U.S. patent application number 13/858750 was filed with the patent office on 2014-04-24 for solid forms of n-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-- (trifluoromethyl)-1,4-dihyroquinoline-3-carboxamide.
This patent application is currently assigned to Vertex Pharmaceuticals Incorporated. The applicant listed for this patent is VERTEX PHARMACEUTICALS INCORPORATED. Invention is credited to Martyn Botfield, Peter D.J. Grootenhuis, Mariusz Krawiec, Fredrick Van Goor, Beili Zhang.
Application Number | 20140113933 13/858750 |
Document ID | / |
Family ID | 41460125 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140113933 |
Kind Code |
A9 |
Zhang; Beili ; et
al. |
April 24, 2014 |
Solid Forms of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihyroquinoline-3-carboxamide
Abstract
The present invention relates to solid state forms, for example,
crystalline forms of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide,
pharmaceutical compositions thereof, and methods therewith.
Inventors: |
Zhang; Beili; (San Diego,
CA) ; Krawiec; Mariusz; (Marlborough, MA) ;
Botfield; Martyn; (Concord, MA) ; Grootenhuis; Peter
D.J.; (San Diego, CA) ; Van Goor; Fredrick;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VERTEX PHARMACEUTICALS INCORPORATED |
Cambridge |
MA |
US |
|
|
Assignee: |
Vertex Pharmaceuticals
Incorporated
Cambridge
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130231368 A1 |
September 5, 2013 |
|
|
Family ID: |
41460125 |
Appl. No.: |
13/858750 |
Filed: |
April 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12605266 |
Oct 23, 2009 |
8436014 |
|
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13858750 |
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61107813 |
Oct 23, 2008 |
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Current U.S.
Class: |
514/312 ;
435/375 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 1/18 20180101; C07D 215/56 20130101; C07D 487/08 20130101;
A61P 19/00 20180101; A61P 25/08 20180101; A61P 11/00 20180101; A61P
1/04 20180101; A61P 3/06 20180101; A61P 11/02 20180101; A61P 5/14
20180101; A61P 11/06 20180101; A61P 13/12 20180101; A61P 25/00
20180101; A61P 11/08 20180101; A61P 37/06 20180101; A61P 27/02
20180101; A61P 7/12 20180101; A61P 15/08 20180101; A61P 19/10
20180101; A61P 1/16 20180101; A61P 27/00 20180101; A61P 1/10
20180101; A61P 21/02 20180101; A61P 19/08 20180101; A61P 3/10
20180101; A61P 25/16 20180101; A61P 7/00 20180101; A61P 37/08
20180101; A61P 25/28 20180101; A61P 1/00 20180101; A61P 31/10
20180101; A61P 7/10 20180101; A61P 25/14 20180101 |
Class at
Publication: |
514/312 ;
435/375 |
International
Class: |
C07D 487/08 20060101
C07D487/08 |
Claims
1-26. (canceled)
27. A method of treating or lessening the severity of a disease in
a patient, wherein said disease is selected from cystic fibrosis,
asthma, smoke induced COPD, chronic bronchitis, rhinosinusitis,
constipation, pancreatitis, pancreatic insufficiency, male
infertility caused by congenital bilateral absence of the vas
deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,
allergic bronchopulmonary aspergillosis (ABPA), liver disease,
hereditary emphysema, hereditary hemochromatosis,
coagulation-fibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases,
such as I-cell disease/pseudo-Hurler, mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, congenital hyperthyroidism,
osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT
deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic
DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,
neurodegenerative diseases such as Alzheimer's disease, Parkinson's
disease, amyotrophic lateral sclerosis, progressive supranuclear
plasy, Pick's disease, several polyglutamine neurological disorders
such as Huntington's, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic
dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease (due to prion protein
processing defect), Fabry disease, Straussler-Scheinker syndrome
(due to Prp processing defect), infertility pancreatitis,
pancreatic insufficiency, osteoporosis, osteopenia, Gorham's
Syndrome, chloride channelopathies, myotonia congenita (Thomson and
Becker forms), Bartter's syndrome type III, Dent's disease,
hyperekplexia, epilepsy, hyperekplexia, lysosomal storage disease,
Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with situs
inversus (also known as Kartagener syndrome), PCD without situs
inversus and ciliary aplasia, and liver disease, said method
comprising the step of administering to said patient an effective
amount of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl-1,4-dihydroquinoline-3-carboxamide characterized
as Form A.
28. The method according to claim 27, wherein said disease is
cystic fibrosis.
29. A method of treating or lessening the severity of a disease in
a patient, wherein said disease is associated with reduced CFTR
function due to mutations in the gene encoding CFTR or
environmental factors, said method comprising the step of
administering to said patient an effective amount of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl-
)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide
characterized as Form A.
30. The method of claim 29, wherein disease is cystic fibrosis,
chronic bronchitis, recurrent bronchitis, acute bronchitis, male
infertility caused by congenital bilateral absence of the vas
deferens (CBAVD), female infertility caused by congenital absence
of the uterus and vagina (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, and asthma.
31. A method of treating or lessening the severity of a disease in
a patient, wherein said disease is associated with normal CFTR
function, said method comprising the step of administering to said
patient an effective amount of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide characterized
as Form A.
32. The method of claim 31, wherein disease is chronic obstructive
pulmonary disease (COPD), chronic bronchitis, recurrent bronchitis,
acute bronchitis, rhinosinusitis, constipation, chronic
pancreatitis, recurrent pancreatitis, and acute pancreatitis,
pancreatic insufficiency, male infertility caused by congenital
bilateral absence of the vas deferens (CBAVD), mild pulmonary
disease, idiopathic pancreatitis, liver disease, hereditary
emphysema, gallstones, gasgtro-esophageal reflux disease,
gastrointestinal malignancies, inflammatory bowel disease,
constipation, diabetes, arthritis, osteoporosis, and
osteopenia.
33. The method of claim 31, wherein the disease is hereditary
hemochromatosis, coagulation-fibrinolysis deficiencies, such as
protein C deficiency, Type 1 hereditary angioedema, lipid
processing deficiencies, such as familial hypercholesterolemia,
Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, congenital hyperthyroidism,
osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT
deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic
DI, Charcot-Marie Tooth syndrome, Perlizaeus-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, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic
dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease (due to prion protein
processing defect), Fabry disease, Straussler-Scheinker syndrome,
Gorham's Syndrome, chloride channelopathies, myotonia congenita
(Thomson and Becker forms), Bartter's syndrome type III, Dent's
disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal storage
disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD
with situs inversus (also known as Kartagener syndrome), PCD
without situs inversus and ciliary aplasia, or Sjogren's
disease
34. (canceled)
35. (canceled)
36. A method of modulating CFTR activity in a biological sample
comprising the step of contacting said CFTR with
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide characterized
as Form A.
37. (canceled)
38. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application Ser. No. 61/107,813, filed Oct. 23, 2008,
entitled "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", the
entire contents of which is incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to solid state forms, for
example, crystalline forms of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide, which is a
modulator of cystic fibrosis transmembrane conductance regulator
("CFTR"). The invention also relates to pharmaceutical compositions
including the crystalline forms of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide, and methods
therewith.
BACKGROUND OF THE INVENTION
[0003] ATP cassette transporters are a family of membrane
transporter proteins that regulate the transport of a wide variety
of pharmacological agents, potentially toxic drugs, and
xenobiotics, as well as anions. They are homologous membrane
proteins that bind and use cellular adenosine triphosphate (ATP)
for their specific activities. Some of these transporters were
discovered as multidrug resistance proteins (like the MDR1-P
glycoprotein, or the multidrug resistance protein, MRP1), defending
malignant cancer cells against chemotherapeutic agents. To date, 48
such transporters have been identified and grouped into 7 families
based on their sequence identity and function.
[0004] One member of the ATP cassette transporters family commonly
associated with disease is the cAMP/ATP-mediated anion channel,
CFTR. CFTR is expressed in a variety of cells types, including
absorptive and secretory epithelia cells, where it regulates anion
flux across the membrane, as well as the activity of other ion
channels and proteins. In epithelial cells, normal functioning of
CFTR is critical for the maintenance of electrolyte transport
throughout the body, including respiratory and digestive tissue.
CFTR is composed of approximately 1480 amino acids that encode a
protein made up of a tandem repeat of transmembrane domains, each
containing six transmembrane helices and a nucleotide binding
domain. The two transmembrane domains are linked by a large, polar,
regulatory (R)-domain with multiple phosphorylation sites that
regulate channel activity and cellular trafficking.
[0005] The gene encoding CFTR has been identified and sequenced
(See Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P.
et al. (1990) Nature 347:358-362), Riordan, J. R. et al. (1989)
Science 245:1066-1073). A defect in this gene causes mutations in
CFTR resulting in cystic fibrosis ("CF"), the most common fatal
genetic disease in humans. Cystic fibrosis affects approximately
one in every 2,500 infants in the United States. Within the general
United States population, up to 10 million people carry a single
copy of the defective gene without apparent ill effects. In
contrast, individuals with two copies of the CF associated gene
suffer from the debilitating and fatal effects of CF, including
chronic lung disease.
[0006] In patients with cystic fibrosis, mutations in CFTR
endogenously expressed in respiratory epithelia lead to reduced
apical anion secretion causing an imbalance in ion and fluid
transport. The resulting decrease in anion transport contributes to
enhanced mucus accumulation in the lung and the accompanying
microbial infections that ultimately cause death in CF patients. In
addition to respiratory disease, CF patients typically suffer from
gastrointestinal problems and pancreatic insufficiency that, if
left untreated, results in death. In addition, the majority of
males with cystic fibrosis are infertile and fertility is decreased
among females with cystic fibrosis. In contrast to the severe
effects of two copies of the CF associated gene, individuals with a
single copy of the CF associated gene exhibit increased resistance
to cholera and to dehydration resulting from diarrhea--perhaps
explaining the relatively high frequency of the CF gene within the
population.
[0007] Sequence analysis of the CFTR gene of CF chromosomes has
revealed a variety of disease causing mutations (Cutting, G. R. et
al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell
61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080;
Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451).
To date, more than 1000 disease causing mutations in the CF gene
have been identified (http://www.genet.sickkids.on.ca/cftr/). The
most prevalent mutation is a deletion of phenylalanine at position
508 of the CFTR amino acid sequence, and is commonly referred to as
.DELTA.F508-CFTR. This mutation occurs in approximately 70 percent
of the cases of cystic fibrosis and is associated with a severe
disease.
[0008] The deletion of residue 508 in .DELTA.F508-CFTR prevents the
nascent protein from folding correctly. This results in the
inability of the mutant protein to exit the ER, and traffic to the
plasma membrane. As a result, the number of channels present in the
membrane is far less than observed in cells expressing wild-type
CFTR. In addition to impaired trafficking, the mutation results in
defective channel gating. Together, the reduced number of channels
in the membrane and the defective gating lead to reduced anion
transport across epithelia, leading to defective ion and fluid
transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studies
have shown, however, that the reduced numbers of .DELTA.F508- CFTR
in the membrane are functional, albeit less than wild-type CFTR.
(Dolmans et al. (1991), Nature Lond. 354: 526-528; Denning et al.,
supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50).
In addition to .DELTA.F508-CFTR, R117H-CFTR and G551D-CFTR, other
disease causing mutations in CFTR that result in defective
trafficking, synthesis, and/or channel gating, could be up- or
down-regulated to alter anion secretion and modify disease
progression and/or severity.
[0009] Although CFTR transports a variety of molecules in addition
to anions, it is clear that this role (the transport of anions,
chloride and bicarbonate) represents one element in an important
mechanism of transporting ions and water across the epithelium. The
other elements include the epithelial Na.sup.+ channel, ENaC,
Na.sup.+/2Cl.sup.-/K.sup.+ co-transporter, Na.sup.+-K.sup.+-ATPase
pump and the basolateral membrane K.sup.+ channels, that are
responsible for the uptake of chloride into the cell.
[0010] These elements work together to achieve directional
transport across the epithelium via their selective expression and
localization within the cell. Chloride absorption takes place by
the coordinated activity of ENaC and CFTR present on the apical
membrane and the Na.sup.+-K.sup.+-ATPase pump and Cl channels
expressed on the basolateral surface of the cell. Secondary active
transport of chloride from the luminal side leads to the
accumulation of intracellular chloride, which can then passively
leave the cell via Cl.sup.- ion channels, resulting in a vectorial
transport. Arrangement of Na.sup.+/2Cl.sup.-/K.sup.+co-transporter,
Na.sup.+-K.sup.+-ATPase pump and the basolateral membrane K.sup.+
channels on the basolateral surface and CFTR on the luminal side
coordinate the secretion of chloride via CFTR on the luminal side.
Because water is probably never actively transported itself, its
flow across epithelia depends on tiny transepithelial osmotic
gradients generated by the bulk flow of sodium and chloride.
[0011] Defective bicarbonate transport due to mutations in CFTR is
hypothesized to cause defects in certain secretory functions. See,
e.g., "Cystic fibrosis: impaired bicarbonate secretion and
mucoviscidosis," Paul M. Quinton, Lancet 2008; 372: 415-417.
[0012] Mutations in CFTR that are associated with moderate CFTR
dysfunction are also evident in patients with conditions that share
certain disease manifestations with CF but do not meet the
diagnostic criteria for CF. These include congenital bilateral
absence of the vas deferens, idiopathic chronic pancreatitis,
chronic bronchitis, and chronic rhinosinusitis. Other diseases in
which mutant CFTR is believed to be a risk factor along with
modifier genes or environmental factors include primary sclerosing
cholangitis, allergic bronchopulmonary aspergillosis, and
asthma.
[0013] Cigarette smoke, hypoxia, and environmental factors that
induce hypoxic signaling have also been demonstrated to impair CFTR
function and may contribute to certain 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-related diseases.
[0014] In addition to cystic fibrosis, modulation of CFTR activity
may be beneficial for other diseases not directly caused by
mutations in CFTR, such as secretory diseases and other protein
folding diseases mediated by CFTR. CFTR regulates chloride and
bicarbonate flux across the epithelia of many cells to control
fluid movement, protein solubilization, mucus viscosity, and enzyme
activity. Defects in CFTR can cause blockage of the airway or ducts
in many organs, including the liver and pancreas. Potentiators are
compounds that enhance the gating activity of CFTR present in the
cell membrane. Any disease which involves thickening of the mucus,
impaired fluid regulation, impaired mucus clearance, or blocked
ducts leading to inflammation and tissue destruction could be a
candidate for potentiators.
[0015] These include, but are not limited to, chronic obstructive
pulmonary disease (COPD), asthma, smoke induced COPD, chronic
bronchitis, rhinosinusitis, constipation, dry eye disease, and
Sjogren's Syndrome, gastro-esophageal reflux disease, gallstones,
rectal prolapse, and inflammatory bowel disease. COPD is
characterized by airflow limitation that is progressive and not
fully reversible. The airflow limitation is due to mucus
hypersecretion, emphysema, and bronchiolitis. Activators of mutant
or wild-type CFTR offer a potential treatment of mucus
hypersecretion and impaired mucociliary clearance that is common in
COPD. Specifically, increasing anion secretion across CFTR may
facilitate fluid transport into the airway surface liquid to
hydrate the mucus and optimized periciliary fluid viscosity. This
would lead to enhanced mucociliary clearance and a reduction in the
symptoms associated with COPD. In addition, by preventing ongoing
infection and inflammation due to improved airway clearance, CFTR
modulators may prevent or slow the parenchimal destruction of the
airway that characterizes emphysema and reduce or reverse the
increase in mucus secreting cell number and size that underlyses
mucus hypersecretion in airway diseases. Dry eye disease is
characterized by a decrease in tear aqueous production and abnormal
tear film lipid, protein and mucin profiles. There are many causes
of dry eye, some of which include age, Lasik eye surgery,
arthritis, medications, chemical/thermal bums, allergies, and
diseases, such as cystic fibrosis and Sjogrens's syndrome.
Increasing anion secretion via CFTR would enhance fluid transport
from the corneal endothelial cells and secretory glands surrounding
the eye to increase corneal hydration. This would help to alleviate
the symptoms associated with dry eye disease. Sjogrens's syndrome
is an autoimmune disease in which the immune system attacks
moisture-producing glands throughout the body, including the eye,
mouth, skin, respiratory tissue, liver, vagina, and gut. Symptoms,
include, dry eye, mouth, and vagina, as well as lung disease. The
disease is also associated with rheumatoid arthritis, systemic
lupus, systemic sclerosis, and polymypositis/dermatomyositis.
Defective protein trafficking is believed to cause the disease, for
which treatment options are limited. Modulators of CFTR activity
may hydrate the various organs afflicted by the disease and may
help to alleviate the associated symptoms. Individuals with cystic
fibrosis have recurrent episodes of intestinal obstruction and
higher incidences of rectal polapse, gallstones, gastro-esophageal
reflux disease, GI malignancies, and inflammatory bowel disease,
indicating that CFTR function may play an important role in
preventing such diseases.
[0016] As discussed above, it is believed that the deletion of
residue 508 in .DELTA.F508-CFTR prevents the nascent protein from
folding correctly, resulting in the inability of this mutant
protein to exit the ER, and traffic to the plasma membrane. As a
result, insufficient amounts of the mature protein are present at
the plasma membrane and chloride transport within epithelial
tissues is significantly reduced. In fact, this cellular phenomenon
of defective ER processing of CFTR by the ER machinery, has been
shown to be the underlying basis not only for CF disease, but for a
wide range of other isolated and inherited diseases. The two ways
that the ER machinery can malfunction is either by loss of coupling
to ER export of the proteins leading to degradation, or by the ER
accumulation of these defective/misfolded proteins [Aridor M, et
al., Nature Med., 5(7), pp 745- 751 (1999); Shastry, B.S., et al.,
Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et
al., Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al.,
TIPS, 21, pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp.
186-198 (1999)]. The diseases associated with the first class of ER
malfunction are cystic fibrosis (due to misfolded .DELTA.F508-CFTR
as discussed above), hereditary emphysema (due to al-antitrypsin;
non Piz variants), hereditary hemochromatosis,
coagulation-fibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases,
such as I-cell disease/pseudo-Hurler, Mucopolysaccharidoses (due to
lysosomal processing enzymes), Sandhof/Tay-Sachs (due to
.beta.-hexosaminidase), Crigler-Najjar type II (due to
UDP-glucuronyl-sialyc-transferase),
polyendocrinopathy/hyperinsulemia, Diabetes mellitus (due to
insulin receptor), Laron dwarfism (due to growth hormone receptor),
myleoperoxidase deficiency, primary hypoparathyroidism (due to
preproparathyroid hormone), melanoma (due to tyrosinase). The
diseases associated with the latter class of ER malfunction are
Glycanosis CDG type 1, hereditary emphysema (due to at-Antitrypsin
(PiZ variant), congenital hyperthyroidism, osteogenesis imperfecta
(due to Type I, II, IV procollagen), hereditary hypofibrinogenemia
(due to fibrinogen), ACT deficiency (due to al-antichymotrypsin),
Diabetes insipidus (DI), neurophyseal DI (due to vasopvessin
hormone/V2-receptor), neprogenic DI (due to aquaporin II),
Charcot-Marie Tooth syndrome (due to peripheral myelin protein 22),
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease (due to PAPP and presenilins), Parkinson's
disease, amyotrophic lateral sclerosis, progressive supranuclear
palsy, Pick's disease, several polyglutamine neurological disorders
such as Huntington's, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic
dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease (due to prion protein
processing defect), Fabry disease (due to lysosomal a-galactosidase
A), Straussler-Scheinker syndrome (due to Prp processing defect),
infertility pancreatitis, pancreatic insufficiency, osteoporosis,
osteopenia, Gorham's Syndrome, chloride channelopathies, myotonia
congenita (Thomson and Becker forms), Bartter's syndrome type III,
Dent's disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal
storage disease, Angelman syndrome, Primary Ciliary Dyskinesia
(PCD), PCD with situs inversus (also known as Kartagener syndrome),
PCD without situs inversus and ciliary aplasia, and liver
disease.
[0017] Other diseases implicated by a mutation in CFTR include male
infertility caused by congenital bilateral absence of the vas
deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,
and allergic bronchopulmonary aspergillosis (ABPA). See,
"CFTR-opathies: disease phenotypes associated with cystic fibrosis
transmembrane regulator gene mutations," Peader G. Noone and
Michael R. Knowles, Respir. Res. 2001, 2: 328-332 (incorporated
herein by reference).
[0018] In addition to up-regulation of CFTR activity, reducing
anion secretion by CFTR modulators may be beneficial for the
treatment of secretory diarrheas, in which epithelial water
transport is dramatically increased as a result of secretagogue
activated chloride transport. The mechanism involves elevation of
cAMP and stimulation of CFTR.
[0019] Although there are numerous causes of diarrhea, the major
consequences of diarrheal diseases, resulting from excessive
chloride transport are common to all, and include dehydration,
acidosis, impaired growth and death. Acute and chronic diarrheas
represent a major medical problem in many areas of the world.
Diarrhea is both a significant factor in malnutrition and the
leading cause of death (5,000,000 deaths/year) in children less
than five years old.
[0020] Secretory diarrheas are also a dangerous condition in
patients with acquired immunodeficiency syndrome (AIDS) and chronic
inflammatory bowel disease (IBD). Sixteen million travelers to
developing countries from industrialized nations every year develop
diarrhea, with the severity and number of cases of diarrhea varying
depending on the country and area of travel.
[0021] Accordingly, there is a need for potent and selective CFTR
potentiators of wild-type and mutant forms of human CFTR. These
mutant CFTR forms include, but are not limited to, .DELTA.F508del,
G551D, R117H, 2789+5G->A.
[0022] There is also a need for modulators of CFTR activity, and
compositions thereof, which can be used to modulate the activity of
the CFTR in the cell membrane of a mammal.
[0023] There is a need for methods of treating diseases caused by
mutation in CFTR using such modulators of CFTR activity.
[0024] There is a need for methods of modulating CFTR activity in
an ex vivo cell membrane of a mammal.
[0025] In addition, there is a need for stable solid forms of said
compound that can be used readily in pharmaceutical compositions
suitable for use as therapeutics.
SUMMARY OF THE INVENTION
[0026] The present invention relates to 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 (hereinafter
"Compound 1") which has the structure below:
##STR00001##
[0027] Compound 1 and pharmaceutically acceptable compositions
thereof are useful for treating 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, such as protein C deficiency, Type 1 hereditary
angioedema, lipid processing deficiencies, such as familial
hypercholesterolemia, Type 1 chylomicronemia, Abetalipoproteinemia,
lysosomal storage diseases, such as I-cell disease/pseudo-hurler,
mucopolysaccharidoses, sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, neprogenic DI, Charcot-Marie tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington's,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as
spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob
disease, Fabry disease, Straussler-Scheinker syndrome, COPD,
dry-eye disease, pancreatic insufficiency, osteoporosis,
osteopenia, Gorham's Syndrome, chloride channelopathies, myotonia
congenita (Thomson and Becker forms), Bartter's syndrome type III,
Dent's disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal
storage disease, Angelman syndrome, Primary Ciliary Dyskinesia
(PCD), PCD with situs inversus (also known as Kartagener syndrome),
PCD without situs inversus and ciliary aplasia, and Sjogren's
disease.
[0028] In one aspect, Compound 1 is in a substantially crystalline,
neat free form Form A.
[0029] Processes described herein can be used to prepare the
compositions of this invention comprising Form A. The amounts and
the features of the components used in the processes are be as
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is an X-ray powder diffraction pattern of Form A.
[0031] FIG. 2 is a conformational picture of Form A based on single
X-ray crystal analysis.
[0032] FIG. 3 provides an FTIR spectrum of Form A.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Definitions
[0034] As used herein, the following definitions shall apply unless
otherwise indicated.
[0035] The term "ABC-transporter" as used herein means an
ABC-transporter protein or a fragment thereof comprising at least
one binding domain, wherein said protein or fragment thereof is
present in vivo or in vitro. The term "binding domain" as used
herein means a domain on the ABC-transporter that can bind to a
modulator. See, e.g., Hwang, T. C. et al., J. Gen. Physiol. (1998):
111(3), 477-90.
[0036] The term "CFTR" as used herein means cystic fibrosis
transmembrane conductance regulator or a mutation thereof capable
of regulator activity, including, but not limited to, AF508 CFTR,
R11711 CFTR, and G551D CFTR (see, e.g.,
http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).
[0037] The term "modulating" as used herein means increasing or
decreasing by a measurable amount.
[0038] The term "normal CFTR" or "normal CFTR function" as used
herein means wild-type like CFTR without any impairment due to
environmental factors such as smoking, pollution, or anything that
produces inflammation in the lungs.
[0039] The term "reduced CFTR" or "reduced CFTR function" as used
herein means less than normal CFTR or less than normal CFTR
function.
[0040] As used herein "crystalline" refers to compounds or
compositions where the structural units are arranged in fixed
geometric patterns or lattices, so that crystalline solids have
rigid long range order. The structural units that constitute the
crystal structure can be atoms, molecules, or ions. Crystalline
solids show definite melting points.
[0041] As used herein the phrase "substantially crystalline", means
a solid material that is arranged in fixed geometric patterns or
lattices that have rigid long range order. For example,
substantially crystalline materials have more than about 85%
crystallinity (e.g., more than about 90% crystallinity or more than
about 95% crystallinity). It is also noted that the term
`substantially crystalline` includes the descriptor `crystalline`,
which is defined in the previous paragraph.
[0042] In one aspect, the invention features a form of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide characterized
as Form A.
[0043] In some embodiments, Form A is characterized by one or more
peaks: from about 7.7 to about 8.1 degrees, for example, about 7.9
degrees; from about 11.7 to about 12.1 degrees, for example, about
11.9 degrees; from about 14.2 to about 14.6 degrees, for example,
about 14.4 degrees; and about 15.6 to about 16.0 degrees, for
example, about 15.8 degrees; in an X-ray powder diffraction
obtained using Cu K alpha radiation.
[0044] In some embodiments, Form A is characterized by one or more
peaks: from about 7.8 to about 8.0 degrees, for example, about 7.9
degrees; from about 11.8 to about 12.0 degrees, for example, about
11.9 degrees; from about 14.3 to about 14.5 degrees, for example,
about 14.4 degrees; and about 15.7 to about 15.9 degrees, for
example, about 15.8 degrees; in an X-ray powder diffraction
obtained using Cu K alpha radiation.
[0045] In other embodiments, Form A is characterized by one or more
peaks from about: 7.7 to about 8.1 degrees, for example, about 7.9
degrees; from about 21.6 to about 22.0 degrees, for example, about
21.8 degrees; and about 23.6 to about 24.0 degrees, for example,
about 23.8 degrees; in an X-ray powder diffraction obtained using
Cu K alpha radiation.
[0046] In still other embodiments, Form A is characterized by one
or more peaks from about: 7.8 to about 8.0 degrees, for example,
about 7.9 degrees; from about 21.7 to about 21.9 degrees, for
example, about 21.8 degrees; and about 23.7 to about 23.9 degrees,
for example, about 23.8 degrees; in an X-ray powder diffraction
obtained using Cu K alpha radiation.
[0047] 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 from about 7.7 to about 8.1 degrees
(e.g., about 7.9 degrees); a peak from about 9.1 to about 9.5
degrees, (e.g., about 9.3 degrees); a peak from about 11.7 to about
12.1 degrees, (e.g., about 11.9 degrees); a peak from about 14.2 to
about 14.6 degrees, (e.g., about 14.4 degrees); a peak from about
14.9 to about 15.3 degrees, (e.g., about 15.1 degrees); a peak from
about 15.6 to about 16.0 degrees, (e.g., about 15.8 degrees); a
peak from about 16.8 to about 17.2 degrees, (e.g., about 17.0
degrees); a peak from about 17.5 to about 17.9 degrees, (e.g.,
about 17.7 degrees); a peak from about 19.1 to about 19.5 degrees,
(e.g., about 19.3 degrees); a peak from about 19.9 to about 20.3
degrees, (e.g., about 20.1 degrees); a peak from about 21.2 to
about 21.6 degrees, (e.g., about 21.4 degrees); a peak from about
21.6 to about 22.0 degrees, (e.g., about 21.8 degrees); a peak from
about 23.2 to about 23.6 degrees, (e.g., about 23.4 degrees); a
peak from about 23.6 to about 24.0 degrees, (e.g., about 23.8
degrees); a peak from about 25.4 to about 25.8 degrees, (e.g.,
about 25.6 degrees); a peak from about 26.6 to about 27.0 degrees,
(e.g., about 26.8 degrees); a peak from about 29.2 to about 29.6
degrees, (e.g., about 29.4 degrees); a peak from about 29.5 to
about 29.9 degrees, (e.g., about 29.7 degrees); a peak from about
29.9 to about 30.3 degrees, (e.g., about 30.1 degrees); and a peak
from about 31.0 to about 31.4 degrees, (e.g., about 31.2
degrees).
[0048] 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 from about 7.8 to about 8.0 degrees
(e.g., about 7.9 degrees); a peak from about 9.2 to about 9.4
degrees, (e.g., about 9.3 degrees); a peak from about 11.8 to about
12.0 degrees, (e.g., about 11.9 degrees); a peak from about 14.3 to
about 14.5 degrees, (e.g., about 14.4 degrees); a peak from about
15.0 to about 15.2 degrees, (e.g., about 15.1 degrees); a peak from
about 15.7 to about 15.9 degrees, (e.g., about 15.8 degrees); a
peak from about 16.9 to about 17.1 degrees, (e.g., about 17.0
degrees); a peak from about 17.6 to about 17.8 degrees, (e.g.,
about 17.7 degrees); a peak from about 19.2 to about 19.4 degrees,
(e.g., about 19.3 degrees); a peak from about 20.0 to about 20.2
degrees, (e.g., about 20.1 degrees); a peak from about 21.3 to
about 21.5 degrees, (e.g., about 21.4 degrees); a peak from about
21.7 to about 21.9 degrees, (e.g., about 21.8 degrees); a peak from
about 23.3 to about 23.5 degrees, (e.g., about 23.4 degrees); a
peak from about 23.7 to about 23.9 degrees, (e.g., about 23.8
degrees); a peak from about 25.5 to about 25.7 degrees, (e.g.,
about 25.6 degrees); a peak from about 26.7 to about 26.9 degrees,
(e.g., about 26.8 degrees); a peak from about 29.3 to about 29.5
degrees, (e.g., about 29.4 degrees); a peak from about 29.6 to
about 29.8 degrees, (e.g., about 29.7 degrees); a peak from about
30.0 to about 30.2 degrees, (e.g., about 30.1 degrees); and a peak
from about 31.1 to about 31.3 degrees, (e.g., about 31.2
degrees).
[0049] In some embodiments, Form A is characterized by a
diffraction pattern as provided in FIG. 1.
[0050] In one aspect, the invention features a pharmaceutical
composition comprising Form A and a pharmaceutically acceptable
adjuvant or carrier.
[0051] In one aspect, the present invention features a method of
treating a CFTR mediated disease in a human comprising
administering to the human an effective amount of Form A.
[0052] In some embodiments, the method comprises administering an
additional therapeutic agent.
[0053] In some embodiments, the disease is selected from cystic
fibrosis, pancreatitis, sinusitis, hereditary emphysema, hereditary
hemochromatosis, coagulation-fibrinolysis deficiencies, such as
protein C deficiency, Type 1 hereditary angioedema, lipid
processing deficiencies, such as familial hypercholesterolemia,
Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington's,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as
spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob
disease, Fabry disease, Straussler-Scheinker syndrome, COPD,
dry-eye disease, pancreatic insufficiency, osteoporosis,
osteopenia, Gorham's Syndrome, chloride channelopathies, myotonia
congenita (Thomson and Becker forms), Bartter's syndrome type III,
Dent's disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal
storage disease, Angelman syndrome, Primary Ciliary Dyskinesia
(PCD), PCD with situs inversus (also known as Kartagener syndrome),
PCD without situs inversus and ciliary aplasia, and Sjogren's
disease.
[0054] In one embodiment, the present invention provides a method
of treating cystic fibrosis in a human, comprising administering to
said human an effective amount of Form A.
[0055] In one aspect, the present invention features a
pharmaceutical pack or kit comprising Form A and a pharmaceutically
acceptable carrier.
[0056] In one aspect, the invention features a crystal form of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide having a
trigonal crystal system, a R-3 space group, and the following unit
cell dimensions: a=19.1670(4) .ANG., b=19.1670(4) .ANG.,
c=33.6572(12) .ANG., .alpha.=90.degree., .beta.=90.degree., and
.gamma.=120.degree..
[0057] In one embodiment, the present invention provides a crystal
form of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide having unit
cell dimensions: a=19.1670(4) .ANG., b=19.1670(4) .ANG.,
c=33.6572(12) .ANG..
[0058] Uses, Formulation and Administration
[0059] Pharmaceutically Acceptable Compositions
[0060] In one aspect of the present invention, pharmaceutically
acceptable compositions are provided, wherein these compositions
comprise Form A as described herein, and optionally comprise a
pharmaceutically acceptable carrier, adjuvant or vehicle. In
certain embodiments, these compositions optionally further comprise
one or more additional therapeutic agents.
[0061] As described above, the pharmaceutically acceptable
compositions of the present invention additionally comprise a
pharmaceutically acceptable carrier, adjuvant, or vehicle, which,
as used herein, includes any and all solvents, diluents, or other
liquid vehicle, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's Pharmaceutical
Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co.,
Easton, Pa., 1980) discloses various carriers used in formulating
pharmaceutically acceptable compositions and known techniques for
the preparation thereof. Except insofar as any conventional carrier
medium is incompatible with the compounds of the invention, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutically acceptable composition, its use is
contemplated to be within the scope of this invention. Some
examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins,
such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, or potassium sorbate, 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 polymers, wool fat, sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; 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 a propylene glycol or polyethylene glycol; esters
such as ethyl oleate and ethyl laurate; agar; buffering agents such
as magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
[0062] Uses of Compounds and Pharmaceutically Acceptable
Compositions
[0063] In yet another aspect, the present invention provides a
method of treating or lessening the severity of a condition,
disease, or disorder implicated by CFTR mutation. In certain
embodiments, the present invention provides a method of treating a
condition, disease, or disorder implicated by a deficiency of the
CFTR activity, the method comprising administering a composition
comprising a compound 1 Form A to a subject, preferably a mammal,
in need thereof.
[0064] In certain embodiments, the present invention provides a
method of treating diseases associated with reduced CFTR function
due to mutations in the gene encoding CFTR or environmental factors
(e.g., smoke). These diseases include, cystic fibrosis, chronic
bronchitis, recurrent bronchitis, acute bronchitis, male
infertility caused by congenital bilateral absence of the vas
deferens (CBAVD), female infertility caused by congenital absence
of the uterus and vagina (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.
[0065] In certain embodiments, the present invention provides a
method for treating diseases 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 caused by congenital
bilateral absence of the vas deferens (CBAVD), mild pulmonary
disease, idiopathic pancreatitis, liver disease, hereditary
emphysema, gallstones, gasgtro-esophageal reflux disease,
gastrointestinal malignancies, inflammatory bowel disease,
constipation, diabetes, arthritis, osteoporosis, and
osteopenia.
[0066] In certain embodiments, the present invention provides a
method for treating diseases associated with normal CFTR function
including hereditary hemochromatosis, coagulation-fibrinolysis
deficiencies, such as protein C deficiency, Type 1 hereditary
angioedema, lipid processing deficiencies, such as familial
hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,
lysosomal storage diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, congenital hyperthyroidism,
osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT
deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic
DI, Charcot-Marie Tooth syndrome, Perlizaeus-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, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic
dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease (due to prion protein
processing defect), Fabry disease, Straussler-Scheinker syndrome,
Gorham's Syndrome, chloride channelopathies, myotonia congenita
(Thomson and Becker forms), Bartter's syndrome type III, Dent's
disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal storage
disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD
with situs inversus (also known as Kartagener syndrome), PCD
without situs inversus and ciliary aplasia, or Sjogren's disease,
comprising the step of administering to said mammal an effective
amount of a composition comprising Form A described herein.
[0067] 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 described herein.
[0068] According to the invention an "effective amount" of Form A
or a pharmaceutically acceptable composition thereof is that amount
effective for treating or lessening the severity of one or more of
the diseases, disorders or conditions as recited above.
[0069] Form A or a pharmaceutically acceptable composition thereof
may be administered using any amount and any route of
administration effective for treating or lessening the severity of
one or more of the diseases, disorders or conditions as recited
above.
[0070] In certain embodiments, Form A or a pharmaceutically
acceptable composition thereof is useful for treating or lessening
the severity of cystic fibrosis in patients who exhibit residual
CFTR activity in the apical membrane of respiratory and
non-respiratory epithelia. The presence of residual CFTR activity
at the epithelial surface can be readily detected using methods
known in the art, e.g., standard electrophysiological, biochemical,
or histochemical techniques. Such methods identify CFTR activity
using in vivo or ex vivo electrophysiological techniques,
measurement of sweat or salivary CF concentrations, or ex vivo
biochemical or histochemical techniques to monitor cell surface
density. Using such methods, residual CFTR activity can be readily
detected in patients heterozygous or homozygous for a variety of
different mutations, including patients homozygous or heterozygous
for the most common mutation, .DELTA.F508.
[0071] In another embodiment, Form A described herein or a
pharmaceutically acceptable composition thereof is useful for
treating or lessening the severity of cystic fibrosis in patients
who have residual CFTR activity induced or augmented using
pharmacological methods or gene therapy. Such methods increase the
amount of CFTR present at the cell surface, thereby inducing a
hitherto absent CFTR activity in a patient or augmenting the
existing level of residual CFTR activity in a patient.
[0072] In one embodiment, Form A described herein or a
pharmaceutically acceptable composition thereof is useful for
treating or lessening the severity of cystic fibrosis in patients
within certain genotypes exhibiting residual CFTR activity, e.g.,
class III mutations (impaired regulation or gating), class IV
mutations (altered conductance), or class V mutations (reduced
synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III,
IV, and V cystic fibrosis Tansmembrane Conductance Regulator
Defects and Opportunities of Therapy; Current Opinion in Pulmonary
Medicine 6:521-529, 2000). Other patient genotypes that exhibit
residual CFTR activity include patients homozygous for one of these
classes or heterozygous with any other class of mutations,
including class I mutations, class II mutations, or a mutation that
lacks classification.
[0073] In one embodiment, Form A described herein or a
pharmaceutically acceptable composition thereof is useful for
treating or lessening the severity of cystic fibrosis in patients
within certain clinical phenotypes, e.g., a moderate to mild
clinical phenotype that typically correlates with the amount of
residual CFTR activity in the apical membrane of epithelia. Such
phenotypes include patients exhibiting pancreatic insufficiency or
patients diagnosed with idiopathic pancreatitis and congenital
bilateral absence of the vas deferens, or mild lung disease.
[0074] 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 agent, its
mode of administration, and the like. The compounds of the
invention are preferably formulated in dosage unit form for ease of
administration and uniformity of dosage. The expression "dosage
unit form" as used herein refers to a physically discrete unit of
agent appropriate for the patient to be treated. It will be
understood, however, that the total daily usage of the compounds
and compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgment. The
specific effective dose level for any particular patient or
organism will depend upon a variety of factors including the
disorder being treated and the severity of the disorder; the
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed, and like
factors well known in the medical arts. The term "patient", as used
herein, means an animal, preferably a mammal, and most preferably a
human.
[0075] The pharmaceutically acceptable compositions of this
invention can be administered to humans and other animals orally,
rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, ointments, drops or
patch), bucally, as an oral or nasal spray, or the like, depending
on the severity of the infection being treated. In certain
embodiments, the compounds of the invention may be administered
orally or parenterally at dosage levels of about 0.01 mg/kg to
about 50 mg/kg and preferably from about 0.5 mg/kg to about 25
mg/kg, of subject body weight per day, one or more times a day, to
obtain the desired therapeutic effect.
[0076] 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.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0077] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed 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 can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0078] The 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 prior to use.
[0079] In order to prolong the effect of a compound of the present
invention, it is often desirable to slow the absorption of the
compound from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the compound then depends upon its rate of
dissolution that, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally administered compound form is accomplished by
dissolving or suspending the compound in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the
compound in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of compound to
polymer and the nature of the particular polymer employed, the rate
of compound release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the compound in liposomes or microemulsions that are
compatible with body tissues.
[0080] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax 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.
[0081] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, 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) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0082] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, 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 can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0083] The active compounds can also be in microencapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a 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
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0084] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, eardrops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
are prepared by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0085] It will also be appreciated that the Form A described herein
or a pharmaceutically acceptable composition thereof can be
employed in combination therapies, that is, Form A described herein
or a pharmaceutically acceptable composition thereof can be
administered concurrently with, prior to, or subsequent to, one or
more other desired therapeutics or medical procedures. The
particular combination of therapies (therapeutics or procedures) to
employ in a combination regimen will take into account
compatibility of the desired therapeutics and/or procedures and the
desired therapeutic effect to be achieved. It will also be
appreciated that the therapies employed may achieve a desired
effect for the same disorder (for example, an inventive compound
may be administered concurrently with another agent used to treat
the same disorder), or they may achieve different effects (e.g.,
control of any adverse effects). As used herein, additional
therapeutic agents that are normally administered to treat or
prevent a particular disease, or condition, are known as
"appropriate for the disease, or condition, being treated."
[0086] In one embodiment, the additional agent is selected from a
mucolytic agent, bronchodialator, an anti-biotic, an anti-infective
agent, an anti-inflammatory agent, a CFTR modulator other than a
compound of the present invention, or a nutritional agent.
[0087] In one embodiment, the additional agent is an antibiotic.
Exemplary antibiotics useful herein include tobramycin, including
tobramycin inhaled powder (TIP), azithromycin, aztreonam, including
the aerosolized form of aztreonam, amikacin, including liposomal
formulations thereof, ciprofloxacin, including formulations thereof
suitable for administration by inhalation, levoflaxacin, including
aerosolized formulations thereof, and combinations of two
antibiotics, e.g., fosfomycin and tobramycin.
[0088] In another embodiment, the additional agent is a mucolyte.
Exemplary mucolytes useful herein includes Pulmozyme.RTM..
[0089] In another embodiment, the additional agent is a
bronchodialator. Exemplary bronchodialtors include albuterol,
metaprotenerol sulfate, pirbuterol acetate, salmeterol, or
tetrabuline sulfate.
[0090] In another embodiment, the additional agent is effective in
restoring lung airway surface liquid. Such agents improve the
movement of salt in and out of cells, allowing mucus in the lung
airway to be more hydrated and, therefore, cleared more easily.
Exemplary such agents include hypertonic saline, denufosol
tetrasodium ([[(3S,
5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyp-
hosphoryl] [[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,
4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]
hydrogen phosphate), or bronchitol (inhaled formulation of
mannitol).
[0091] In another embodiment, the additional agent is an
anti-inflammatory agent, i.e., an agent that can reduce the
inflammation in the lungs. Exemplary such agents useful herein
include ibuprofen, docosahexanoic acid (DHA), sildenafil, inhaled
glutathione, pioglitazone, hydroxychloroquine, or simavastatin.
[0092] In another embodiment, the additional agent reduces the
activity of the epithelial sodium channel blocker (ENaC) either
directly by blocking the channel or indirectly by modulation of
proteases that lead to an increase in ENaC activity (e.g., seine
proteases, channel-activating proteases). Exemplary such agents
include camostat (a trypsin-like protease inhibitor), QAU145,
552-02, GS-9411, INO-4995, Aerolytic, and amiloride. Additional
agents that reduce the activity of the epithelial sodium channel
blocker (ENaC) can be found, for example, in PCT Publication No.
W02009/074575, the entire contents of which are incorporated herein
in their entirety.
[0093] Amongst other diseases described herein, combinations of
CFTR modulators, such as Form A, and agents that reduce the
activity of ENaC are use for treating Liddle's syndrome, an
inflammatory or allergic condition including cystic fibrosis,
primary ciliary dyskinesia, chronic bronchitis, chronic obstructive
pulmonary disease, asthma, respiratory tract infections, lung
carcinoma, xerostomia and keratoconjunctivitis sire, respiratory
tract infections (acute and chronic; viral and bacterial) and lung
carcinoma.
[0094] Combinations of CFTR modulators, such as Form A, and agents
that reduce the activity of ENaC are also useful for treating
diseases mediated by blockade of the epithelial sodium channel also
include diseases other than respiratory diseases that are
associated with abnormal fluid regulation across an epithelium,
perhaps involving abnormal physiology of the protective surface
liquids on their surface, e.g., xerostomia (dry mouth) or
keratoconjunctivitis sire (dry eye). Furthermore, blockade of the
epithelial sodium channel in the kidney could be used to promote
diuresis and thereby induce a hypotensive effect.
[0095] Asthma includes both intrinsic (non-allergic) asthma and
extrinsic (allergic) asthma, mild asthma, moderate asthma, severe
asthma, bronchitic asthma, exercise-induced asthma, occupational
asthma and asthma induced following bacterial infection. Treatment
of asthma is also to be understood as embracing treatment of
subjects, e.g., of less than 4 or 5 years of age, exhibiting
wheezing symptoms and diagnosed or diagnosable as "wheezy infants",
an established patient category of major medical concern and now
often identified as incipient or early-phase asthmatics. (For
convenience this particular asthmatic condition is referred to as
"wheezy-infant syndrome".) Prophylactic efficacy in the treatment
of asthma will be evidenced by reduced frequency or severity of
symptomatic attack, e.g., of acute asthmatic or bronchoconstrictor
attack, improvement in lung function or improved airways
hyperreactivity. It may further be evidenced by reduced requirement
for other, symptomatic therapy, i.e., therapy for or intended to
restrict or abort symptomatic attack when it occurs, e.g.,
anti-inflammatory (e.g., cortico-steroid) or bronchodilatory.
Prophylactic benefit in asthma may, in particular, be apparent in
subjects prone to "morning dipping". "Morning dipping" is a
recognized asthmatic syndrome, common to a substantial percentage
of asthmatics and characterized by asthma attack, e.g., between the
hours of about 4-6 am, i.e., at a time normally substantially
distant from any previously administered symptomatic asthma
therapy.
[0096] Chronic obstructive pulmonary disease includes chronic
bronchitis or dyspnea associated therewith, emphysema, as well as
exacerbation of airways hyperreactivity consequent to other drug
therapy, in particular, other inhaled drug therapy. In some
embodiments, the combinations of CFTR modulators, such as Form A,
and agents that reduce the activity of ENaC are useful for the
treatment of bronchitis of whatever type or genesis including,
e.g., acute, arachidic, catarrhal, croupus, chronic or phthinoid
bronchitis.
[0097] In another embodiment, the additional agent is a CFTR
modulator other than compound 1 Form A, i.e., an agent that has the
effect of modulating CFTR activity. Exemplary such agents include
ataluren ("PTC124.RTM.";
3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid),
sinapultide, lancovutide, depelestat (a human recombinant
neutrophil elastase inhibitor), cobiprostone (7-{(2R, 4aR, 5R,
7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclope-
nta[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 agent is
(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxo1-5-yl)
cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.
[0098] In another embodiment, the additional agent is a nutritional
agent. Exemplary such agents include pancrelipase (pancreating
enzyme replacement), including Pancrease.RTM., Pancreacarb.RTM.,
Ultrase.RTM., or Creon.RTM., Liprotomase.RTM. (formerly
Trizytek.RTM.), Aquadeks.RTM., or glutathione inhalation. In one
embodiment, the additional nutritional agent is pancrelipase.
[0099] In one embodiment, the additional agent is a CFTR modulator
other than a compound of the present invention.
[0100] The amount of additional therapeutic agent present in the
compositions of this invention will be no more than the amount that
would normally be administered in a composition comprising that
therapeutic agent as the only active agent. Preferably the amount
of additional therapeutic agent in the presently disclosed
compositions will range from about 50% to 100% of the amount
normally present in a composition comprising that agent as the only
therapeutically active agent.
[0101] Form A described herein or a pharmaceutically acceptable
composition thereof may also be incorporated into compositions for
coating an implantable medical device, such as prostheses,
artificial valves, vascular grafts, stents and catheters.
Accordingly, the present invention, in another aspect, includes a
composition for coating an implantable device comprising a compound
of the present invention as described generally above, and in
classes and subclasses herein, and a carrier suitable for coating
said implantable device. In still another aspect, the present
invention includes an implantable device coated with a composition
comprising a compound of the present invention as described
generally above, and in classes and subclasses herein, and a
carrier suitable for coating said implantable device. Suitable
coatings and the general preparation of coated implantable devices
are described in U.S. Pat. Nos. 6,099,562; 5,886,026; and
5,304,121. The coatings are typically biocompatible polymeric
materials such as a hydrogel polymer, polymethyldisiloxane,
polycaprolactone, polyethyleneglycol, polylactic acid, ethylene
vinyl acetate, and mixtures thereof. The coatings may optionally be
further covered by a suitable topcoat of fluorosilicone,
polysaccarides, polyethylene glycol, phospholipids or combinations
thereof to impart controlled release characteristics in the
composition.
[0102] Another aspect of the invention relates to modulating CFTR
activity in a biological sample or a patient (e.g., in vitro or in
vivo), which method comprises administering to the patient, or
contacting said biological sample with Form A described herein or a
pharmaceutically acceptable composition thereof. The term
"biological sample", as used herein, includes, without limitation,
cell cultures or extracts thereof; biopsied material obtained from
a mammal or extracts thereof; and blood, saliva, urine, feces,
semen, tears, or other body fluids or extracts thereof.
[0103] Modulation of CFTR in a biological sample is useful for a
variety of purposes that are known to one of skill in the art.
Examples of such purposes include, but are not limited to, the
study of CFTR in biological and pathological phenomena; and the
comparative evaluation of new modulators of CFTR.
[0104] In yet another embodiment, a method of modulating activity
of an anion channel in vitro or in vivo, is provided comprising the
step of contacting said channel with Form A described herein or a
pharmaceutically acceptable composition thereof. In preferred
embodiments, the anion channel is a chloride channel or a
bicarbonate channel. In other preferred embodiments, the anion
channel is a chloride channel.
[0105] According to an alternative embodiment, the present
invention provides a method of increasing the number of functional
CFTR in a membrane of a cell, comprising the step of contacting
said cell with Form A described herein or a pharmaceutically
acceptable composition thereof.
[0106] According to another preferred embodiment, the activity of
the CFTR is measured by measuring the transmembrane voltage
potential. Means for measuring the voltage potential across a
membrane in the biological sample may employ any of the known
methods in the art, such as optical membrane potential assay or
other electrophysiological methods.
[0107] The optical membrane potential assay utilizes
voltage-sensitive FRET sensors described by Gonzalez and Tsien
(See.sub.., Gonzalez, J. E. and R. Y. Tsien (1995) "Voltage sensing
by fluorescence resonance energy transfer in single cells."Biophys
J 69(4): 1272-80, and Gonzalez, J. E. and R. Y. Tsien (1997);
"Improved indicators of cell membrane potential that use
fluorescence resonance energy transfer" Chem Biol 4(4): 269-77) in
combination with instrumentation for measuring fluorescence changes
such as the Voltage/Ion Probe Reader (VIPR) (See.sub.s Gonzalez, J.
E., K. Oades, et al. (1999) "Cell-based assays and instrumentation
for screening ion-channel targets" Drug Discov Today 4(9):
431-439).
[0108] These voltage sensitive assays are based on the change in
fluorescence resonant energy transfer (FRET) between the
membrane-soluble, voltage-sensitive dye, DiSBAC.sub.2(3), and a
fluorescent phospholipid, CC2-DMPE, which is attached to the outer
leaflet of the plasma membrane and acts as a FRET donor. Changes in
membrane potential (V.sub.m) cause the negatively charged
DiSBAC.sub.2(3) to redistribute across the plasma membrane and the
amount of energy transfer from CC2-DMPE changes accordingly. The
changes in fluorescence emission can be monitored using VIPR.TM.
II, which is an integrated liquid handler and fluorescent detector
designed to conduct cell-based screens in 96- or 384-well
microtiter plates.
[0109] In another aspect the present invention provides a kit for
use in measuring the activity of CFTR or a fragment thereof in a
biological sample in vitro or in vivo 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) measuring activity of said CFTR or a fragment
thereof. In one embodiment, the kit further comprises instructions
for a) contacting an additional composition with the biological
sample; b) measuring the activity of said CFTR or a fragment
thereof in the presence of said additional compound, and c)
comparing the activity of the CFTR in the presence of the
additional compound with the density of the CFTR in the presence of
Form A described herein. In preferred embodiments, the kit is used
to measure the density of CFTR.
[0110] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
EXAMPLES
[0111] Methods & Materials
[0112] XRPD (X-ray Powder Diffraction)
[0113] The X-ray powder diffraction (XRPD) data were recorded at
room temperature using a Rigaku/MSC MiniFlex Desktop Powder X-ray
Diffractometer (Rigaku, The Woodlands, Tex.). The X-Ray was
generated using Cu tube operated at 30 kV and 15 mA with KB
suppression filter. The divergence slit was variable with the
scattering and receiving slits set at 4.2 degree and slit 0.3 mm,
respectively. The scan mode was fixed time (FT) with 0.02 degree
step width and count time of 2.0 seconds. The Powder X-ray
Diffractometer was calibrated using reference standard: 75%
Sodalite (Na.sub.3Al.sub.4Si.sub.4O.sub.12C1) and 25% Silicon
(Rigaku, Cat# 2100/ALS). The six samples stage was used with zero
background sample holders (SH-LBSI511-RNDB). The powder sample was
placed on the indented area and flattened with glass slide.
[0114] FTIR (Fourier Transform Infrared) Spectroscopy
[0115] FTIR spectra were collected from a Thermo Scientific,
Nicolet 6700 FT-IR spectrometer, with smart orbit sampling
compartment, diamond window, using Software: Omnic, 7.4. The powder
sample was placed directly on the diamond crystal and pressure was
added to conform the surface of the sample to the surface of the
diamond crystal. The background spectrum was collected and then the
sample spectrum was collected. The collection settings were as
follows: [0116] Detector: DTGS KBr; [0117] Beamsplitter: KBr;
[0118] Source: IR; [0119] Scan range: 4000-400 cm.sup.-1; [0120]
Gain: 8.0; [0121] Optical velocity: 0.6329 cm/sec; [0122] Aperture:
100; [0123] No. of scans: 32; and [0124] Resolution: 4
cm.sup.-1.
Example 1
Preparation of
4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic Acid
(7).
##STR00002##
[0126] 2-Chloro-5-(trifluoromethyl)aniline 2 (200 g, 1.023 mol),
diethyl 2-(ethoxymethylene)malonate 3 (276 g, 1.3 mol) and toluene
(100 mL) were combined under a nitrogen atmosphere in a three-neck,
1-L round bottom flask equipped with Dean-Stark condenser. The
solution was heated with stirring to 140.degree. C. and the
temperature was maintained for 4 h. The reaction mixture was cooled
to 70.degree. C. and hexane (600 mL) was slowly added. 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 (2.times.400 mL) and then dried under
vacuum to provide a white solid (350 g, 94% yield) as the desired
condensation product diethyl
24(2-chloro-5-(trifluoromethyl)phenylamino) methylene) malonate 4.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.28 (d, J=13.0 Hz, 1
H), 8.63 (d, J=13.0 Hz, 1 H), 8.10 (s, 1 H), 7.80 (d, J=8.3 Hz, 1
H), 7.50 (dd, J=1.5, 8.4 Hz, 1H), 4.24 (q, J=7.1 Hz, 2H), 4.17 (q,
J=7.1 Hz,2 H),1.27 (m, 6H).
[0127] Preparation of ethyl
8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate
(5). A 3-neck, 1-L flask was charged with Dowtherm.RTM. (200 mL, 8
mL/g), which was degassed at 200.degree. C. for 1 h. The solvent
was heated to 260.degree. C. and charged in portions over 10 min
with diethyl 2-((2-chloro-5-(trifluoromethyl)phenylamino)
methylene)malonate 4 (25 g, 0.07 mol). The resulting mixture was
stirred at 260.degree. C. for 6.5 hours (h) and the resulting
ethanol byproduct removed by distillation. The mixture was allowed
to slowly cool to 80.degree. C. Hexane (150 mL) was slowly added
over 30 minutes (min), followed by an additional 200 mL of hexane
added in one portion. The slurry was stirred until it had reached
room temperature. The solid was filtered, washed with hexane
(3.times.150 mL), and then dried under vacuum to provide ethyl
8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate
5 as a tan solid (13.9 g, 65% yield). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta.11.91 (s, 1H), 8.39 (s, 1H), 8.06 (d, J=8.3
Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 4.24 (q, J=7.1 Hz, 2H), 1.29 (t,
J=7.1 Hz, 3H).
[0128] Preparation of ethyl
4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate (6). A 3-neck,
5-L flask was charged with of ethyl
8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate
5 (100 g, 0.3 mol), ethanol (1250 mL, 12.5 mL/g) and triethylamine
(220 mL, 1.6 mol). The vessel was then charged with 10 g of 10%
Pd/C (50% wet) at 5.degree. C. The reaction was stirred vigorously
under hydrogen atmosphere for 20 h at 5.degree. C., after which
time the reaction mixture was concentrated to a volume of
approximately 150 mL. The product, ethyl
4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate 6, as a slurry
with Pd/C, was taken directly into the next step.
[0129] Preparation of
4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid
(7). Ethyl 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate 6
(58 g, 0.2 mol, crude reaction slurry containing Pd/C) was
suspended in NaOH (814 mL of 5 M, 4.1 mol) in a 1-L flask with a
reflux condenser and heated at 80.degree. C. for 18 h, followed by
further heating at 100.degree. C. for 5 h. The reaction was
filtered warm through packed Celite to remove Pd/C and the Celite
was rinsed with 1 N NaOH. The filtrate was acidified to about pH 1
to obtain a thick, white precipitate. The precipitate was filtered
then rinsed with water and cold acetonitrile. The solid was then
dried under vacuum to provide
4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid 7
as a white solid (48 g, 92% yield). .sup.1H NMR (400.0 MHz,
DMSO-d.sub.6) .delta. 15.26 (s, 1H), 13.66 (s, 1H), 8.98 (s, 1H),
8.13 (dd, J=1.6, 7.8 Hz, 1H), 8.06-7.99 (m, 2H).
Example 2
Preparation of
4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethypaniline
##STR00003##
[0131] Preparation of 7-[4-nitro-3-(trifluoromethyl)
phenyl]-7-azabicyclo[2.2.1]heptane (20). To a flask containing
7-azabicyclo[2.2.1]heptane hydrochloride 9 (4.6 g, 34.43 mmol,
obtained from under a nitrogen atmosphere was added a solution of
4-fluoro-1-nitro-2-(trifluoromethyl)benzene 8 (6.0 g, 28.69 mmol)
and triethylamine (8.7 g, 12.00 mL, 86.07 mmol) in acetonitrile (50
mL). The reaction flask was heated at 80.degree. C. under a
nitrogen atmosphere for 16 h. The reaction mixture was allowed to
cool and then was partitioned between water and dichloromethane.
The organic layer was washed with 1 M HCl, dried over
Na.sub.2SO.sub.4, filtered, and concentrated to dryness.
Purification by silica gel chromatography (0-10% ethyl acetate in
hexanes) yielded 7-[4-nitro-3-(trifluoromethyl)
phenyl]-7-azabicyclo[2.2.1]heptane 10 (7.2 g, 88% yield) as a
yellow solid. .sup.1H NMR (400.0 MHz, DMSO-d.sub.6) .delta. 8.03
(d, J =9.1 Hz, 1 H), 7.31 (d, J=2.4 Hz, 1H), 7.25 (dd, J=2.6, 9.1
Hz, 1H), 4.59 (s, 2H), 1.69-1.67 (m, 4H), 1.50 (d, J=7.0 Hz,
4H).
[0132] Preparation of 4-(7-azabicyclo[2.2.1]
heptan-7-yl)-2-(trifluoromethyl)aniline (11). A flask charged with
7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane 10
(7.07 g, 24.70 mmol) and 10% Pd/C (0.71 g, 6.64 mmol) was evacuated
and then flushed with nitrogen. Ethanol (22 mL) was added and the
reaction flask was fitted with a hydrogen balloon. After stirring
vigorously for 12 h, the reaction mixture was purged with nitrogen
and Pd/C was removed by filtration. The filtrate was concentrated
to a dark oil under reduced pressure and the residue purified by
silica gel chromatography (0-15% ethyl acetate in hexanes) to
provide 4-(7-azabicyclo[2.2.1]
heptan-7-yl)-2-(trifluoromethyl)aniline 11 as a purple solid (5.76
g, 91% yield). .sup.1HNMR (400.0 MHz, DMSO-d.sub.6) .delta. 6.95
(dd, J=2.3, 8.8 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.72 (d, J=8.8 Hz,
1H), 4.89 (s, 2H), 4.09 (s, 2H), 1.61-1.59 (m, 4H) and 1.35 (d,
J=6.8 Hz, 4H).
Example 3
Preparation of
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide (Compound
1).
##STR00004##
[0134] To a solution of
4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylic acid 7 (9.1 g,
35.39 mmol) and
4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethypaniline 11
(9.2 g, 35.74 mmol) in 2-methyltetrahydrofuran (91.00 mL) was added
propyl phosphonic acid cyclic anhydride (50% solution in ethyl
acetate, 52.68 mL, 88.48 mmol) and pyridine (5.6 g, 5.73 mL, 70.78
mmol) at room temperature. The reaction flask heated at 65.degree.
C. for 10 h under a nitrogen atmosphere. After cooling to room
temperature the reaction was then diluted with ethyl acetate and
quenched with saturated Na.sub.2CO.sub.3 solution (50 mL). The
layers were separated, and the aqueous layer was extracted twice
more with ethyl acetate. The combined organic layers were washed
with water, dried over Na.sub.2SO.sub.4, filtered and concentrated
to a tan solid. The crude solid product was slurried in ethyl
acetate/diethyl ether (2:1), collected by vacuum filtration, and
washed twice more with ethyl acetate/diethyl ether (2:1) to provide
the product as a light yellow crystalline powder. The powder was
dissolved in warm ethyl acetate and absorbed onto Celite.
[0135] Purification by silica gel chromatography (0-50% ethyl
acetate in dichloromethane) provided
N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5--
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide (Compound 1)
as a white crystalline solid (Form A) (13.5 g, 76% yield). LC/MS
m/z 496.0 [M+H].sup.+, retention time 1.48 min (RP-C.sub.18,10-99%
CH.sub.3CN/0.05% TFA over 3 min). .sup.111 NMR (400.0 MHz,
DMSO-d.sub.6) .delta. 13.08 (s, 1H), 12.16 (s, 111), 8.88 (s, 1H),
8.04 (dd, J=2.1, 7.4 Hz, 1H), 7.95-7.88 (m, 3H), 7.22 (dd, 2.5, 8.9
Hz, 1H), 7.16 (d, J=2.5 Hz, 1 H), 4.33 (s, 2H), 1.67 (d, J=6.9 Hz,
4H), 1.44 (d, J=6.9 Hz, 4H).
[0136] The powder diffractogram of Form A is shown in FIG. 1.
[0137] Table 1, below provides representative XRPD peaks of Form
A.
TABLE-US-00001 TABLE 1 Form A XRPD Peaks 2 Theta (degrees)
Intensity (%) 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
[0138] Conformational pictures of Form A based on single crystal
X-ray analysis are shown in FIG. 2. Diffraction data were acquired
on a Bruker Apex II Diffractometer equipped with sealed tube
CuK-alpha source and an Apex II CCD detector. The structure was
solved and refined using SHELX program (Sheldrick, G. M., Acta
Cryst. A64, pp.112-122 (2008)). Based on intensities, statistics
and symmetry, the structure was solved and refined in a trigonal
crystal system and an R-3 space group. Form A has the following
unit cell dimensions: a=19.1670(4) .ANG., b=19.1670(4) .ANG.,
c=33.6572(12) .ANG., .alpha.=90.degree., .beta.=90.degree. , and
.gamma.=120.degree..
[0139] An FTIR spectra of From A is provided in FIG. 3.
[0140] Table 2, below provides representative FTIR peaks of Form
A.
TABLE-US-00002 TABLE 2 Form A FTIR Peaks Position (cm.sup.-1)
Intensity 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 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
[0141] Assays for Detecting and Measuring .DELTA.F508-CFTR
Potentiation Properties of Compounds
[0142] Membrane potential optical methods for assaying
.DELTA.F508-CFTR modulation properties of compounds
[0143] The assay utilizes fluorescent voltage sensing dyes to
measure changes in membrane potential using a fluorescent plate
reader (e.g., FLIPR III, Molecular Devices, Inc.) as a readout for
increase in functional .DELTA.F508-CFTR in NIH 3T3 cells. The
driving force for the response is the creation of a chloride ion
gradient in conjunction with channel activation by a single liquid
addition step after the cells have previously been treated with
compounds and subsequently loaded with a voltage sensing dye.
[0144] Identification of Potentiator Compounds
[0145] To identify potentiators of AF508-CFTR, a double-addition
HTS assay format was developed. This HTS assay utilizes fluorescent
voltage sensing dyes to measure changes in membrane potential on
the FLIPR III as a measurement for increase in gating (conductance)
of .DELTA.F508 CFTR in temperature-corrected .DELTA.F508 CFTR NIH
3T3 cells. The driving force for the response is a Cl.sup.31 ion
gradient in conjunction with channel activation with forskolin in a
single liquid addition step using a fluoresecent plate reader such
as FLIPR III after the cells haw previously been treated with
potentiator compounds (or DMSO vehicle control) and subsequently
loaded with a redistribution dye.
[0146] Solutions Bath Solution #1: (in mM) NaCl 160, KCl4.5,
CaCl.sub.2 2, MgCl.sub.2 1, HEPES 10, pH 7.4 with NaOH.
[0147] Chloride-free bath solution: chloride salts in bath solution
#1 are substituted with gluconate salts.
[0148] Cell Culture
[0149] NIH3T3 mouse fibroblasts stably expressing .DELTA.F508-CFTR
are used for optical measurements of membrane potential. The cells
are maintained at 37.degree. C. in 5% CO.sub.2 and 90% humidity in
Dulbecco's modified Eagle's medium supplemented with 2 mM
glutamine, 10% fetal bovine serum, 1 X NEAR, .beta.-ME, 1 X
pen/strep, and 25 mM HEPES in 175 cm.sup.2 culture flasks. For all
optical assays, the cells were seeded at 20,000/well in 384-well
matrigel-coated plates and cultured for 2 hrs at 37.degree. C.
before culturing at 27.degree. C. for 24 hrs. for the potentiator
assay. For the correction assays, the cells are cultured at
27.degree. C. or 37.degree. C. with and without compounds for 16-24
hours. Electrophysiological Assays for assaying .DELTA.-CFTR
modulation properties of compounds.
[0150] Using Chamber Assay
[0151] Ussing chamber experiments were performed on polarized
airway epithelial cells expressing .DELTA.F508-CFTR to further
characterize the .DELTA.F508-CFTR modulators identified in the
optical assays. Non-CF and CF airway epithelia were isolated from
bronchial tissue, cultured as previously described (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), and plated onto Costar.RTM. Snapwell.TM. filters that
were precoated with NIH3T3-conditioned media. After four days the
apical media was removed and the cells were grown at an air liquid
interface for >14 days prior to use. This resulted in a
monolayer of fully differentiated columnar cells that were
ciliated, features that are characteristic of airway epithelia.
Non-CF HBE were isolated from non-smokers that did not have any
known lung disease. CF-HBE were isolated from patients homozygous
for .DELTA.F508-CFTR.
[0152] HBE grown on Costar.RTM. Snapwell.TM. cell culture inserts
were mounted in an using chamber (Physiologic Instruments, Inc.,
San Diego, Calif.), and the transepithelial resistance and
short-circuit current in the presence of a basolateral to apical
Cl.sup.- gradient (I.sub.SC) were measured using a voltage-clamp
system (Department of Bioengineering, University of Iowa, Iowa).
Briefly, HBE were examined under voltage-clamp recording conditions
(V.sub.hold=0 mV) at 37.degree. C. The basolateral solution
contained (in mM) 145 NaCl, 0.83 K.sub.2HPO.sub.4, 3.3
KH.sub.2PO.sub.4, 1.2 MgCl.sub.2, 1.2 CaCl.sub.2, 10 Glucose, 10
HEPES (pH adjusted to 7.35 with NaOH) and the apical solution
contained (in mM) 145 NaGluconate, 1.2 MgCl.sub.2, 1.2 CaCl.sub.2,
10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).
[0153] Identification of Potentiator Compounds
[0154] Typical protocol utilized a basolateral to apical membrane
Cl.sup.- concentration gradient. To set up this gradient, normal
ringers was used on the basolateral membrane, whereas apical NaCl
was replaced by equimolar sodium gluconate (titrated to pH 7.4 with
NaOH) to give a large Cl.sup.- concentration gradient across the
epithelium. Forskolin (10 .mu.M) and all test compounds were added
to the apical side of the cell culture inserts. The efficacy of the
putative .DELTA.F508-CFTR potentiators was compared to that of the
known potentiator, genistein.
[0155] Patch-clamp Recordings
[0156] Total Cl.sup.- current in .DELTA.F508-NIH3T3 cells was
monitored using the perforated-patch recording configuration as
previously described (Rae, J., Cooper, K., Gates, P., & Watsky,
M. (1991) J Neurosci. Methods 37, 15-26). Voltage-clamp recordings
were performed at 22.degree. C. using an Axopatch 200B patch-clamp
amplifier (Axon Instruments Inc., Foster City, Calif.). The pipette
solution contained (in mM) 150 N-methyl-D-glucamine (NMDG)-Cl, 2
MgCl.sub.2, 2 CaCl.sub.2, 10 EGTA, 10 HEPES, and 240
.mu.g/mlamphotericin-B (pH adjusted to 7.35 with HCl). The
extracellular medium contained (in mM) 150 NMDG-Cl, 2
M.sub.gC1.sub.2, 2 CaCl.sub.2, 10 HEPES (pH adjusted to 7.35 with
HCl). Pulse generation, data acquisition, and analysis were
performed using a PC equipped with a Digidata 1320 A/D interface in
conjunction with Clampex 8 (Axon Instruments Inc.). To activate
.DELTA.F508-CFTR, 10 .mu.M forskolin and 20 .mu.M genistein were
added to the bath and the current-voltage relation was monitored
every 30 sec.
[0157] Identification of Potentiator Compounds
[0158] The ability of .DELTA.F508-CFTR potentiators to increase the
macroscopic .DELTA.F508-CFTR Cl.sup.- current (I.sub..DELTA.F508)
in NIH3T3 cells stably expressing .DELTA.F508-CFTR was also
investigated using perforated-patch-recording techniques. The
potentiators identified from the optical assays evoked a
dose-dependent increase in I.DELTA..sub.F508 with similar potency
and efficacy observed in the optical assays. In all cells examined,
the reversal potential before and during potentiator application
was around -30 mV, which is the calculated E.sub.Cl (-28 mV).
[0159] Cell Culture
[0160] NIH3T3 mouse fibroblasts stably expressing .DELTA.F508-CFTR
are used for whole-cell recordings. The cells are maintained at
37.degree. C. in 5% CO.sub.2 and 90% humidity in Dulbecco's
modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal
bovine serum, 1 X NEAA, .beta.-ME, 1 X pen/strep, and 25 mM HEPES
in 175 cm.sup.2 culture flasks. For whole-cell recordings,
2,500-5,000 cells were seeded on poly-L-lysine-coated glass
coverslips and cultured for 24-48 hrs at 27.degree. C. before use
to test the activity of potentiators; and incubated with or without
the correction compound at 37.degree. C. for measuring the activity
of correctors.
[0161] Single-Channel Recordings
[0162] Gating activity of wt-CFTR and temperature-corrected
.DELTA.F508-CFTR expressed in NIH3T3 cells was observed using
excised inside-out membrane patch recordings as previously
described (Dalemans, W., Barbry, P., Champigny, G., Jallat, S.,
Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J-P.,
Lazdunski, M. (1991) Nature 354, 526-528) using an Axopatch 200B
patch-clamp amplifier (Axon Instruments Inc.). The pipette
contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl.sub.2, 2
MgCl.sub.2, and 10 HEPES (pH adjusted to 7.35 with Tris base). The
bath contained (in mM): 150 NMDG-Cl, 2 MgCl.sub.2, 5 EGTA, 10 TES,
and 14 Tris base (pH adjusted to 7.35 with HCl). After excision,
both wt- and .DELTA.F508-CFTR were activated by adding 1 mM Mg-ATP,
75 nM of the catalytic subunit of cAMP-dependent protein kinase
(PKA; Promega Corp. Madison, Wis.), and 10 mM NaF to inhibit
protein phosphatases, which prevented current rundown. The pipette
potential was maintained at 80 mV. Channel activity was analyzed
from membrane patches containing 2 active channels. The maximum
number of simultaneous openings determined the number of active
channels during the course of an experiment. To determine the
single-channel current amplitude, the data recorded from 120 sec of
.DELTA.F508-CFTR activity was filtered "off-line" at 100 Hz and
then used to construct all-point amplitude histograms that were
fitted with multigaussian functions using Bio-Patch Analysis
software (Bio-Logic Comp. France). The total microscopic current
and open probability (P.sub.o) were determined from 120 sec of
channel activity. The P.sub.o was determined using the Bio-Patch
software or from the relationship P.sub.o=I/i(N), where I=mean
current, i=single-channel current amplitude, and N=number of active
channels in patch.
[0163] Cell Culture
[0164] NIH3T3 mouse fibroblasts stably expressing .DELTA.F508-CFTR
are used for excised-membrane patch-clamp recordings. The cells are
maintained at 37.degree. C. in 5% CO.sub.2 and 90% humidity in
Dulbecco's modified Eagle's medium supplemented with 2 mM
glutamine, 10% fetal bovine serum, 1 X NEAA, .beta.-ME, 1 X
pen/strep, and 25 mM HEPES in 175 cm.sup.2 culture flasks. For
single channel recordings, 2,500-5,000 cells were seeded on
poly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at
27.degree. C. before use.
[0165] Compound 1 Form A is useful as a modulator of ATP binding
cassette transporters. The EC.sub.50 (.mu.m) of Compound 1 Form A
was measured to be less than 2.0 .mu.M. The efficacy of Compound 1
Form A was calculated to be from 100% to 25%. It should be noted
that 100% efficacy is the maximum response obtained with
4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol.
* * * * *
References