3	35.70
1072.5	53.76

Position (cm)-1)	Strength of
		1091.2	43.79
1113.4	28.46
		1131.4	30.00
1153.0	34.61
		1168.3	40.13
1199.3	74.26
		1221.8	48.07
1253.1	47.84
		1277.6	36.67
1291.7	48.07
		1310.8	55.99
1329.1	63.21
		1352.8	42.30
1433.2	42.45
		1463.0	63.68
1526.0	35.86
		1574.0	60.60
1607.5	60.30
		1662.6	55.12
1740.9	86.74
		2870.0	81.63
2947.7	75.12
		2963.8	75.30
3092.7	84.58

Assays for detecting and measuring the Δ F508-CFTR enhancing properties of compounds

Transmembrane potential optical method for determining Δ F508-CFTR modulating properties of compounds

The assay uses a fluorescent voltage sensing dye to determine transmembrane potential changes as read-outs of increased functional Δ F508-CFTR in NIH3T3 cells using a fluorescent plate reader (e.g., FLIPR III, Molecular Devices, Inc.). The driving force for this response is that after pre-treating the cells with compounds and then loading the voltage sensing dye, a chloride ion gradient associated with channel activation is generated by a single liquid addition step.

Identification of enhancer compounds

To identify af 508-CFTR enhancers, a double-addition HTS assay format was developed. The HTS assay uses a fluorescent voltage sensing dye to determine the change in transmembrane potential on FLIPR III as a measure of increased gating of af 508CFTR in temperature-calibrated af 508CFTR NIH3T3 cells (conductance). The driving force for this response is the Cl-ion gradient associated with channel activation with forskolin in a single liquid addition step using a fluorescent plate reader such as FLIPR III, after pre-treatment of cells with the enhancer compound (or DMSO vehicle control) followed by loading of the redistribution dye.

Solutions of

Bath solution # 1: (in mM) NaCl 160, KCl 4.5, CaCl₂2，MgCl₂1, HEPES 10, pH 7.4, NaOH.

Chloride ion-free bath solution: replacement of hydrochloride salt in bath solution #1 with gluconate

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for optical measurement of transmembrane potential. Cells were maintained at 37 ℃ with 5% CO₂And 175cm in 90% humidity²In Dulbecco's modified Eagle's Medium in flasks, the medium was supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, beta-ME, 1X pen/strep and 25mM HEPES. For all optical assays, cells were seeded at-20,000/well on 384-well matrigel-coated plates, incubated at 37 ℃ for 2hrs, then at 27 ℃ for 24hrs for enhancer analysis. For calibration assays, cells were incubated with and without compounds at 27 ℃ or 37 ℃ for 16-24 hours. Electrophysiological analysis was used to analyze compounds for Δ F508-CFTR modulating properties.

Ussing Chamber analysis

Ussing laboratory experiments were performed on polarized airway epithelial cells expressing Δ F508-CFTR to further characterize modulators of Δ F508-CFTR identified in the optical assay. non-CF and CF airway epithelium was isolated from bronchial tissue and cultured as described above (Galietta, l.j.v., Lantero, s., Gazzolo, a., Sacco, o., Romano, l.,Rossi，G.A.，&Zegarra-Moran, O. (1998) In Vitro cell. Dev. biol.34, 478-481) In pre-coating with NIH3T 3-conditioned mediumSnapwell^TMSpread flat on the filter. After 4 days, the top medium was removed and the cells were allowed to grow on an air liquid interface for > 14 days before use. This results in a monolayer of fully differentiated columnar cells that are ciliated and characterized by airway epithelium. non-CF HBEs were isolated from non-smokers who did not have any known lung disease. CF-HBE was isolated from patients homozygous for Δ F508-CFTR.

Will grow inHBE on Snapwell cell culture inserts was fixed in a Ussing Chamber (physiological Instruments, Inc., San Diego, Calif.) and Cl was measured outside the substrate to the apical end using a voltage clamp system (Department of Bioengineering, University of Iowa, IA)^-Gradient (I)_SC) Transepithelial resistance and short circuit current in the presence of (c). Briefly, under voltage clamp recording conditions (V)_Holding0mV), HBE was tested at 37 ℃. The solution on the outside of the substrate contained (in mM) 145 NaCl, 0.83K₂HPO₄、3.3 KH₂PO₄、1.2 MgClX、1.2CaCl₂10 glucose, 10HEPES (pH adjusted to 7.35 with NaOH), apical solution containing (in mM) 145 sodium gluconate, 1.2MgCl₂、1.2CaCl₂10 glucose, 10HEPES (pH adjusted to 7.35 with NaOH).

Identification of enhancer compounds

Typical protocols use Cl from the outside of the substrate to the top film^-A concentration gradient. To create this gradient, normal ringer's solution (ringer) was used for the substrate outer membrane. In addition, large trans-epithelial Cl was obtained by replacing the apical membrane NaCl with equimolar sodium gluconate (titrated to pH 7.4 with NaOH)^-A concentration gradient. Forskolin (10. mu.M) and all test compounds were added to the cellsThe top surface of the culture insert. The effect of the putative af 508-CFTR enhancer was compared to that of the known enhancer genistein.

Patch clamp record

Monitoring of total Cl in AF 508-NIH3T3 cells using a perforated patch recording configuration as previously described^-Electric current (Rae, j., Cooper, k., Gates, p.,&watsky, m. (1991) j. neurosci. methods 37, 15-26). Voltage clamp recordings were performed at 22 ℃ using an Axopatch 200B patch clamp amplifier (AxonInstruments inc., Foster City, CA). The pipette solution contained (in mM) 150N-methyl-D-glucosamine (NMDG) -Cl, 2MgCl₂、2CaCl₂10EGTA, 10HEPES and 240. mu.g/ml amphotericin-B (pH adjusted to 7.35 with HCl). The extracellular medium contains (in mM) 150NMDG-Cl, 2MgCl₂、2CaCl₂10HEPES (pH adjusted to 7.35 with HCl). Pulsing, data acquisition and analysis were performed using a PC equipped with a Digidata 1320A/D interface and Clampex 8(Axon Instruments Inc.). To activate af 508-CFTR, 10 μ M forskolin and 20 μ M genistein were added to the bath and the current-voltage dependence was monitored every 30 seconds.

Identification of enhancer compounds

The enhancement of the macroscopic Δ F508-CFTR Cl-current (I) in NIH3T3 cells stably expressing Δ F508-CFTR was also investigated by using a perforated patch recording technique_ΔF508) The ability of the cell to perform. Enhancers identified from the optical assay result in I with similar potency and efficacy observed in the optical assay_ΔF508Is increased in a dose-dependent manner. In all cells tested, the reversal potential before and during the application of the enhancing agent was about-30 mV, which is the calculated E_cl(-28mV)。

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for whole cell recordings. Cells were maintained at 37 ℃ in 5% CO₂And 175cm in 90% humidity²Dulbecco's modified Eagle's Medium in culture flasks supplemented with2mM glutamine, 10% fetal bovine serum, 1X NEAA, beta-ME, 1X pen/strep and 25mM HEPES. For whole cell recording, 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and used to test the activity of the enhancer after incubation at 27 ℃ for 24-48 hrs; and incubated with or without a correction compound at 37 ℃ to determine the activity of the correction agent.

Single channel recording

The gating activity of temperature-calibrated Δ F508-CFTR and wt-CFTR expressed in NIH3T3 cells was observed using excised membrane inside-out patches (dalemanns, w., Barbry, p., champgny, g., jalat, s., Dott, k., Dreyer, d., crytal, r.g., Pavirani, a., Lecocq, J-p., Lazdunski, M. (1991) Nature 354, 526-. The removal tube contained (in mM): 150NMDG, 150 aspartic acid, 5CaCl₂、2MgCl₂And 10HEPES (pH adjusted to 7.35 with Tris base). The bath contained (in mM): 150NMDG-Cl, 2MgCl₂5EGTA, 10TES and 14Tris base (pH adjusted to 7.35 with HCl). After excision, wt-and Δ F508-CFTR were activated by the addition of 1mM Mg-ATP, 75nM cAMP-dependent protein kinase catalytic subunit (PKA; Promega Corp. Madison, Wis.) and 10mM NaF to inhibit protein phosphatase, which prevented the current from dropping. The pipette potential was maintained at 80 mV. Channel activity was analyzed from membranes containing ≦ 2 active channels. The maximum number of simultaneous openings determines the number of active channels during the experiment. To determine single channel current amplitude, data recorded from 120 sec Δ F508-CFTR activity was filtered "off-line" at 100Hz and then used to construct an amplitude histogram for all points, fitted with a multiple Gaussian function using Bio-Patch analysis software (Bio-Logic Comp. France). Total microscopic Current and open probability (P) were determined from 120 seconds of channel Activity_o). Using Bio-Patch software or according to the relation P_oDetermining P_oWhere I is the average current, I is the single channel current amplitude, and N is the number of active channels in the membrane.

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for patch clamp recordings of incised membranes. Cells were maintained at 37 ℃ in 5% CO₂And 175cm in 90% humidity²Dulbecco's modified Eagle's Medium in flasks supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, beta-ME, 1X pen/strep and 25mM HEPES. For single channel recording, 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and used after incubation at 27 ℃ for 24-48 hrs.

Compound 1 form a is useful as a modulator of ATP-binding cassette transporter. Determination of the EC of Compound 1 form A₅₀(mum) less than 2.0. mu.M. The efficiency of compound 1 form a was calculated to be 100% to 25%. It should be noted that 100% efficiency is the maximum response obtained using 4-methyl-2- (5-phenyl-1H-pyrazol-3-yl) phenol.

Claims (40)

1. Form A of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide, wherein the form A is characterized by the following peaks in an X-ray powder diffraction pattern: a peak at about 7.9 degrees, a peak at about 9.3 degrees, a peak at about 11.9 degrees, a peak at about 14.4 degrees, a peak at about 15.1 degrees, a peak at about 15.8 degrees, a peak at about 17.0 degrees, a peak at about 17.7 degrees, a peak at about 19.3 degrees, a peak at about 20.1 degrees, a peak at about 21.4 degrees, a peak at about 21.8 degrees, a peak at about 23.4 degrees, a peak at about 23.8 degrees, a peak at about 25.6 degrees, a peak at about 26.8 degrees, a peak at about 29.4 degrees, a peak at about 29.7 degrees, a peak at about 30.1 degrees, and a peak at about 31.2 degrees.
2. 2. Form a of claim 1, wherein the form a is characterized by a diffraction pattern as shown in figure 1.
3. 3. A pharmaceutical composition comprising form a of claim 1 or 2 and a pharmaceutically acceptable adjuvant or carrier.
4. 4. The pharmaceutical composition of claim 3, further comprising an additional active agent selected from a mucolytic, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than form A, or a nutritional agent.
5. 5. The pharmaceutical composition of claim 4, wherein the additional agent is a CFTR modulator other than form A.
6. 6. Use of form a according to claim 1 or 2 in the manufacture of a medicament for treating or lessening the severity of a disease in a patient, wherein the disease is selected from the group consisting of cystic fibrosis, asthma, smoking-induced chronic obstructive pulmonary disease, chronic bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency, mild lung disease, allergic bronchopulmonary aspergillosis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiency, lipid processing deficiency, lysosomal storage disease, polyendocrinopathy, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, type 1 Glycanosis CDG disease, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus, charcot-marie syndrome, pelizaeus-merzbacher disease, diabetes mellitus, chronic bronchitis, rhinosinusitis, constipation, pancreatitis, chronic pulmonary disease, pancreatic insufficiency, mild pulmonary disease, allergic bronchopulmonary aspergillosis, hereditary emphysema, multiple sclerosis, Neurodegenerative diseases, polyglutamine neurological disorders, spongiform encephalopathy, Fabry's disease, Straussler-Scheinker syndrome, infertility, osteoporosis, osteopenia, Gorham syndrome, chloride channel pathology, Bart syndrome type III, Dent's disease, startle syndrome, epilepsy, Angelman's syndrome, primary ciliary dyskinesia, and liver disease.
7. 7. The use of claim 6, wherein the disease is cystic fibrosis.
8. 8. The use of claim 6, wherein the disease is protein C deficiency or hereditary angioedema type 1.
9. 9. The use of claim 6, wherein the disease is selected from the group consisting of familial hypercholesterolemia, chylomicronemia type 1, and betalipoproteinemia.
10. 10. The use of claim 6, wherein the disease is selected from the group consisting of I-cell disease, pseudohuler's disease, mucopolysaccharidosis, Mulberry Hoff's disease, Tay-Sasa disease, and Kerr-Na syndrome type II.
11. 11. The use according to claim 6, wherein the disease is selected from the group consisting of neurogenic diabetes insipidus and renal diabetes insipidus.
12. 12. The use of claim 6, wherein the disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy and pick's disease.
13. 13. The use of claim 6, wherein the disease is selected from Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and myotonic dystrophy.
14. 14. The use of claim 6, wherein the disease is hereditary Creutzfeld-Jacob disease.
15. 15. The use of claim 6, wherein the disease is selected from Thomson-type congenital myotonia and Becker-type congenital myotonia.
16. 16. The use of claim 6, wherein the disease is selected from the group consisting of primary ciliary dyskinesia with left and right translocation, primary ciliary dyskinesia without left and right translocation, and ciliary dysplasia.
17. 17. The use of claim 6, wherein said disease is selected from the group consisting of hyperinsulinemia, diabetes, and larcen dwarfism.
18. 18. Use of form a of claim 1 or 2 in the preparation of a medicament for treating or lessening the severity of a disease in a patient, wherein the disease is associated with reduced CFTR function due to a mutation in the gene encoding CFTR or environmental factors.
19. 19. The use of claim 18, wherein the disease is selected from the group consisting of cystic fibrosis, chronic bronchitis, recurrent bronchitis, acute bronchitis, infertility, idiopathic chronic pancreatitis, idiopathic recurrent pancreatitis, idiopathic acute pancreatitis, chronic rhinosinusitis, primary sclerosing cholangitis, allergic bronchopulmonary aspergillosis, diabetes, dry eye, constipation, bone disease, and asthma.
20. 20. Use of form a of claim 1 or 2 in the preparation of a medicament for treating or lessening the severity of a disease in a patient, wherein the disease is associated with normal CFTR function.
21. 21. The use of claim 20, wherein the disease is selected from the group consisting of chronic obstructive pulmonary disease, chronic bronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis, constipation, chronic pancreatitis, recurrent pancreatitis and acute pancreatitis, pancreatic insufficiency, infertility, mild pulmonary disease, idiopathic pancreatitis, liver disease, hereditary emphysema, gallstones, gastroesophageal reflux disease, gastrointestinal malignancies, inflammatory bowel disease, diabetes, arthritis, osteoporosis, and osteopenia.
22. 22. The use of claim 20, wherein the disease is hereditary hemochromatosis, coagulation-fibrinolysis deficiency, lipid processing deficiency, lysosomal storage disease, polyendocrinopathy, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, type 1 Glycanosis CDG disease, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus, charcot-marie-tooth syndrome, pelizaeus-merzschia, neurodegenerative diseases, polyglutamine neurological disorders, spongiform encephalopathy, fabry disease, Straussler-scheinner syndrome, Gorham syndrome, chloride tunnel disease, barter syndrome type III, Dent's disease, epilepsy, startle syndrome, Angelman syndrome, primary ciliary dyskinesia, or sjogren's disease.
23. 23. The use of claim 22, wherein the disease is protein C deficiency or hereditary angioedema type 1.
24. 24. The use of claim 22, wherein the disease is selected from the group consisting of familial hypercholesterolemia, chylomicronemia type 1, and betalipoproteinemia.
25. 25. The use of claim 22, wherein the disease is selected from the group consisting of I-cell disease, pseudohuler's disease, mucopolysaccharidosis, sandhoff's disease, tay-sachs disease and cro-na syndrome type II.
26. 26. The use according to claim 22, wherein the disease is selected from the group consisting of neurogenic diabetes insipidus and renal diabetes insipidus.
27. 27. The use of claim 22, wherein the disease is selected from alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy and pick's disease.
28. 28. The use of claim 22, wherein the disease is selected from huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and myotonic dystrophy.
29. 29. The use of claim 22, wherein the disease is hereditary Creutzfeld-Jacob disease.
30. 30. The use of claim 22, wherein the disease is selected from Thomson-type congenital myotonia and Becker-type congenital myotonia.
31. 31. The use of claim 22, wherein the disease is selected from the group consisting of primary ciliary dyskinesia with left and right translocation, primary ciliary dyskinesia without left and right translocation, and ciliary dysplasia.
32. 32. The use of claim 22, wherein said disease is selected from the group consisting of hyperinsulinemia, diabetes, and larcen dwarfism.
33. 33. A kit for determining the activity of CFTR or a fragment thereof in a biological sample in vitro or in vivo, said kit comprising:

    (i) a composition comprising form a of claim 1 or 2; and

    (ii) instructions for the following:

    a) contacting the composition with the biological sample; and

    b) determining the activity of the CFTR or fragment thereof.
34. 34. The kit of claim 33, further comprising instructions for:

    a) contacting an additional compound with the biological sample;

    b) determining the activity of said CFTR or fragment thereof in the presence of said additional compound; and

    c) comparing the activity of CFTR or fragment thereof in the presence of a further compound with the activity of CFTR or fragment thereof in the presence of form a according to claim 1 or 2.
35. 35. The kit of claim 34, wherein the step of comparing the activity of said CFTR or fragment thereof provides a determination of the density of said CFTR or fragment thereof.
36. 36. Use of form a of claim 1 or 2 in the preparation of a formulation for modulating CFTR activity in a biological sample.
37. A crystalline form of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide having a trigonal system, an R-3 space group, and the following unit cell dimensions:

    α =90 °; β =90 °; and γ =120 °.
38. 38. Use according to claim 6 or 21, wherein the infertility is male infertility due to congenital bilateral loss of vas deferens.
39. 39. Use according to claim 19, wherein the infertility is male infertility due to congenital bilateral loss of vas deferens, or female infertility due to congenital loss of uterus and vagina.
40. 40. The use according to claim 6, wherein the pancreatitis is idiopathic pancreatitis.