U.S. patent application number 14/027791 was filed with the patent office on 2014-04-03 for formulations of (r)-1-(2,2-difluorobenzo[d] [1,3] dioxol-5-yl)-n-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpro- pan-2-yl)-1h-indol-5-yl)cyclopropanecarboxamide.
This patent application is currently assigned to VERTEX PHARMACEUTICALS INCORPORATED. The applicant listed for this patent is VERTEX PHARMACEUTICALS INCORPORATED. Invention is credited to ROSSITZA GUEORGUIEVA ALARGOVA, Irina Nikolaevna Kadiyala, Noreen Tasneem Zaman.
Application Number | 20140094499 14/027791 |
Document ID | / |
Family ID | 45607527 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140094499 |
Kind Code |
A1 |
ALARGOVA; ROSSITZA GUEORGUIEVA ;
et al. |
April 3, 2014 |
FORMULATIONS OF (R)-1-(2,2-DIFLUOROBENZO[D] [1,3]
DIOXOL-5-YL)-N-(1-(2,3-DIHYDROXYPROPYL)-6-FLUORO-2-(1-HYDROXY-2-METHYLPRO-
PAN-2-YL)-1H-INDOL-5-YL)CYCLOPROPANECARBOXAMIDE
Abstract
The present invention relates to formulations of
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarbox-
amide (Compound 1), pharmaceutical packs or kits thereof, and
methods of treatment therewith.
Inventors: |
ALARGOVA; ROSSITZA GUEORGUIEVA;
(Brighton, MA) ; Kadiyala; Irina Nikolaevna;
(Newton, MA) ; Zaman; Noreen Tasneem; (Acton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VERTEX PHARMACEUTICALS INCORPORATED |
CAMBRIDGE |
MA |
US |
|
|
Assignee: |
VERTEX PHARMACEUTICALS
INCORPORATED
CAMBRIDGE
MA
|
Family ID: |
45607527 |
Appl. No.: |
14/027791 |
Filed: |
September 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13155420 |
Jun 8, 2011 |
8563593 |
|
|
14027791 |
|
|
|
|
61352512 |
Jun 8, 2010 |
|
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Current U.S.
Class: |
514/414 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 19/10 20180101; A61K 45/06 20130101; A61K 47/10 20130101; A61K
31/404 20130101; A61K 9/0095 20130101; A61P 27/02 20180101 |
Class at
Publication: |
514/414 |
International
Class: |
A61K 31/404 20060101
A61K031/404; A61K 45/06 20060101 A61K045/06 |
Claims
1-37. (canceled)
38. A method of treating a CFTR mediated disease selected from male
infertility and pancreatic insufficiency in a subject comprising
administering to the subject an effective amount of a formulation
comprising
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarbox-
amide (Compound 1), water, a surfactant which is vitamin E TPGS,
and a polyethylene glycol (PEG) which is PEG 400.
39-42. (canceled)
43. The method of claim 38, wherein the concentration of Compound 1
in the formulation is from about 0.05 to about 3% by weight.
44. The method of claim 43, wherein the concentration of Compound 1
in the formulation is from about 0.1 to about 2% by weight.
45. The method of claim 43, wherein the concentration of Compound 1
in the formulation is about 0.12, 0.25, or 0.50% by weight.
46. The method of claim 43, wherein the concentration of
polyethylene glycol in the formulation is from about 40 to about
60% by weight.
47. The method of claim 43, wherein the concentration of
polyethylene glycol in the formulation is from about 45 to about
55% by weight.
48. The method of claim 43, wherein the concentration of
polyethylene glycol in the formulation is about 50% by weight.
49. The method of claim 43, wherein the concentration of Compound 1
in the formulation is from about 0.05 to about 3% by weight; and
the concentration of polyethylene glycol is from about 40 to about
60% by weight.
50. The method of claim 43, wherein the concentration of Compound 1
in the formulation is from about 0.1 to about 2% by weight; and the
concentration of polyethylene glycol is from about 45 to about 55%
by weight.
51. The method of claim 43, wherein the concentration of Compound 1
in the formulation is about 0.12, 0.25, or 0.50% by weight; and the
concentration of polyethylene glycol is about 50% by weight.
52. The method of claim 43, wherein the concentration of surfactant
in the formulation is from about 5 to about 15% by weight.
53. The method of claim 43, wherein the concentration of surfactant
in the formulation is from about 10 to about 12% by weight.
54. The method of claim 43, wherein the concentration of surfactant
in the formulation is about 11% by weight.
55. The method of claim 43, wherein the concentration of Compound 1
in the formulation is from about 0.05 to about 3% by weight, the
concentration of polyethylene glycol is from about 40 to about 60%
by weight, and the concentration of surfactant is from about 5 to
about 15% by weight.
56. The method of claim 55, wherein the concentration of Compound 1
in the formulation is from about 0.05 to about 3% by weight, the
polyethylene glycol is PEG 400 at about 40 to about 60% by weight,
and the surfactant is vitamin E TPGS at about 5 to 15% by
weight.
57. The method of claim 43, wherein the formulation is about 30 to
50 g.
58. The method of claim 43, wherein the formulation is about 35 to
45 g.
59. The method of claim 43, wherein the formulation is about 40
g.
60. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00015 Component Amount (g) Compound 1 0.05 PEG
400 19.95 Vitamin E TPGS 4.48 Water 15.52
61. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00016 Component Amount (g) Compound 1 0.10 PEG
400 19.90 Vitamin E TPGS 4.48 Water 15.52
62. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00017 Component Amount (g) Compound 1 0.20 PEG
400 19.80 Vitamin E TPGS 4.48 Water 15.52
63. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00018 Component Amount (g) Compound 1 0.30 PEG
400 19.70 Vitamin E TPGS 4.48 Water 15.52
64. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00019 Component Amount (% by weight) Compound 1
0.10-0.80 PEG 400 48-51 Vitamin E TPGS 10-12 Water 37-40
65. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00020 Component Amount (% by weight) Compound 1
0.20-0.60 PEG 400 49-50 Vitamin E TPGS 10-12 Water 37-40
66. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00021 Component Amount (% by weight) Compound 1
0.25 PEG 400 49.75 Vitamin E TPGS 11.20 Water 38.80
67. The method of claim 43, wherein the formulation comprises the
following: TABLE-US-00022 Component Amount (% by weight) Compound 1
0.50 PEG 400 49.50 Vitamin E TPGS 11.20 Water 38.80
68. The method of claim 43, wherein the formulation further
comprises a taste masker.
69. The method of claim 43, wherein the formulation further
comprises an additional therapeutic agent.
70. The method of claim 43, wherein the additional therapeutic
agent is selected from a mucolytic agent, bronchodialator, an
anti-biotic, an anti-infective agent, an anti-inflammatory agent, a
CFTR modulator other than Compound 1, or a nutritional agent.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/155,420, filed Jun. 8, 2011, which claims the benefit under
35 U.S.C. .sctn.119 of U.S. Provisional Application Ser. No.
61/352,512, filed Jun. 8, 2010, the entire contents of the
aforementioned applications are incorporated herein by
reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to an oral formulation
comprising
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarbox-
amide (Compound 1) as described herein, water, and a polyethylene
glycol (PEG). The oral formulation may additionally comprise a
surfactant, and additionally a taste masker. The invention further
relates to a method of treating a CFTR mediated disease such as
cystic fibrosis with such a formulation.
BACKGROUND OF THE INVENTION
[0003] CFTR is a cAMP/ATP-mediated anion channel that 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
epithelia 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.
[0004] 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.
[0005] 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
enhance 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.
[0006] 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, greater than 1000 disease-causing mutations in the CF gene
have been identified as reported by the scientific and medical
literature. 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. Other mutations include the R117H
and G551D.
[0007] 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.
(Dalemans 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, 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.
[0008] Although CFTR transports a variety of molecules in addition
to anions, it is clear that this role (the transport of anions)
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.
[0009] 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.- 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.
[0010] 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 endoplasmic reticulum (ER) processing of ATP-binding
cassette (ABC) transporters 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)].
[0011]
(R)-1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypro-
pyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropane-
carboxamide is disclosed in International PCT Publication WO
2010054138 (said publication being incorporated herein by reference
in its entirety) as a modulator of CFTR activity and thus as a
useful treatment for CFTR-mediated diseases such as cystic
fibrosis. A need remains, however, for pharmaceutical compositions
comprising
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarbox-
amide that are readily prepared and that are suitable for use as
therapeutics.
SUMMARY OF THE INVENTION
[0012] The present invention relates to oral formulations of
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarbox-
amide which has the structure below:
##STR00001##
[0013] Compound 1 is useful for treating or lessening the severity
of a variety of CFTR mediated diseases.
[0014] In one aspect, the invention features a formulation
comprising
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarbox-
amide (Compound 1), water, and a polyethylene glycol (PEG).
[0015] In another embodiment, the polyethylene glycol is selected
from PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900,
or PEG 1000. In another embodiment, the polyethylene glycol is PEG
400.
[0016] In another embodiment, the concentration of Compound 1 is
from about 0.05 to about 3% by weight. In another embodiment, the
concentration of Compound 1 is from about 0.1 to about 2% by
weight. In another embodiment, the concentration of Compound 1 is
about 0.12, 0.25, or 0.50% by weight.
[0017] In another embodiment, the concentration of polyethylene
glycol is from about 40 to about 60% by weight. In another
embodiment, the concentration of polyethylene glycol is from about
45 to about 55% by weight. In another embodiment, the concentration
of polyethylene glycol is about 50% by weight.
[0018] In another embodiment, the concentration of Compound 1 is
from about 0.05 to about 3% by weight; and the concentration of
polyethylene glycol is from about 40 to about 60% by weight. In
another embodiment, the concentration of Compound 1 is from about
0.1 to about 2% by weight; and the concentration of polyethylene
glycol is from about 45 to about 55% by weight. In another
embodiment, the concentration of Compound 1 is about 0.12, 0.25, or
0.50% by weight; and the concentration of ethylene glycol is about
50% by weight.
[0019] In another embodiment, the concentration of Compound 1 is
about 0.12, 0.25, or 0.50% by weight; and the polyethylene glycol
is PEG 400 at about 50% by weight.
[0020] In another embodiment the formulation of Compound 1 further
comprises a surfactant. In another embodiment, the surfactant is an
anionic, cationic, or nonionic surfactant. In another embodiment,
the surfactant is a nonionic surfactant selected from the group
consisting of vitamin E d-.alpha.-tocopheryl PEG 1000 succinate
(vitamin E TPGS), polysorbate 20, polysorbate 40, polysorbate 60,
polysorbate 65, polysorbate 80, alkyl poly(ethylene oxide),
poloxamine, alkyl polyglucosides, octyl glucoside, decyl maltoside,
fatty alcohol, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide
DEA, Solutol surfactants such as Solutol HS 15, and cocamide TEA.
In another embodiment, the surfactant is vitamin E TPGS.
[0021] In another embodiment, the concentration of surfactant is
from about 5 to about 15% by weight. In another embodiment, the
concentration of surfactant is from about 10 to about 12% by
weight. In another embodiment, the concentration of surfactant is
about 11% by weight. In another embodiment, the surfactant is
vitamin E TPGS at about 11% by weight.
[0022] In another embodiment, the concentration of Compound 1 is
from about 0.05 to about 3% by weight, the concentration of
polyethylene glycol is from about 40 to about 60% by weight, and
the concentration of surfactant is from about 5 to about 15% by
weight.
[0023] In another embodiment, the concentration of Compound 1 is
from about 0.05 to about 3% by weight, the polyethylene glycol is
PEG 400 at about 40 to about 60% by weight, and the surfactant is
vitamin E TPGS at about 5 to 15% by weight.
[0024] In another embodiment the formulation of Compound 1 is about
30 to 50 g. In another embodiment, the formulation is about 35 to
45 g. In another embodiment, the formulation is about 40 g.
[0025] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00001 Component Amount (g) Compound 1 0.05 PEG 400 19.95
Vitamin E TPGS 4.48 Water 15.52
[0026] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00002 Component Amount (g) Compound 1 0.10 PEG 400 19.90
Vitamin E TPGS 4.48 Water 15.52
[0027] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00003 Component Amount (g) Compound 1 0.20 PEG 400 19.80
Vitamin E TPGS 4.48 Water 15.52
[0028] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00004 Component Amount (g) Compound 1 0.30 PEG 400 19.70
Vitamin E TPGS 4.48 Water 15.52
[0029] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00005 Component Amount (% by weight) Compound 1 0.10-0.80
PEG 400 48-51 Vitamin E TPGS 10-12 Water 37-40
[0030] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00006 Component Amount (% by weight) Compound 1 0.20-0.60
PEG 400 49-50 Vitamin E TPGS 10-12 Water 37-40
[0031] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00007 Component Amount (% by weight) Compound 1 0.25 PEG
400 49.75 Vitamin E TPGS 11.20 Water 38.80
[0032] In another embodiment, the formulation of Compound 1
comprises the following:
TABLE-US-00008 Component Amount (% by weight) Compound 1 0.50 PEG
400 49.50 Vitamin E TPGS 11.20 Water 38.80
[0033] In another embodiment, the invention features any of the
above described formulations further comprising a taste masker. In
another embodiment, the invention features any of the above
described formulations further comprising an additional therapeutic
agent. In another embodiment, the additional therapeutic agent is
selected from a mucolytic agent, bronchodialator, an anti-biotic,
an anti-infective agent, an anti-inflammatory agent, a CFTR
modulator other than Compound 1, or a nutritional agent.
[0034] In another aspect, the invention features a method of
treating a CFTR mediated disease 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, protein C deficiency, Type 1
hereditary angioedema, lipid processing deficiencies, familial
hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,
lysosomal storage diseases, 1-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, Alzheimer's disease, Parkinson's
disease, amyotrophic lateral sclerosis, progressive supranuclear
plasy, Pick's disease, several polyglutamine neurological
disorders, Huntington's, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, myotonic
dystrophy, spongiform encephalopathies, hereditary
Creutzfeldt-Jakob disease (due to prion protein processing defect),
Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eye
disease, Sjogren's disease, 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), inherited
disorders of the structure and/or function of cilia, PCD with situs
inversus (also known as Kartagener syndrome), PCD without situs
inversus, or ciliary aplasia.
[0035] In another embodiment, the invention features a method of
treating a CFTR mediated disease selected from cystic fibrosis,
COPD, emphysema, dry-eye disease, or osteoporosis in a subject
comprising administering to the subject an effective amount of any
of the above formulations of Compound 1. In another embodiment, the
CFTR mediated disease is cystic fibrosis.
[0036] In another embodiment, the method comprises administering an
additional therapeutic agent. In another embodiment, the additional
therapeutic agent is selected from a mucolytic agent,
bronchodialator, an anti-biotic, an anti-infective agent, an
anti-inflammatory agent, a CFTR modulator other than Compound 1, or
a nutritional agent.
[0037] In another aspect, the invention features a pharmaceutical
pack or kit comprising any of the above formulations of Compound 1
and instructions for use thereof.
[0038] Processes described herein can be used to prepare the
compositions of this invention. The amounts and the features of the
components used in the processes would be as described herein.
SUMMARY OF THE FIGURES
[0039] FIG. 1 depicts a flow chart for preparing formulations of
Compound 1 with PEG, wherein the surfactant is added as an aqueous
solution.
[0040] FIG. 2 depicts a flow chart for preparing formulations of
Compound 1 with PEG 400, wherein the surfactant, vitamin E, is
added as an aqueous solution.
[0041] FIG. 3 depicts a flow chart for preparing formulations of
Compound 1 with PEG, wherein the surfactant is dissolved in the
PEG, followed by dilution.
[0042] FIG. 4 depicts a flow chart for preparing formulations of
Compound 1 with PEG 400, wherein the surfactant, vitamin E, is
dissolved in the PEG 400, followed by dilution.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0043] As used herein, the following definitions shall apply unless
otherwise indicated.
[0044] 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, .DELTA.F508
CFTR and G551D CFTR (see, e.g.,
http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).
[0045] The term "modulating" as used herein means increasing or
decreasing, e.g. activity, by a measurable amount.
[0046] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
invention. All tautomeric forms of the Compound 1 are included
herein. For example, Compound 1 may exist as tautomers, both of
which are included herein:
##STR00002##
[0047] Additionally, unless otherwise stated, structures depicted
herein are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms. For example,
Compound 1, wherein one or more hydrogen atoms are replaced
deuterium or tritium, or one or more carbon atoms are replaced by a
.sup.13C- or .sup.14C-enriched carbon are within the scope of this
invention. Such compounds are useful, for example, as analytical
tools, probes in biological assays, or compounds with improved
therapeutic profile.
[0048] The term "protecting group," abbreviated as P, as used
herein refers to any chemical group introduced into a molecule by
chemical modification of a functional group in order to obtain
chemoselectivity in a subsequent chemical reaction. Non-limiting
examples of alcohol protecting groups include acetyl (Ac), benzoyl
(Bz), benzyl (Bn), .beta.-methoxyethoxymethyl ether (MEM),
dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl
(MMT), p-methoxybenzyl ether (PMB), pivaloyl (Piv),
tetrahydropyranyl (THP), trityl (Tr), and trimethylsilyl (TMS). In
one embodiment, the protecting group is Bn which has the structure
--CH.sub.2C.sub.6H.sub.5.
[0049] The abbreviation "DCM" stands for dichloromethane. The
abbreviation "IPA" stands for isopropyl alcohol. The abbreviation
"DMSO" stands for dimethylsulfoxide. The abbreviation "MTBE" stands
for methyl t-butyl ether. The abbreviation "THF" stands for
tetrahydrofuran. The abbreviation "TEA" stands for triethylamine.
The abbreviation "dba" as in Pd(dba).sub.2 stands for
dibenzylideneacetone. The abbreviation "dppf" as in
Pd(dppf)Cl.sub.2 stands for stands for 1,1'-bis(diphenylphosphino)
ferrocene.
[0050] Methods of Preparing Compound 1.
[0051] Compound 1 is
(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-
-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropane
carboxamide and, in one embodiment, is prepared by coupling an acid
chloride moiety with an amine moiety according to Schemes 1-3.
##STR00003##
[0052] Wherein compound 2 is prepared according to Scheme 2.
##STR00004##
[0053] Wherein compound 3 is prepared according to Scheme 3.
##STR00005##
[0054] Formulations of Compound 1 can be prepared according to FIG.
1.
[0055] In one embodiment, formulations of Compound 1 can be
prepared according to FIG. 2.
[0056] In one embodiment, the surfactant is added as an aqueous
solution prepared beforehand. In another embodiment, the
concentration of the aqueous surfactant solution is about 20 to 25%
by weight. In another embodiment, the concentration of the aqueous
surfactant solution is about 22% by weight. In another embodiment,
the surfactant is vitamin E TPGS and the contration is about 22% by
weight.
[0057] In another embodiment, the elevated temperature is about 30
to 50.degree. C. In another embodiment, the elevated temperature is
about 35 to 45.degree. C. In another embodiment, the elevated
temperature is about 40.degree. C.
[0058] Formulations of Compound 1 may also be prepared according to
FIG. 3.
[0059] In another embodiment, formulations of Compound 1 can be
prepared according to FIG. 4.
[0060] In this set of embodiments, Compound 1 and the surfactant,
e.g. vitamin E TPGS, are dissolved in the PEG, e.g. PEG400,
followed by dilution. Previously, the surfactant was added as an
aqueous solution which provided the dilution.
[0061] In another embodiment, the elevated temperature is about 30
to 50.degree. C. In another embodiment, the elevated temperature is
about 35 to 45.degree. C. In another embodiment, the elevated
temperature is about 40.degree. C.
[0062] The Compound 1 formulations of the invention demonstrate
good in vivo exposure in dog studies. The PEG cosolvent and vitamin
E TPGS induced micelle formation increase Compound 1 solubility
from 7 micrograms to several milligrams per g of solution.
[0063] Uses, Formulation and Administration
[0064] Aqueous Formulations
[0065] In one aspect of the invention, aqueous formulations are
provided comprising Compound 1 as described herein, water, and a
polyethylene glycol, and optionally comprising other agents such as
a taste masker and/or flavorant, and additional pharmaceutically
acceptable carriers, adjuvants or vehicles. In certain embodiments,
these formulations optionally further comprise one or more
additional therapeutic agents.
[0066] It will also be appreciated that Compound 1 can exist as a
pharmaceutically acceptable derivative or a prodrug thereof.
According to the invention, a pharmaceutically acceptable
derivative or a prodrug includes, but is not limited to esters,
salts of such esters, or any other adduct or derivative which upon
administration to a patient in need thereof is capable of
providing, directly or indirectly, a compound as otherwise
described herein, or a metabolite or residue thereof.
[0067] 1. Polyethylene Glycol
[0068] Polyethylene glycol (PEG) is a polyether compound and
includes polyethylene oxide (PEO) and polyoxyethylene (POE). PEG,
PEO, and POE refer to an oligomer or polymer of ethylene oxide. The
three names are chemically synonymous, but historically PEG has
tended to refer to oligomers and polymers with a molecular mass
below 20,000 g/mol, PEO to polymers with a molecular mass above
20,000 g/mol, and POE to a polymer of any molecular mass. As used
herein, PEG refers to a polyethylene glycol of any molecular mass
that is amenable for use in a pharmaceutical formulations. Such
suitable polyethylene glycols are well known in the art; see, e.g.,
http://www.medicinescomplete.com/mc/excipients/current, which is
incorporated herein by reference. Exemplary PEGs include low
molecular weight PEGs such as PEG 200, PEG 300, PEG 400, etc. The
number that follows the term "PEG" indicates the average molecular
weight of that particular polymer. E.g., PEG 400 is a polyethylene
glycol polymer wherein the average molecular weight of the polymer
therein is about 400.
[0069] In one embodiment, the PEG has an average molecular weight
of from about 200 to about 600. In another embodiment, PEG is PEG
400 (for example a PEG having a molecular weight of from about 380
to about 420 g/mol).
[0070] 2. Surfactants
[0071] Surfactants reduce the interfacial tension between water and
an organic compound such as Compound 1 by adsorbing at the
water-Compound 1 interface, thus facilitating Compound 1
wettability and dissolution. Surfactants increase the solubility of
Compound 1 via micelle formation and contribute to the stability of
the aqueous solution. Surfactants are often classified into four
primary groups; anionic, cationic, non-ionic, and zwitterionic
(dual charge). In a preferred embodiment, the surfactant is an
anionic, cationic, or nonionic surfactant.
[0072] Anionic surfactants may be chosen from salts of dodecyl
sulfate, lauryl sulfate, laureth sulfate, alkyl benzene sulfonates,
butanoic acid, hexanoic acid, octanoic acid, decanoic acid, lauric
acid, myristic acid, palmitic acid, stearic acid, arachidic acid,
behenic acid, myristoleic acid, palmitoleic acid, oleic acid,
linoleic acid, alpha-linolenic acid, arachidonic acid,
eicosapentaenoic acid, erucic acid, or docosahexaenoic acid.
[0073] Cationic surfactants may be chosen from cetyl
trimethylammonium bromide, cetylpyridinium chloride, polethoxylated
tallow amine, benzalkonium chloride, and benzethonium chloride.
[0074] Nonionic surfactants may be chosen from vitamine E
derivatives, polysorbates, alkyl poly(ethylene oxide), poloxamine,
alkyl polyglucosides, octyl glucoside, decyl maltoside, fatty
alcohol, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide DEA,
and cocamide TEA. In particular, the nonionic surfactant is the
vitamine E derivative vitamin E, d-.alpha.-tocopheryl polyethylene
glycol 1000 succinate (vitamin E TPGS). Non-ionic surfactants also
include the PEG derivatives under the brand name Solutol. For
example Solutol HS 15 is prepared by reacting 15 moles of ethylene
oxide with 1 mole of hydroxyl stearic acid.
[0075] The oral formulations of the invention generally comprise
from about 5 to about 15% by weight surfactant. In another
embodiment, the concentration of surfactant is from about 10 to
about 12% by weight. In another embodiment, the concentration of
surfactant is about 11% by weight.
[0076] 3. Taste Masker and/or Flavoring Agent
[0077] As previously stated, it is advantageous to include a taste
masking agent in the oral formulations of Compound 1. Such taste
masking agents are alkali metal and alkaline earth metal chlorides
including sodium chloride, lithium chloride, potassium chloride,
magnesium chloride, and calcium chloride. Sodium chloride is
preferred. The taste masking agent is generally included in the
suspension in a taste-masking amount, generally an amount of about
0.5 to about 2.0 weight % as taste masker based on the weight of
the suspension. For other salts, equivalent molar amounts can be
calculated. Other taste maskers include sugars, with or without the
presence of other sweetening and/or flavoring agents. When used,
flavoring agents may be chosen from synthetic flavor oils and
flavoring aromatics and/or natural oils, extracts from plant
leaves, flowers, fruits, and so forth and combinations thereof.
These may include cinnamon oil, oil of wintergreen, peppermint
oils, clove oil, bay oil, anise oil, eucalyptus, thyme oil, cedar
leaf oil, oil of nutmeg, oil of sage, oil of bitter almonds, and
cassia oil. Also useful as flavors are vanilla, citrus oil,
including lemon, orange, grape, lime and grapefruit, and fruit
essence, including apple, banana, pear, peach, strawberry,
raspberry, cherry, plum, pineapple, apricot, and so forth. The
amount of flavoring may depend on a number of factors including the
organoleptic effect desired. Generally the flavoring will be
present in an amount of from about 0.01 to about 1.0 percent by
weight based on the total suspension weight.
[0078] As described above, the formulations of the present
invention can comprise a pharmaceutically acceptable carrier,
adjuvant, or vehicle, additional to water 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, partial glyceride mixtures of
saturated vegetable fatty acids, 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; 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; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; and ethyl
alcohol, 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, perfuming
agents, preservatives and antioxidants can also be present in the
composition, according to the judgment of the formulator.
[0079] Uses of Compounds and Pharmaceutically Acceptable
Compositions
[0080] In yet another aspect, the present invention provides a
method of treating a condition, disease, or disorder implicated by
CFTR. In certain embodiments, the present invention provides a
method of treating a condition, disease, or disorder implicated by
a deficiency of CFTR activity, the method comprising administering
an oral formulation comprising Compound 1 described herein to a
subject, preferably a mammal, in need thereof.
[0081] A "CFTR-mediated disease" as used herein is a disease
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, protein C
deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases,
1-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, Alzheimer's disease, Parkinson's
disease, amyotrophic lateral sclerosis, progressive supranuclear
plasy, Pick's disease, several polyglutamine neurological
disorders, Huntington's, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, myotonic
dystrophy, spongiform encephalopathies, hereditary
Creutzfeldt-Jakob disease (due to prion protein processing defect),
Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eye
disease, Sjogren's disease, 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), inherited
disorders of the structure and/or function of cilia, PCD with situs
inversus (also known as Kartagener syndrome), PCD without situs
inversus, or ciliary aplasia.
[0082] In certain embodiments, the present invention provides a
method of treating a CFTR-mediated disease in a subject comprising
the step of administering to the subject an effective amount of an
oral formulation comprising Compound 1 described herein.
[0083] According to another embodiment, the invention provides a
method of treating cystic fibrosis, emphysema, COPD, dry-eye
disease, or osteoporosis in a subject comprising the step of
administering to the subject an oral formulation comprising
Compound 1 described herein.
[0084] According to the invention an "effective amount" of an oral
formulation of Compound 1 is that amount effective for treating or
lessening the severity of any of the diseases recited above.
[0085] In certain embodiments, an oral formulation of Compound 1
described herein is useful for treating or lessening the severity
of cystic fibrosis in subjects 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.
[0086] In one embodiment, an oral formulation of Compound 1
described herein 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.
[0087] In one embodiment, an oral formulation of Compound 1
described herein 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.
[0088] 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 terms "patient" and
"subject" are used synonymously herein, and refer to an animal,
preferably a mammal, and most preferably a human.
[0089] In certain embodiments, Compound 1 may be administered
orally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and
preferably from about 1 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.
[0090] In certain embodiments, the dosage amount of Compound 1 in
the dosage unit form is from about 50 mg to about 2,000 mg. In
another embodiment, the dosage amount of Compound 1 is from about
200 mg to about 900 mg. In another embodiment, the dosage amount of
Compound 1 is from about 300 mg to about 800 mg. In another
embodiment, the dosage amount of Compound 1 is from about 400 mg to
about 700 mg. In another embodiment, the dosage amount of Compound
1 is from about 500 mg to about 600 mg.
[0091] It will also be appreciated that the oral formulations of
Compound 1 described herein can be employed in combination
therapies, that is, the oral formulations of Compound 1 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".
[0092] 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.
[0093] In one embodiment, the additional therapeutic 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.
[0094] In another embodiment, the additional agent is a mucolyte.
Exemplary mucolytes useful herein includes Pulmozyme.RTM..
[0095] In another embodiment, the additional agent is a
bronchodialator. Exemplary bronchodialtors include albuterol,
metaprotenerol sulfate, pirbuterol acetate, salmeterol, or
tetrabuline sulfate.
[0096] 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-h-
ydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydrox-
yoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogen
phosphate), or bronchitol (inhaled formulation of mannitol).
[0097] 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.
[0098] In another embodiment, the additional agent is a CFTR
modulator other than Compound 1, 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-oxoo-
ctahydrocyclopenta[b]pyran-5-yl}heptanoic acid), and
N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.
[0099] In another embodiment, the additional agent is a nutritional
agent. Exemplary nutritional 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.
[0100] In another embodiment, the additional agent is a compound
selected from gentamicin, curcumin, cyclophosphamide,
4-phenylbutyrate, miglustat, felodipine, nimodipine, Philoxin B,
geniestein, Apigenin, cAMP/cGMP modulators such as rolipram,
sildenafil, milrinone, tadalafil, aminone, isoproterenol,
albuterol, and almeterol, deoxyspergualin, HSP 90 inhibitors, HSP
70 inhibitors, proteosome inhibitors such as epoxomicin,
lactacystin, etc.
[0101] In another embodiment, the additional agent is a compound
disclosed in WO 2004028480, WO 2004110352, WO 2005094374, WO
2005120497, or WO 2006101740.
[0102] In another embodiment, the additiona agent is a
benzo(c)quinolizinium derivative that exhibits CFTR modulation
activity or a benzopyran derivative that exhibits CFTR modulation
activity.
[0103] In another embodiment, the additional agent is a compound
disclosed in U.S. Pat. No. 7,202,262, U.S. Pat. No. 6,992,096,
US20060148864, US20060148863, US20060035943, US20050164973,
WO2006110483, WO2006044456, WO2006044682, WO2006044505,
WO2006044503, WO2006044502, or WO2004091502.
[0104] In another embodiment, the additional agent is a compound
disclosed in WO2004080972, WO2004111014, WO2005035514,
WO2005049018, WO2006099256, WO2006127588, or WO2007044560.
[0105] These combinations are useful for treating the diseases
described herein including cystic fibrosis. These combinations are
also useful in the kits described herein.
[0106] 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.
[0107] 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
Methods & Materials
[0108] Vitride.RTM. (sodium bis(2-methoxyethoxy)aluminum hydride
[or NaAlH.sub.2(OCH.sub.2CH.sub.2OCH.sub.3).sub.2], 65 wgt %
solution in toluene) was purchased from Aldrich Chemicals.
3-Fluoro-4-nitroaniline was purchased from Capot Chemicals.
5-Bromo-2,2-difluoro-1,3-benzodioxole was purchased from Alfa
Aesar. 2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was
purchased from Saltigo (an affiliate of the Lanxess
Corporation).
[0109] Anywhere in the present application where a name of a
compound may not correctly describe the structure of the compound,
the structure supersedes the name and governs.
[0110] Synthesis of Compound 1
[0111] Acid Moiety
Synthesis of
(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile
##STR00006##
[0113] A reactor was purged with nitrogen and charged with 900 mL
of toluene. The solvent was degassed via nitrogen sparge for no
less than 16 h. To the reactor was then charged Na.sub.3PO.sub.4
(155.7 g, 949.5 mmol), followed by bis(dibenzylideneacetone)
palladium (0) (7.28 g, 12.66 mmol). A 10% w/w solution of
tert-butylphosphine in hexanes (51.23 g, 25.32 mmol) was charged
over 10 min at 23.degree. C. from a nitrogen purged addition
funnel. The mixture was allowed to stir for 50 min, at which time
5-bromo-2,2-difluoro-1,3-benzodioxole (75 g, 316.5 mmol) was added
over 1 min. After stirring for an additional 50 min, the mixture
was charged with ethyl cyanoacetate (71.6 g, 633.0 mmol) over 5 min
followed by water (4.5 mL) in one portion. The mixture was heated
to 70.degree. C. over 40 min and analyzed by HPLC every 1-2 h for
the percent conversion of the reactant to the product. After
complete conversion was observed (typically 100% conversion after
5-8 h), the mixture was cooled to 20-25.degree. C. and filtered
through a celite pad. The celite pad was rinsed with toluene
(2.times.450 mL) and the combined organics were concentrated to 300
mL under vacuum at 60-65.degree. C. The concentrate was charged
with 225 mL DMSO and concentrated under vacuum at 70-80.degree. C.
until active distillation of the solvent ceased. The solution was
cooled to 20-25.degree. C. and diluted to 900 mL with DMSO in
preparation for Step 2. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.
7.16-7.10 (m, 2H), 7.03 (d, J=8.2 Hz, 1H), 4.63 (s, 1H), 4.19 (m,
2H), 1.23 (t, J=7.1 Hz, 3H).
Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile
##STR00007##
[0115] The DMSO solution of
(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile
from above was charged with 3 N HCl (617.3 mL, 1.85 mol) over 20
min while maintaining an internal temperature <40.degree. C. The
mixture was then heated to 75.degree. C. over 1 h and analyzed by
HPLC every 1-2 h for % conversion. When a conversion of >99% was
observed (typically after 5-6 h), the reaction was cooled to
20-25.degree. C. and extracted with MTBE (2.times.525 mL), with
sufficient time to allow for complete phase separation during the
extractions. The combined organic extracts were washed with 5% NaCl
(2.times.375 mL). The solution was then transferred to equipment
appropriate for a 1.5-2.5 Torr vacuum distillation that was
equipped with a cooled receiver flask. The solution was
concentrated under vacuum at <60.degree. C. to remove the
solvents. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was then
distilled from the resulting oil at 125-130.degree. C. (oven
temperature) and 1.5-2.0 Ton.
(2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was isolated as a
clear oil in 66% yield from 5-bromo-2,2-difluoro-1,3-benzodioxole
(2 steps) and with an HPLC purity of 91.5% AUC (corresponds to a
w/w assay of 95%). .sup.1H NMR (500 MHz, DMSO) .delta. 7.44 (br s,
1H), 7.43 (d, J=8.4 Hz, 1H), 7.22 (dd, J=8.2, 1.8 Hz, 1H), 4.07 (s,
2H).
Synthesis of
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile
##STR00008##
[0117] A stock solution of 50% w/w NaOH was degassed via nitrogen
sparge for no less than 16 h. An appropriate amount of MTBE was
similarly degassed for several hours. To a reactor purged with
nitrogen was charged degassed MTBE (143 mL) followed by
(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (40.95 g, 207.7
mmol) and tetrabutylammonium bromide (2.25 g, 10.38 mmol). The
volume of the mixture was noted and the mixture was degassed via
nitrogen sparge for 30 min. Enough degassed MTBE is charged to
return the mixture to the original volume prior to degassing. To
the stirring mixture at 23.0.degree. C. was charged degassed 50%
w/w NaOH (143 mL) over 10 min followed by 1-bromo-2-chloroethane
(44.7 g, 311.6 mmol) over 30 min. The reaction was analyzed by HPLC
in 1 h intervals for % conversion. Before sampling, stirring was
stopped and the phases allowed to separate. The top organic phase
was sampled for analysis. When a % conversion >99% was observed
(typically after 2.5-3 h), the reaction mixture was cooled to
10.degree. C. and was charged with water (461 mL) at such a rate as
to maintain a temperature <25.degree. C. The temperature was
adjusted to 20-25.degree. C. and the phases separated. Note:
sufficient time should be allowed for complete phase separation.
The aqueous phase was extracted with MTBE (123 mL), and the
combined organic phase was washed with 1 N HCl (163 mL) and 5% NaCl
(163 mL). The solution of
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in
MTBE was concentrated to 164 mL under vacuum at 40-50.degree. C.
The solution was charged with ethanol (256 mL) and again
concentrated to 164 mL under vacuum at 50-60.degree. C. Ethanol
(256 mL) was charged and the mixture concentrated to 164 mL under
vacuum at 50-60.degree. C. The resulting mixture was cooled to
20-25.degree. C. and diluted with ethanol to 266 mL in preparation
for the next step. .sup.1H NMR (500 MHz, DMSO) .delta. 7.43 (d,
J=8.4 Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 7.30 (dd, J=8.4, 1.9 Hz,
1H), 1.75 (m, 2H), 1.53 (m, 2H).
Synthesis of
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic
acid
##STR00009##
[0119] The solution of
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in
ethanol from the previous step was charged with 6 N NaOH (277 mL)
over 20 min and heated to an internal temperature of 77-78.degree.
C. over 45 min. The reaction progress was monitored by HPLC after
16 h. Note: the consumption of both
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile and
the primary amide resulting from partial hydrolysis of
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile were
monitored. When a % conversion >99% was observed (typically 100%
conversion after 16 h), the reaction mixture was cooled to
25.degree. C. and charged with ethanol (41 mL) and DCM (164 mL).
The solution was cooled to 10.degree. C. and charged with 6 N HCl
(290 mL) at such a rate as to maintain a temperature <25.degree.
C. After warming to 20-25.degree. C., the phases were allowed to
separate. The bottom organic phase was collected and the top
aqueous phase was back extracted with DCM (164 mL). Note: the
aqueous phase was somewhat cloudy before and after the extraction
due to a high concentration of inorganic salts. The organics were
combined and concentrated under vacuum to 164 mL. Toluene (328 mL)
was charged and the mixture condensed to 164 mL at 70-75.degree. C.
The mixture was cooled to 45.degree. C., charged with MTBE (364 mL)
and stirred at 60.degree. C. for 20 min. The solution was cooled to
25.degree. C. and polish filtered to remove residual inorganic
salts. MTBE (123 mL) was used to rinse the reactor and the
collected solids. The combined organics were transferred to a clean
reactor in preparation for the next step.
Isolation of
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic
acid
##STR00010##
[0121] The solution of
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid
from the previous step is concentrated under vacuum to 164 mL,
charged with toluene (328 mL) and concentrated to 164 mL at
70-75.degree. C. The mixture was then heated to 100-105.degree. C.
to give a homogeneous solution. After stirring at that temperature
for 30 min, the solution was cooled to 5.degree. C. over 2 hours
and maintained at 5.degree. C. for 3 hours. The mixture was then
filtered and the reactor and collected solid washed with cold 1:1
toluene/n-heptane (2.times.123 mL). The material was dried under
vacuum at 55.degree. C. for 17 hours to provide
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid
as an off-white crystalline solid.
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid
was isolated in 79% yield from
(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (3 steps including
isolation) and with an HPLC purity of 99.0% AUC. ESI-MS m/z calc.
242.04. found 241.58 (M+1).sup.+; .sup.1H NMR (500 MHz, DMSO)
.delta. 12.40 (s, 1H), 7.40 (d, J=1.6 Hz, 1H), 7.30 (d, J=8.3 Hz,
1H), 7.17 (dd, J=8.3, 1.7 Hz, 1H), 1.46 (m, 2H), 1.17 (m, 2H).
[0122] Alternative Synthesis of the Acid Moiety
Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol
##STR00011##
[0124] Commercially available
2,2-difluoro-1,3-benzodioxole-5-carboxylic acid (1.0 eq) is
slurried in toluene (10 vol). Vitride.RTM. (2 eq) is added via
addition funnel at a rate to maintain the temperature at
15-25.degree. C. At the end of addition the temperature is
increased to 40.degree. C. for 2 h then 10% (w/w) aq. NaOH (4.0 eq)
is carefully added via addition funnel maintaining the temperature
at 40-50.degree. C. After stirring for an additional 30 minutes,
the layers are allowed to separate at 40.degree. C. The organic
phase is cooled to 20.degree. C. then washed with water
(2.times.1.5 vol), dried (Na.sub.2SO.sub.4), filtered, and
concentrated to afford crude
(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that is used directly
in the next step.
Synthesis of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole
##STR00012##
[0126] (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) is
dissolved in MTBE (5 vol). A catalytic amount of DMAP (1 mol %) is
added and SOCl.sub.2 (1.2 eq) is added via addition funnel. The
SOCl.sub.2 is added at a rate to maintain the temperature in the
reactor at 15-25.degree. C. The temperature is increased to
30.degree. C. for 1 hour then cooled to 20.degree. C. then water (4
vol) is added via addition funnel maintaining the temperature at
less than 30.degree. C. After stirring for an additional 30
minutes, the layers are allowed to separate. The organic layer is
stirred and 10% (w/v) aq. NaOH (4.4 vol) is added. After stirring
for 15 to 20 minutes, the layers are allowed to separate. The
organic phase is then dried (Na.sub.2SO.sub.4), filtered, and
concentrated to afford crude
5-chloromethyl-2,2-difluoro-1,3-benzodioxole that is used directly
in the next step.
Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile
##STR00013##
[0128] A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole
(1 eq) in DMSO (1.25 vol) is added to a slurry of NaCN (1.4 eq) in
DMSO (3 vol) maintaining the temperature between 30-40.degree. C.
The mixture is stirred for 1 hour then water (6 vol) is added
followed by MTBE (4 vol). After stirring for 30 min, the layers are
separated. The aqueous layer is extracted with MTBE (1.8 vol). The
combined organic layers are washed with water (1.8 vol), dried
(Na.sub.2SO.sub.4), filtered, and concentrated to afford crude
(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) that is used
directly in the next step.
[0129] The remaining steps are the same as described above for the
synthesis of the acid moiety.
[0130] Amine Moiety
Synthesis of 2-bromo-5-fluoro-4-nitroaniline
##STR00014##
[0132] A flask was charged with 3-fluoro-4-nitroaniline (1.0 equiv)
followed by ethyl acetate (10 vol) and stirred to dissolve all
solids. N-Bromosuccinimide (1.0 equiv) was added as a portion-wise
as to maintain internal temperature of 22.degree. C. At the end of
the reaction, the reaction mixture was concentrated in vacuo on a
rotavap. The residue was slurried in distilled water (5 vol) to
dissolve and remove succinimide. (The succinimide can also be
removed by water workup procedure.) The water was decanted and the
solid was slurried in 2-propanol (5 vol) overnight. The resulting
slurry was filtered and the wetcake was washed with 2-propanol,
dried in vacuum oven at 50.degree. C. overnight with N.sub.2 bleed
until constant weight was achieved. A yellowish tan solid was
isolated (50% yield, 97.5% AUC). Other impurities were a
bromo-regioisomer (1.4% AUC) and a di-bromo adduct (1.1% AUC).
.sup.1H NMR (500 MHz, DMSO)
[0133] 8.19 (1H, d, J=8.1 Hz), 7.06 (br. s, 2H), 6.64 (d, 1H,
J=14.3 Hz).
Synthesis of p-toluenesulfonic acid salt of
(R)-1-(4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol
##STR00015##
[0135] A thoroughly dried flask under N.sub.2 was charged with the
following: Activated powdered 4A molecular sieves (50 wt % based on
2-bromo-5-fluoro-4-nitroaniline), 2-Bromo-5-fluoro-4-nitroaniline
(1.0 equiv), zinc perchlorate dihydrate (20 mol %), and toluene (8
vol). The mixture was stirred at room temperature for NMT 30 min.
Lastly, (R)-benzyl glycidyl ether (2.0 equiv) in toluene (2 vol)
was added in a steady stream. The reaction was heated to 80.degree.
C. (internal temperature) and stirred for approximately 7 hours or
until 2-Bromo-5-fluoro-4-nitroaniline was <5% AUC.
[0136] The reaction was cooled to room temperature and Celite (50
wt %) was added, followed by ethyl acetate (10 vol). The resulting
mixture was filtered to remove Celite and sieves and washed with
ethyl acetate (2 vol). The filtrate was washed with ammonium
chloride solution (4 vol, 20% w/v). The organic layer was washed
with sodium bicarbonate solution (4 vol.times.2.5% w/v). The
organic layer was concentrated in vacuo on a rotovap. The resulting
slurry was dissolved in isopropyl acetate (10 vol) and this
solution was transferred to a Buchi hydrogenator.
[0137] The hydrogenator was charged with 5 wt % Pt(S)/C (1.5 mol %)
and the mixture was stirred under N.sub.2 at 30.degree. C.
(internal temperature). The reaction was flushed with N.sub.2
followed by hydrogen. The hydrogenator pressure was adjusted to 1
Bar of hydrogen and the mixture was stirred rapidly (>1200 rpm).
At the end of the reaction, the catalyst was filtered through a pad
of Celite and washed with dichloromethane (10 vol). The filtrate
was concentrated in vacuo. Any remaining isopropyl acetate was
chased with dichloromethane (2 vol) and concentrated on a rotavap
to dryness.
[0138] The resulting residue was dissolved in dichloromethane (10
vol). p-Toluenesulfonic acid monohydrate (1.2 equiv) was added and
stirred overnight. The product was filtered and washed with
dichloromethane (2 vol) and suction dried. The wetcake was
transferred to drying trays and into a vacuum oven and dried at
45.degree. C. with N.sub.2 bleed until constant weight was
achieved. p-Toluenesulfonic acid salt of
(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol
was isolated as an off-white solid.
[0139] Chiral purity was determined to be >97% ee.
Synthesis of (3-Chloro-3-methylbut-1-ynyl)trimethylsilane
##STR00016##
[0141] Propargyl alcohol (1.0 equiv) was charged to a vessel.
Aqueous hydrochloric acid (37%, 3.75 vol) was added and stirring
begun. During dissolution of the solid alcohol, a modest endotherm
(5-6.degree. C.) is observed. The resulting mixture was stirred
overnight (16 h), slowly becoming dark red. A 30 L jacketed vessel
is charged with water (5 vol) which is then cooled to 10.degree. C.
The reaction mixture is transferred slowly into the water by
vacuum, maintaining the internal temperature of the mixture below
25.degree. C. Hexanes (3 vol) is added and the resulting mixture is
stirred for 0.5 h. The phases were settled and the aqueous phase
(pH<1) was drained off and discarded. The organic phase was
concentrated in vacuo using a rotary evaporator, furnishing the
product as red oil.
Synthesis of
(4-(Benzyloxy)-3,3-dimethylbut-1-ynyl)trimethylsilane
##STR00017##
[0143] Method A
[0144] All equivalent and volume descriptors in this part are based
on a 250 g reaction. Magnesium turnings (69.5 g, 2.86 mol, 2.0
equiv) were charged to a 3 L 4-neck reactor and stirred with a
magnetic stirrer under nitrogen for 0.5 h. The reactor was immersed
in an ice-water bath. A solution of the propargyl chloride (250 g,
1.43 mol, 1.0 equiv) in THF (1.8 L, 7.2 vol) was added slowly to
the reactor, with stirring, until an initial exotherm
(.about.10.degree. C.) was observed. The Grignard reagent formation
was confirmed by IPC using .sup.1H-NMR spectroscopy. Once the
exotherm subsided, the remainder of the solution was added slowly,
maintaining the batch temperature <15.degree. C. The addition
required .about.3.5 h. The resulting dark green mixture was
decanted into a 2 L capped bottle.
[0145] All equivalent and volume descriptors in this part are based
on a 500 g reaction. A 22 L reactor was charged with a solution of
benzyl chloromethyl ether (95%, 375 g, 2.31 mol, 0.8 equiv) in THF
(1.5 L, 3 vol). The reactor was cooled in an ice-water bath. Two
Grignard reagent batches prepared as described above were combined
and then added slowly to the benzyl chloromethyl ether solution via
an addition funnel, maintaining the batch temperature below
25.degree. C. The addition required 1.5 h. The reaction mixture was
stirred overnight (16 h).
[0146] All equivalent and volume descriptors in this part are based
on a 1 kg reaction. A solution of 15% ammonium chloride was
prepared in a 30 L jacketed reactor (1.5 kg in 8.5 kg of water, 10
vol). The solution was cooled to 5.degree. C. Two Grignard reaction
mixtures prepared as described above were combined and then
transferred into the ammonium chloride solution via a header
vessel. An exotherm was observed in this quench, which was carried
out at a rate such as to keep the internal temperature below
25.degree. C. Once the transfer was complete, the vessel jacket
temperature was set to 25.degree. C. Hexanes (8 L, 8 vol) was added
and the mixture was stirred for 0.5 h. After settling the phases,
the aqueous phase (pH 9) was drained off and discarded. The
remaining organic phase was washed with water (2 L, 2 vol). The
organic phase was concentrated in vacuo using a 22 L rotary
evaporator, providing the crude product as an orange oil.
[0147] Method B
[0148] Magnesium turnings (106 g, 4.35 mol, 1.0 eq) were charged to
a 22 L reactor and then suspended in THF (760 mL, 1 vol). The
vessel was cooled in an ice-water bath such that the batch
temperature reached 2.degree. C. A solution of the propargyl
chloride (760 g, 4.35 mol, 1.0 equiv) in THF (4.5 L, 6 vol) was
added slowly to the reactor. After 100 mL was added, the addition
was stopped and the mixture stirred until a 13.degree. C. exotherm
was observed, indicating the Grignard reagent initiation. Once the
exotherm subsided, another 500 mL of the propargyl chloride
solution was added slowly, maintaining the batch temperature
<20.degree. C. The Grignard reagent formation was confirmed by
IPC using .sup.1H-NMR spectroscopy. The remainder of the propargyl
chloride solution was added slowly, maintaining the batch
temperature <20.degree. C. The addition required .about.1.5 h.
The resulting dark green solution was stirred for 0.5 h. The
Grignard reagent formation was confirmed by IPC using .sup.1H-NMR
spectroscopy. Neat benzyl chloromethyl ether was charged to the
reactor addition funnel and then added dropwise into the reactor,
maintaining the batch temperature below 25.degree. C. The addition
required 1.0 h. The reaction mixture was stirred overnight. The
aqueous work-up and concentration was carried out using the same
procedure and relative amounts of materials as in Method A to give
the product as an orange oil.
Syntheisis of 4-Benzyloxy-3,3-dimethylbut-1-yne
##STR00018##
[0150] A 30 L jacketed reactor was charged with methanol (6 vol)
which was then cooled to 5.degree. C. Potassium hydroxide (85%, 1.3
equiv) was added to the reactor. A 15-20.degree. C. exotherm was
observed as the potassium hydroxide dissolved. The jacket
temperature was set to 25.degree. C. A solution of
4-benzyloxy-3,3-dimethyl-1-trimethylsilylbut-1-yne (1.0 equiv) in
methanol (2 vol) was added and the resulting mixture was stirred
until reaction completion, as monitored by HPLC. Typical reaction
time at 25.degree. C. is 3-4 h. The reaction mixture is diluted
with water (8 vol) and then stirred for 0.5 h. Hexanes (6 vol) was
added and the resulting mixture was stirred for 0.5 h. The phases
were allowed to settle and then the aqueous phase (pH 10-11) was
drained off and discarded. The organic phase was washed with a
solution of KOH (85%, 0.4 equiv) in water (8 vol) followed by water
(8 vol). The organic phase was then concentrated down using a
rotary evaporator, yielding the title material as a yellow-orange
oil. Typical purity of this material is in the 80% range with
primarily a single impurity present .sup.1H NMR (400 MHz,
C.sub.6D.sub.6) .delta. 7.28 (d, 2H, J=7.4 Hz), 7.18 (t, 2H, J=7.2
Hz), 7.10 (d, 1H, J=7.2 Hz), 4.35 (s, 2H), 3.24 (s, 2H), 1.91 (s,
1H), 1.25 (s, 6H).
Synthesis of
N-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindo-
le
Method A
Synthesis of
(R)-1-((4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl)-5-fluoropheny-
l)amino)-3-(benzyloxy)propan-2-ol
##STR00019##
[0152] p-Toluenesulfonic acid salt of
(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol
was freebased by stirring the solid in dichloromethane (5 vol) and
saturated NaHCO.sub.3 solution (5 vol) until clear organic layer
was achieved. The resulting layers were separated and the organic
layer was washed with saturated NaHCO.sub.3 solution (5 vol)
followed by brine and concentrated in vacuo to obtain
(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol
free base as an oil.
[0153] Palladium acetate (0.01 eq), dppb (0.015 eq), CuI (0.015 eq)
and potassium carbonate (3 eq) are suspended in acetonitrile (1.2
vol). After stirring for 15 minutes, a solution of
4-benzyloxy-3,3-dimethylbut-1-yne (1.1 eq) in acetonitrile (0.2
vol) is added. The mixture is sparged with nitrogen gas for 1 h and
then a solution of
(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol
free base (1 eq) in acetonitrile (4.1 vol) is added. The mixture is
sparged with nitrogen gas for another hour and then is heated to
80.degree. C. Reaction progress is monitored by HPLC and the
reaction is usually complete within 3-5 h. The mixture is cooled to
room temperature and then filtered through Celite. The cake is
washed with acetonitrile (4 vol). The combined filtrates are
azeotroped to dryness and then the mixture is polish filtered into
the next reactor. The acetonitrile solution of
(R)-1-((4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl)-5-fluoropheny-
l)amino)-3-(benzyloxy)propan-2-ol thus obtained is used directly in
the next procedure (cyclization) without further manipulation.
Synthesis of
N-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindo-
le
##STR00020##
[0155] Bis-acetonitriledichloropalladium (0.1 eq) and CuI (0.1 eq)
are charged to the reactor and then suspended in a solution of
(R)-1-(4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl)-5-fluorophenyp-
amino)-3-(benzyloxy)propan-2-ol obtained above (1 eq) in
acetonitrile (9.5 vol total). The mixture is sparged with nitrogen
gas for 1 h and then is heated to 80.degree. C. The reaction
progress is monitored by HPLC and the reaction is typically
complete within 1-3 h. The mixture is filtered through Celite and
the cake is washed with acetonitrile. A solvent swap into ethyl
acetate (7.5 vol) is performed. The ethyl acetate solution is
washed with aqueous NH.sub.3--NH.sub.4Cl solution (2.times.2.5 vol)
followed by 10% brine (2.5 vol). The ethyl acetate solution is then
stirred with silica gel (1.8 wt eq) and Si-TMT (0.1 wt eq) for 6 h.
After filtration, the resulting solution is concentrated down. The
residual oil is dissolved in DCM/heptane (4 vol) and then purified
by column chromatography. The oil thus obtained is then
crystallized from 25% EtOAc/heptane (4 vol). Crystalline
(R)-1-(5-amino-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-1--
yl)-3-(benzyloxy)propan-2-ol is typically obtained in 27-38% yield.
.sup.1H NMR (400 MHz, DMSO) .delta. 7.38-7.34 (m, 4H), 7.32-7.23
(m, 6H), 7.21 (d, 1H, J=12.8 Hz), 6.77 (d, 1H, J=9.0 Hz), 6.06 (s,
1H), 5.13 (d, 1H, J=4.9 Hz), 4.54 (s, 2H), 4.46 (br. s, 2H), 4.45
(s, 2H), 4.33 (d, 1H, J=12.4 Hz), 4.09-4.04 (m, 2H), 3.63 (d, 1H,
J=9.2 Hz), 3.56 (d, 1H, J=9.2 Hz), 3.49 (dd, 1H, J=9.8, 4.4 Hz),
3.43 (dd, 1H, J=9.8, 5.7 Hz), 1.40 (s, 6H).
Synthesis of
N-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindo-
le
Method B
##STR00021##
[0157] Palladium acetate (33 g, 0.04 eq), dppb (94 g, 0.06 eq), and
potassium carbonate (1.5 kg, 3.0 eq) are charged to a reactor. The
free based oil benzylglocolated 4-ammonium-2-bromo-5-fluoroaniline
(1.5 kg, 1.0 eq) was dissolved in acetonitrile (8.2 L, 4.1 vol) and
then added to the reactor. The mixture was sparged with nitrogen
gas for NLT 1 h. A solution of 4-benzyloxy-3,3-dimethylbut-1-yne
(70%, 1.1 kg, 1.05 eq) in acetonitrile was added to the mixture
which was then sparged with nitrogen gas for NLT 1 h. The mixture
was heated to 80.degree. C. and then stirred overnight. IPC by HPLC
is carried out and the reaction is determined to be complete after
16 h. The mixture was cooled to ambient temperature and then
filtered through a pad of Celite (228 g). The reactor and Celite
pad were washed with acetonitrile (2.times.2 L, 2 vol). The
combined phases are concentrated on a 22 L rotary evaporator until
8 L of solvent have been collected, leaving the crude product in 7
L (3.5 vol) of acetonitrile.
[0158] Bis-acetonitriledichloropalladium (144 g, 0.15 eq) was
charged to the reactor. The crude solution was transferred back
into the reactor and the roto-vap bulb was washed with acetonitrile
(4 L, 2 vol). The combined solutions were sparged with nitrogen gas
for NLT 1 h. The reaction mixture was heated to 80.degree. C. for
NLT 16 h. In process control by HPLC shows complete consumption of
starting material. The reaction mixture was filtered through Celite
(300 g). The reactor and filter cake were washed with acetonitrile
(3 L, 1.5 vol). The combined filtrates were concentrated to an oil
by rotary evaporation. The oil was dissolved in ethyl acetate (8.8
L, 4.4 vol). The solution was washed with 20% ammonium chloride (5
L, 2.5 vol) followed by 5% brine (5 L, 2.5 vol). Silica gel (3.5
kg, 1.8 wt. eq.) of silica gel was added to the organic phase,
which was stirred overnight. Deloxan THP II metal scavenger (358 g)
and heptane (17.6 L) were added and the resulting mixture was
stirred for NLT 3 h. The mixture was filtered through a sintered
glass funnel. The filter cake was washed with 30% ethyl acetate in
heptane (25 L). The combined filtrates were concentrated under
reduced pressure to give
N-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindo-
le as a brown paste (1.4 kg).
[0159] Synthesis of Compound 1
[0160] Synthesis of Benzyl Protected Compound 1.
##STR00022##
[0161] 1-(2,2-Difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic
acid (1.3 equiv) was slurried in toluene (2.5 vol, based on
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid).
Thionyl chloride (SOCl.sub.2, 1.7 equiv) was added via addition
funnel. and the mixture was heated to 60.degree. C. The resulting
mixture was stirred for 2 h. The toluene and the excess SOCl2 were
distilled off using rotavop. Additional toluene (2.5 vol, based on
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid)
was added and the mixture was distilled down to 1 vol of toluene. A
solution of
(R)-1-(5-amino-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-
-1-yl)-3-(benzyloxy)propan-2-ol (1 eq) and triethylamine (3 eq) in
DCM (4 vol) is cooled to 0.degree. C. The acid chloride solution in
toluene (1 vol) is added while maintaining the batch temperature
below 10.degree. C. The reaction progress is monitored by HPLC, and
the reaction is usually complete within minutes. After warming to
25.degree. C., the reaction mixture is washed with 5% NaHCO.sub.3
(3.5 vol), 1 M NaOH (3.5 vol) and 1 M HCl (5 vol). A solvent swap
to into methanol (2 vol) is performed and the resulting solution of
(R)--N-(1-(3-(benzyloxy)-2-hydroxypropyl)-2-(1-(benzyloxy)-2-methylpropan-
-2-yl)-6-fluoro-1H-indol-5-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyc-
lopropanecarboxamide in methanol is used without further
manipulation in the next step (hydrogenolysis).
[0162] Synthesis of Compound 1.
##STR00023##
[0163] 5% palladium on charcoal (.about.50% wet, 0.01 eq) is
charged to an appropriate hydrogenation vessel. The
(R)--N-(1-(3-(benzyloxy)-2-hydroxypropyl)-2-(1-(benzyloxy)-2-methylpropan-
-2-yl)-6-fluoro-1H-indol-5-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyc-
lopropanecarboxamide solution in methanol (2 vol) obtained above is
added carefully, followed by a 3 M solution of HCl in methanol. The
vessel is purged with nitrogen gas and then with hydrogen gas. The
mixture is stirred vigorously until the reaction is complete, as
determined by HPLC analysis. Typical reaction time is 3-5 h. The
reaction mixture is filtered through Celite and the cake is washed
with methanol (2 vol). A solvent swap into isopropanol (3 vol) is
performed. Crude VX-661 is crystallized from 75% IPA-heptane (4
vol, ie. 1 vol heptane added to the 3 vol of IPA) and the resulting
crystals are matured in 50% IPA-heptane (ie. 2 vol of heptane added
to the mixture). Typical yields of compound 4 from the two-step
acylation/hydrogenolysis procedure range from 68% to 84%. Compound
4 can be recrystallized from IPA-heptane following the same
procedure just described.
[0164] Compound 1 may also be prepared by one of several synthetic
routes disclosed in US published patent application US20090131492,
incorporated herein by reference.
[0165] Table 10 below recites analytical data for Compound 1.
TABLE-US-00009 TABLE 10 Cmpd. LC/MS LC/RT No. M + 1 min NMR 1 521.5
1.69 1H NMR (400.0 MHz, CD.sub.3CN) d 7.69 (d, J = 7.7 Hz, 1H),
7.44 (d, J = 1.6 Hz, 1H), 7.39 (dd, J = 1.7, 8.3 Hz, 1H), 7.31 (s,
1H), 7.27 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 12.0 Hz, 1H), 6.34 (s,
1H), 4.32 (d, J = 6.8 Hz, 2H), 4.15-4.09 (m, 1H), 3.89 (dd, J =
6.0, 11.5 Hz, 1H), 3.63-3.52 (m, 3H), 3.42 (d, J = 4.6 Hz, 1H),
3.21 (dd, J = 6.2, 7.2 Hz, 1H), 3.04 (t, J = 5.8 Hz, 1H), 1.59 (dd,
J = 3.8, 6.8 Hz, 2H), 1.44 (s, 3H), 1.33 (s, 3H) and 1.18 (dd, J =
3.7, 6.8 Hz, 2H) ppm.
[0166] Preparation of Compound 1 Formulations
[0167] Preparation of a 40 g Formulation Comprising 50 mg of
Compound 1
[0168] A 40 g aqueous formulation is prepared with the following
components and amounts:
TABLE-US-00010 Component Amount (g) Compound 1 0.05 PEG 400 19.95
Vitamin E TPGS 4.48 Water 15.52
[0169] A 22.4% by weight vitamin E TPGS solution is prepared
separately by adding vitamin E TPGS (4.48 g) to a clear glass jar
equipped with a magnetic stirring bar. Water (15.52 mL) is added
and the jar is sealed and stirred at a rate such that the vortex
depth is approximately 2/3 of the liquid depth. Stir the mixture of
vitamin E TPGS and water at room temperature until all of the
vitamin E TPGS is dissolved and there are no visible solid
particles. Depending on the size of the vitamin E TPGS solid
particles, this step may take more than 4 hours. Stirring of the
solution in the closed container can be done overnight. To speed up
the vitamin E TPGS dissolution process, it can be carried out in a
water bath at 30-35.degree. C. Remove the solution from stirring
and filter using a disposable filter unit.
[0170] Weigh 19.95 g of PEG 400 into a separate jar and place the
jar in a water bath at 40.degree. C..+-.5.degree. C. to warm up.
Increase the stirring rate until a vortex forms in the middle of
the liquid. Weigh in 50 mg of Compound 1, close the jar, and stir
the formulation at 40.degree. C..+-.5.degree. C. until all of
Compound 1 dissolves completely (about 1 hour). Remove the
formulation from the water bath and stir for at least 1 hour to
equilibrate at room temperature. Add the vitamin E TPGS solution
from the first jar and mix the formulation by hand until a
homogeneous solution forms.
[0171] Preparation of a 40 g Formulation Comprising 100 mg of
Compound 1
[0172] A 40 g aqueous formulation is prepared with the following
components and amounts:
TABLE-US-00011 Component Amount (g) Compound 1 0.10 PEG 400 19.90
Vitamin E TPGS 4.48 Water 15.52
[0173] A 22.4% by weight vitamin E TPGS solution is prepared as
described above.
[0174] Weigh 19.90 g of PEG 400 into a separate jar and place the
jar in a water bath at 40.degree. C..+-.5.degree. C. to warm up.
Increase the stirring rate until a vortex forms in the middle of
the liquid. Weigh in 100 mg of Compound 1, close the jar, and stir
the formulation at 40.degree. C..+-.5.degree. C. until all of
Compound 1 dissolves completely (about 1 hour). Remove the
formulation from the water bath and stir for at least 1 hour to
equilibrate at room temperature. Add the vitamin E TPGS solution
from the first jar and mix the formulation by hand until a
homogeneous solution forms.
[0175] Preparation of a 40 g Formulation Comprising 200 mg of
Compound 1
[0176] A 40 g aqueous formulation is prepared with the following
components and amounts:
TABLE-US-00012 Component Amount (g) Compound 1 0.20 PEG 400 19.80
Vitamin E TPGS 4.48 Water 15.52
[0177] A 22.4% by weight vitamin E TPGS solution is prepared as
described above.
[0178] Weigh 19.80 g of PEG 400 into a separate jar and place the
jar in a water bath at 40.degree. C..+-.5.degree. C. to warm up.
Increase the stirring rate until a vortex forms in the middle of
the liquid. Weigh in 200 mg of Compound 1, close the jar, and stir
the formulation at 40.degree. C..+-.5.degree. C. until all of
Compound 1 dissolves completely (about 1 hour). Remove the
formulation from the water bath and stir for at least 1 hour to
equilibrate at room temperature. Add the vitamin E TPGS solution
from the first jar and mix the formulation by hand until a
homogeneous solution forms.
[0179] Preparation of a 40 g Formulation Comprising 300 mg of
Compound 1
[0180] A 40 g aqueous formulation is prepared with the following
components and amounts:
TABLE-US-00013 Component Amount (g) Compound 1 0.30 PEG 400 19.70
Vitamin E TPGS 4.48 Water 15.52
[0181] A 22.4% by weight vitamin E TPGS solution is prepared as
described above.
[0182] Weigh 19.70 g of PEG 400 into a separate jar and place the
jar in a water bath at 40.degree. C..+-.5.degree. C. to warm up.
Increase the stirring rate until a vortex forms in the middle of
the liquid. Weigh in 300 mg of Compound 1, close the jar, and stir
the formulation at 40.degree. C..+-.5.degree. C. until all of
Compound 1 dissolves completely (about 1 hour). Remove the
formulation from the water bath and stir for at least 1 hour to
equilibrate at room temperature. Add the vitamin E TPGS solution
from the first jar and mix the formulation by hand until a
homogeneous solution forms.
[0183] Assays
[0184] Assays for Detecting and Measuring .DELTA.F508-CFTR
Correction Properties of Compounds
[0185] Membrane Potential Optical Methods for Assaying
.DELTA.F508-CFTR Modulation Properties of Compounds
[0186] The optical membrane potential assay utilized
voltage-sensitive FRET sensors described by Gonzalez and Tsien
(See, 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., 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).
[0187] 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 were 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.
[0188] 1. Identification of Correction Compounds
[0189] To identify small molecules that correct the trafficking
defect associated with .DELTA.F508-CFTR; a single-addition HTS
assay format was developed. The cells were incubated in serum-free
medium for 16 hrs at 37.degree. C. in the presence or absence
(negative control) of test compound. As a positive control, cells
plated in 384-well plates were incubated for 16 hrs at 27.degree.
C. to "temperature-correct" .DELTA.F508-CFTR. The cells were
subsequently rinsed 3.times. with Krebs Ringers solution and loaded
with the voltage-sensitive dyes. To activate .DELTA.F508-CFTR, 10
.mu.M forskolin and the CFTR potentiator, genistein (20 .mu.M),
were added along with Cl.sup.--free medium to each well. The
addition of Cl.sup.--free medium promoted Cl.sup.- efflux in
response to .DELTA.F508-CFTR activation and the resulting membrane
depolarization was optically monitored using the FRET-based
voltage-sensor dyes.
[0190] 2. Identification of Potentiator Compounds
[0191] To identify potentiators of .DELTA.F508-CFTR, a
double-addition HTS assay format was developed. During the first
addition, a Cl.sup.--free medium with or without test compound was
added to each well. After 22 sec, a second addition of Cr-free
medium containing 2-10 .mu.M forskolin was added to activate
.DELTA.F508-CFTR. The extracellular Cr concentration following both
additions was 28 mM, which promoted Cl.sup.- efflux in response to
.DELTA.F508-CFTR activation and the resulting membrane
depolarization was optically monitored using the FRET-based
voltage-sensor dyes.
[0192] 3. Solutions [0193] Bath Solution #1: (in mM) NaCl 160, KCl
4.5, CaCl.sub.2 2, MgCl.sub.2 1, HEPES 10, pH 7.4 with NaOH. [0194]
Chloride-free bath solution: Chloride salts in Bath Solution #1 are
substituted with gluconate salts. [0195] CC2-DMPE: Prepared as a 10
mM stock solution in DMSO and stored at -20.degree. C. [0196]
DiSBAC.sub.2(3): Prepared as a 10 mM stock in DMSO and stored at
-20.degree. C.
[0197] 4. Cell Culture
[0198] 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.times.NEAA, .beta.-ME,
1.times. pen/strep, and 25 mM HEPES in 175 cm.sup.2 culture flasks.
For all optical assays, the cells were seeded at 30,000/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.
[0199] Electrophysiological Assays for Assaying .DELTA.F508-CFTR
Modulation Properties of Compounds
[0200] 1. Ussing Chamber Assay
[0201] Using chamber experiments were performed on polarized
epithelial cells expressing .DELTA.F508-CFTR to further
characterize the .DELTA.F508-CFTR modulators identified in the
optical assays. FRT.sup..DELTA.F508-CFTR epithelial cells grown on
Costar Snapwell cell culture inserts were mounted in an Ussing
chamber (Physiologic Instruments, Inc., San Diego, Calif.), and the
monolayers were continuously short-circuited using a Voltage-clamp
System (Department of Bioengineering, University of Iowa, Iowa,
and, Physiologic Instruments, Inc., San Diego, Calif.).
Transepithelial resistance was measured by applying a 2-mV pulse.
Under these conditions, the FRT epithelia demonstrated resistances
of 4 K.OMEGA./cm.sup.2 or more. The solutions were maintained at
27.degree. C. and bubbled with air. The electrode offset potential
and fluid resistance were corrected using a cell-free insert. Under
these conditions, the current reflects the flow of Cl.sup.- through
.DELTA.F508-CFTR expressed in the apical membrane. The I.sub.SC was
digitally acquired using an MP100A-CE interface and AcqKnowledge
software (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).
[0202] 2. Identification of Correction Compounds
[0203] Typical protocol utilized a basolateral to apical membrane
Cl.sup.- concentration gradient. To set up this gradient, normal
ringer 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. All experiments were performed with intact monolayers.
To fully activate .DELTA.F508-CFTR, forskolin (10 .mu.M) and the
PDE inhibitor, IBMX (100 .mu.M), were applied followed by the
addition of the CFTR potentiator, genistein (50 .mu.M).
[0204] As observed in other cell types, incubation at low
temperatures of FRT cells stably expressing .DELTA.F508-CFTR
increases the functional density of CFTR in the plasma membrane. To
determine the activity of correction compounds, the cells were
incubated with 10 .mu.M of the test compound for 24 hours at
37.degree. C. and were subsequently washed 3.times. prior to
recording. The cAMP- and genistein-mediated I.sub.SC in
compound-treated cells was normalized to the 27.degree. C. and
37.degree. C. controls and expressed as percentage activity.
Preincubation of the cells with the correction compound
significantly increased the cAMP- and genistein-mediated I.sub.SC
compared to the 37.degree. C. controls.
[0205] 3. Identification of Potentiator Compounds
[0206] 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 and was permeabilized
with nystatin (360 .mu.g/ml), 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. All
experiments were performed 30 min after nystatin permeabilization.
Forskolin (10 .mu.M) and all test compounds were added to both
sides of the cell culture inserts. The efficacy of the putative
.DELTA.F508-CFTR potentiators was compared to that of the known
potentiator, genistein.
[0207] 4. Solutions [0208] Basolateral solution (in mM): NaCl
(135), CaCl.sub.2 (1.2), MgCl.sub.2 (1.2), K.sub.2HPO.sub.4 (2.4),
KHPO.sub.4 (0.6), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) (10), and dextrose (10). The solution was titrated to
pH 7.4 with NaOH. [0209] Apical solution (in mM): Same as
basolateral solution with NaCl replaced with Na Gluconate
(135).
[0210] 5. Cell Culture
[0211] Fisher rat epithelial (FRT) cells expressing
.DELTA.F508-CFTR (FRT.sup..DELTA.F508-CFTR) were used for Ussing
chamber experiments for the putative .DELTA.F508-CFTR modulators
identified from our optical assays. The cells were cultured on
Costar Snapwell cell culture inserts and cultured for five days at
37.degree. C. and 5% CO.sub.2 in Coon's modified Ham's F-12 medium
supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100
.mu.g/ml streptomycin. Prior to use for characterizing the
potentiator activity of compounds, the cells were incubated at
27.degree. C. for 16-48 hrs to correct for the .DELTA.F508-CFTR. To
determine the activity of corrections compounds, the cells were
incubated at 27.degree. C. or 37.degree. C. with and without the
compounds for 24 hours.
[0212] 6. Whole-Cell Recordings
[0213] The macroscopic .DELTA.F508-CFTR current (I.sub..DELTA.F508)
in temperature- and test compound-corrected NIH3T3 cells stably
expressing .DELTA.F508-CFTR were monitored using the
perforated-patch, whole-cell recording. Briefly, voltage-clamp
recordings of I.sub..DELTA.F508 were performed at room temperature
using an Axopatch 200B patch-clamp amplifier (Axon Instruments
Inc., Foster City, Calif.). All recordings were acquired at a
sampling frequency of 10 kHz and low-pass filtered at 1 kHz.
Pipettes had a resistance of 5-6 M.OMEGA. when filled with the
intracellular solution. Under these recording conditions, the
calculated reversal potential for Cl.sup.- (E.sub.Cl) at room
temperature was -28 mV. All recordings had a seal resistance >20
G.OMEGA. and a series resistance <15 M.OMEGA.. 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.). The bath contained <250 .mu.l of saline
and was continuously perifused at a rate of 2 ml/min using a
gravity-driven perfusion system.
[0214] 7. Identification of Correction Compounds
[0215] To determine the activity of correction compounds for
increasing the density of functional .DELTA.F508-CFTR in the plasma
membrane, we used the above-described perforated-patch-recording
techniques to measure the current density following 24-hr treatment
with the correction compounds. To fully activate .DELTA.F508-CFTR,
10 .mu.M forskolin and 20 .mu.M genistein were added to the cells.
Under our recording conditions, the current density following 24-hr
incubation at 27.degree. C. was higher than that observed following
24-hr incubation at 37.degree. C. These results are consistent with
the known effects of low-temperature incubation on the density of
.DELTA.F508-CFTR in the plasma membrane. To determine the effects
of correction compounds on CFTR current density, the cells were
incubated with 10 .mu.M of the test compound for 24 hours at
37.degree. C. and the current density was compared to the
27.degree. C. and 37.degree. C. controls (% activity). Prior to
recording, the cells were washed 3.times. with extracellular
recording medium to remove any remaining test compound.
Preincubation with 10 .mu.M of correction compounds significantly
increased the cAMP- and genistein-dependent current compared to the
37.degree. C. controls.
[0216] 8. Identification of Potentiator Compounds
[0217] 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.sub..DELTA.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).
[0218] 9. Solutions [0219] Intracellular solution (in mM):
Cs-aspartate (90), CsCl (50), MgCl.sub.2 (1), HEPES (10), and 240
.mu.g/ml amphotericin-B (pH adjusted to 7.35 with CsOH). [0220]
Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl
(150), MgCl.sub.2 (2), CaCl.sub.2 (2), HEPES (10) (pH adjusted to
7.35 with HCl).
[0221] 10. Cell Culture
[0222] 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.times.NEAA, .beta.-ME, 1.times. 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.
[0223] 11. Single-Channel Recordings
[0224] The single-channel activities of temperature-corrected
.DELTA.F508-CFTR stably expressed in NIH3T3 cells and activities of
potentiator compounds were observed using excised inside-out
membrane patch. Briefly, voltage-clamp recordings of single-channel
activity were performed at room temperature with an Axopatch 200B
patch-clamp amplifier (Axon Instruments Inc.). All recordings were
acquired at a sampling frequency of 10 kHz and low-pass filtered at
400 Hz. Patch pipettes were fabricated from Corning Kovar Sealing
#7052 glass (World Precision Instruments, Inc., Sarasota, Fla.) and
had a resistance of 5-8 M.OMEGA. when filled with the extracellular
solution. The .DELTA.F508-CFTR was activated after excision, by
adding 1 mM Mg-ATP, and 75 nM of the cAMP-dependent protein kinase,
catalytic subunit (PKA; Promega Corp. Madison, Wis.). After channel
activity stabilized, the patch was perifused using a gravity-driven
microperfusion system. The inflow was placed adjacent to the patch,
resulting in complete solution exchange within 1-2 sec. To maintain
.DELTA.F508-CFTR activity during the rapid perifusion, the
nonspecific phosphatase inhibitor F (10 mM NaF) was added to the
bath solution. Under these recording conditions, channel activity
remained constant throughout the duration of the patch recording
(up to 60 min). Currents produced by positive charge moving from
the intra- to extracellular solutions (anions moving in the
opposite direction) are shown as positive currents. The pipette
potential (V.sub.p) was maintained at 80 mV.
[0225] 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.
[0226] 12. Solutions [0227] Extracellular solution (in mM): NMDG
(150), aspartic acid (150), CaCl.sub.2 (5), MgCl.sub.2(2), and
HEPES (10) (pH adjusted to 7.35 with Tris base). [0228]
Intracellular solution (in mM): NMDG-Cl (150), MgCl.sub.2 (2), EGTA
(5), TES (10), and Tris base (14) (pH adjusted to 7.35 with
HCl).
[0229] 13. Cell Culture
[0230] 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.times.NEAA, .beta.-ME,
1.times. 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.
[0231] Using the procedures described above, the activity, i.e.,
IC50s, of Compound 1 has been measured and is shown in Table
11.
TABLE-US-00014 TABLE 11 IC50 Bins: +++ <= 2.0 < ++ <= 5.0
< + PercentActivity Bins: + <= 25.0 < ++ <= 100.0 <
+++ Cmpd. No. Binned IC50 Binned MaxEfficacy 1 +++ +++
* * * * *
References