U.S. patent application number 14/069571 was filed with the patent office on 2014-06-12 for pharmaceutical compositions for the treatment of cftr mediated diseases.
This patent application is currently assigned to Vertex Pharmaceuticals Incorporated. The applicant listed for this patent is Vertex Pharmaceuticals Incorporated. Invention is credited to Radhika Karkare, Michael Douglas Moore, Marinus Jacobus Verwijs.
Application Number | 20140163068 14/069571 |
Document ID | / |
Family ID | 50628072 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140163068 |
Kind Code |
A1 |
Verwijs; Marinus Jacobus ;
et al. |
June 12, 2014 |
Pharmaceutical Compositions for the Treatment of CFTR Mediated
Diseases
Abstract
Pharmaceutical compositions comprising
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid (Compound 1) in Form I and a solid
dispersion comprising substantially amorphous
N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide
(Compound 2), methods of treating, lessening the severity of, or
symptomatically treating CFTR mediated diseases, such as cystic
fibrosis, methods of manufacturing, methods of administering, and
kits thereof are disclosed.
Inventors: |
Verwijs; Marinus Jacobus;
(Framingham, MA) ; Karkare; Radhika; (Medford,
MA) ; Moore; Michael Douglas; (Charlestown,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vertex Pharmaceuticals Incorporated |
Cambridge |
MA |
US |
|
|
Assignee: |
Vertex Pharmaceuticals
Incorporated
Cambridge
MA
|
Family ID: |
50628072 |
Appl. No.: |
14/069571 |
Filed: |
November 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61721622 |
Nov 2, 2012 |
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61728328 |
Nov 20, 2012 |
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61770668 |
Feb 28, 2013 |
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61824005 |
May 16, 2013 |
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61840668 |
Jun 28, 2013 |
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Current U.S.
Class: |
514/312 ;
206/570; 264/145 |
Current CPC
Class: |
A61P 1/16 20180101; A61P
1/18 20180101; A61P 43/00 20180101; A61K 31/47 20130101; A61K
9/2054 20130101; A61K 9/2013 20130101; A61K 9/2027 20130101; A61K
9/10 20130101; A61K 9/14 20130101; A61K 9/2095 20130101; A61K
9/0053 20130101; A61P 1/00 20180101; A61P 11/00 20180101; A61K 9/20
20130101; A61P 3/12 20180101; A61K 31/443 20130101; A61K 9/2077
20130101; A61K 31/47 20130101; A61K 2300/00 20130101; A61K 31/443
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/312 ;
264/145; 206/570 |
International
Class: |
A61K 31/47 20060101
A61K031/47; A61K 31/443 20060101 A61K031/443 |
Claims
1. A pharmaceutical composition comprising a fixed dosage amount of
3-(6-(1-(2,2-difluorobenzo[d][1,3]
dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic
acid (Compound 1) Form I and a solid dispersion comprising
substantially amorphous
N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carb-
oxamide (Compound 2).
2. The pharmaceutical composition of claim 1 further comprising: a.
a filler; b. a disintegrant; c. a surfactant; and d. a binder;
referred to as PC-I.
3. The pharmaceutical composition of claim 1, comprising 30 to 55
percent by weight Compound 1 Form I, and 10 to 45 percent by weight
solid dispersion comprising substantially amorphous Compound 2.
4. The pharmaceutical composition of claim 2 having the following
formulation: TABLE-US-00033 % by wgt. Compound 1 Form I 35-50 Solid
dispersion comprising 25-40 substantially amorphous Compound 2
Microcrystalline cellulose 10-20 Croscarmellose sodium 1-3 Sodium
lauryl sulfate 0.5-2 Polyvinylpyrrolidone 0-5
referred to as PC-II.
5. A pharmaceutical composition comprising: a. Compound 1 Form I;
b. a solid dispersion comprising substantially amorphous Compound
2; c. a filler; d. a disintegrant; e. a surfactant; f. a binder;
and g. a lubricant; referred to as PC-III.
6. The pharmaceutical composition of claim 5, comprising about 100
to 250 mg of Compound 1 Form I, and about 80 to 150 mg of
substantially amorphous Compound 2.
7. The pharmaceutical composition of claim 5, comprising about 200
mg of Compound 1 Form I, and about 125 mg of substantially
amorphous Compound 2.
8. The pharmaceutical composition of claim 5, comprising about 200
mg of Compound 1 Form I, and about 83 mg of substantially amorphous
Compound 2.
9. The pharmaceutical composition of claim 5, comprising about 150
mg of Compound 1 Form I, and about 125 mg of substantially
amorphous Compound 2.
10. The pharmaceutical composition of claim 5, comprising 25 to 50
percent by weight Compound 1 Form I, and 15 to 35 percent by weight
a solid dispersion comprising substantially amorphous Compound
2.
11. The pharmaceutical composition of claim 5 having the following
formulation: TABLE-US-00034 % by wgt. Compound 1 Form I 25-50 A
solid dispersion comprising 15-35 substantially amorphous Compound
2 Microcrystalline cellulose 20-30 Croscarmellose sodium 3-10
Sodium lauryl sulfate 0.5-2 Polyvinylpyrrolidone 0-5 Magnesium
stearate 0.5-2
referred to as PC-IV.
12. The pharmaceutical composition of claim 5, further comprising a
colorant and a wax.
13. The pharmaceutical composition of claim 2, wherein the
pharmaceutical composition is a solid oral pharmaceutical
composition.
14. The pharmaceutical composition claim 13, wherein the solid oral
pharmaceutical composition is a granule.
15. The granule of claim 14 having the following formulation:
TABLE-US-00035 % by wgt. Compound 1 Form I 43 Solid dispersion
comprising 34 substantially amorphous Compound 2 Microcrystalline
cellulose 17 Croscarmellose sodium 2 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3
referred to as PC-V.
16. The granule of claim 14 having the following formulation:
TABLE-US-00036 % by wgt. Compound 1 Form I 38 Solid dispersion
comprising 40 substantially amorphous Compound 2 Microcrystalline
cellulose 16 Croscarmellose sodium 2 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3
referred to as PC-VI.
17. The granule of claim 14 having the following formulation:
TABLE-US-00037 % by wgt. Compound 1 Form I 51 Solid dispersion
comprising 27 substantially amorphous Compound 2 Microcrystalline
cellulose 16 Croscarmellose sodium 2 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3
referred to as PC-VII.
18. The pharmaceutical composition of claim 5, wherein the
pharmaceutical composition is a solid oral pharmaceutical
composition.
19. The pharmaceutical composition claim 18, wherein the solid oral
pharmaceutical composition is a tablet.
20. The tablet claim 19 having the following formulation:
TABLE-US-00038 % by wgt. Compound 1 Form I 35 Solid dispersion
comprising 28 substantially amorphous Compound 2 Microcrystalline
cellulose 26 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1
referred to as PC-VIII.
21. The tablet of claim 19 having the following formulation:
TABLE-US-00039 % by wgt. Compound 1 Form I 31 Solid dispersion
comprising 32 substantially amorphous Compound 2 Microcrystalline
cellulose 26 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1
referred to as PC-IX.
22. The tablet of claim 19 having the following formulation:
TABLE-US-00040 % by wgt. Compound 1 Form I 41 Solid dispersion
comprising 22 substantially amorphous Compound 2 Microcrystalline
cellulose 26 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1
referred to as PC-X.
23. The tablet of claim 19 having the following formulation:
TABLE-US-00041 mg Compound 1 Form I 200 Solid dispersion comprising
156 substantially amorphous Compound 2 Microcrystalline cellulose
150 Croscarmellose sodium 34 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 15 Magnesium stearate 6
referred to as PC-XI.
24. The tablet of claim 19 having the following formulation:
TABLE-US-00042 mg Compound 1 Form I 150 Solid dispersion comprising
156 substantially amorphous Compound 2 Microcrystalline cellulose
129 Croscarmellose sodium 30 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5
referred to as PC-XII.
25. The tablet of claim 19 having the following formulation:
TABLE-US-00043 mg Compound 1 Form I 200 Solid dispersion comprising
104 substantially amorphous Compound 2 Microcrystalline cellulose
128 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5
referred to as PC-XIII.
26. The tablet of claim 19 having the following formulation:
TABLE-US-00044 % by wgt. Compound 1 Form I 34 Solid dispersion
comprising 27 substantially amorphous Compound 2 Microcrystalline
cellulose 25 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1 Colorant 3
referred to as PC-XIV.
27. The tablet of claim 19 having the following formulation:
TABLE-US-00045 % by wgt. Compound 1 Form I 30 Solid dispersion
comprising 31 substantially amorphous Compound 2 Microcrystalline
cellulose 25 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1 Colorant 3
referred to as PC-XV.
28. The tablet of claim 19 having the following formulation:
TABLE-US-00046 % by wgt. Compound 1 Form I 40 Solid dispersion
comprising 21 substantially amorphous Compound 2 Microcrystalline
cellulose 25 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1 Colorant 3
referred to as PC-XVI.
29. The tablet claim 19 having the following formulation:
TABLE-US-00047 mg Compound 1 Form I 200 Solid dispersion comprising
156 substantially amorphous Compound 2 Micro crystalline cellulose
150 Croscarmellose sodium 34 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 15 Magnesium stearate 6 Colorant 17
referred to as PC-XVII.
30. The tablet of claim 19 having the following formulation:
TABLE-US-00048 mg Compound 1 Form I 200 Substantially amorphous 125
Compound 2 Microcrystalline cellulose 150 Croscarmellose sodium 34
Sodium lauryl sulfate 4 Polyvinylpyrrolidone 15 Magnesium stearate
6 Colorant 17
referred to as PC-XVIII.
31. The tablet of claim 19 having the following formulation:
TABLE-US-00049 mg Compound 1 Form I 150 Solid dispersion comprising
156 substantially amorphous Compound 2 Microcrystalline cellulose
129 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5 Colorant 15
referred to as PC-XIX.
32. The tablet of claim 19 having the following formulation:
TABLE-US-00050 mg Compound 1 Form I 200 Solid dispersion comprising
104 substantially amorphous Compound 2 Microcrystalline cellulose
128 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5 Colorant 14
referred to as PC-XX.
33. The tablet of claim 19 having the following formulation:
TABLE-US-00051 mg Compound 1 Form I 200 Solid dispersion comprising
83 substantially amorphous Compound 2 Microcrystalline cellulose
128 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5 Colorant 14
referred to as PC-XXI.
34. The tablet of claim 19 having the following formulation:
TABLE-US-00052 Component % by wgt. Compound 1 Form I 20-40 Solid
dispersion comprising 30-40 substantially amorphous Compound 2
Microcrystalline cellulose 20-30 Croscarmellose sodium 1-10
Polyvinylpyrrolidone 1-5 Sodium lauryl sulfate 0.1-1 Magnesium
stearate 0.5-1.5
referred to as PC-XXII.
35. The tablet of claim 19 having the following formulation:
TABLE-US-00053 Component mg/Tablet Compound 1 Form I 100 Solid
dispersion comprising 156 substantially amorphous Compound 2
Microcrystalline cellulose 55 Croscarmellose sodium 7
Polyvinylpyrrolidone 11 Sodium lauryl sulfate 3 Total Granules 332
Croscarmellose sodium 18 Microcrystalline cellulose 53 Magnesium
stearate 4 Total Tablet 407
referred to as PC-XXIII.
36. The tablet of claim 19 having the following formulation:
TABLE-US-00054 Component mg/Tablet Compound 1 Form I 150 Solid
dispersion comprising 156 substantially amorphous Compound 2
Microcrystalline cellulose 65 Croscarmellose sodium 8
Polyvinylpyrrolidone 13 Sodium lauryl sulfate 4 Total Granules 396
Croscarmellose sodium 22 Microcrystalline cellulose 64 Magnesium
stearate 5 Total Tablet 487
referred to as PC-XXIV.
37. The tablet of claim 19 having the following formulation:
TABLE-US-00055 Component mg/Tablet Compound 1 Form I 75 Solid
dispersion 156 comprising substantially amorphous Compound 2
Microcrystalline 49 cellulose Croscarmellose 6 sodium
Polyvinylpyrrolidone 10 Sodium lauryl sulfate 3 Total Granules 299
Croscarmellose 17 sodium Microcrystalline 48 cellulose Magnesium
stearate 4 Core Tablet 368 Pink Opadry 11 Total Tablet 379
referred to as PC-XXV.
38. A method of treating, lessening the severity of, or
symptomatically treating cystic fibrosis in a patient comprising
administering to the patient an effective amount of the
pharmaceutical composition of claim 1.
39. The method of claim 38, wherein the pharmaceutical composition
has the formulation of any one of PC-I to PC-XXV.
40. The method of claim 38, wherein the patient has a .DELTA.F508
CFTR mutation.
41. The method of claim 40, wherein the patient is homozygous in
.DELTA.F508.
42. The method of claim 40, wherein the patient is heterozygous in
.DELTA.F508.
43. A method of preparing a granule comprising wet granulating the
following components: a. Compound 1 Form I; b. a solid dispersion
comprising substantially amorphous Compound 2; c. a filler; d. a
disintegrant; e. a surfactant; and f. a binder.
44. A method of preparing a tablet comprising compressing: i) a
plurality of granular pharmaceutical compositions comprising the
following components: a. Compound 1 Form I; b. a solid dispersion
comprising substantially amorphous Compound 2; c. a filler; d. a
disintegrant; e. a surfactant; and f. a binder; ii) a disintegrant;
iii) a filler; and iv) a lubricant.
45. A continuous process for preparing a tablet comprising Compound
1 Form I and a solid dispersion comprising substantially amorphous
Compound 2 comprising the steps of: a) mixing Compound 1 Form I, a
solid dispersion comprising substantially amorphous Compound 2, a
filler, and a disintegrant in a blender to form a blend; b)
preparing a granulation solution with water, a binder, and a
surfactant; c) feeding the blend from step a) into a continuous
twin screw granulator while adding the granulation solution from
step b) to produce granules; d) drying the granules from step c)
and milling them; e) blending the milled granules from step d) with
a filler, disintegrant, and lubricant to form a blend; and f)
compressing the blend from step e) into a tablet.
46. A kit comprising the pharmaceutical composition of claim 1 and
a separate therapeutic agent.
47. The kit of claim 46, wherein the pharmaceutical composition and
the therapeutic agent are in separate containers.
48. The kit of claim 47, wherein the containers are bottles, vials,
or blister packs, or combination thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/721,622, filed Nov. 2, 2012; 61/728,328,
filed Nov. 20, 2012; 61/770,668, filed Feb. 28, 2013; 61/824,005,
filed May 16, 2013; and 61/840,668, filed Jun. 28, 2013, the entire
contents of all applications are incorporated herein by reference
in their entirety.
TECHNICAL FIELD OF INVENTION
[0002] The invention relates to pharmaceutical compositions
comprising 3-(6-(1-(2,2-difluorobenzo[d][1,3]
dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic
acid (Compound 1) Form I and a solid dispersion comprising
substantially amorphous
N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carb-
oxamide (Compound 2), methods of treatment, methods of
manufacturing, methods of administering, and kits thereof.
BACKGROUND
[0003] Cystic fibrosis (CF) is a recessive genetic disease that
affects approximately 30,000 children and adults in the United
States and approximately 30,000 children and adults in Europe.
Despite progress in the treatment of CF, there is no cure.
[0004] In patients with CF, mutations in CFTR endogenously
expressed in respiratory epithelia leads to reduced apical anion
secretion causing an imbalance in ion and fluid transport. The
resulting decrease in anion transport contributes to enhanced mucus
accumulation in the lung and the accompanying microbial infections
that ultimately cause death in CF patients. In addition to
respiratory disease, CF patients typically suffer from
gastrointestinal problems and pancreatic insufficiency that, if
left untreated, results in death. In addition, the majority of
males with cystic fibrosis are infertile and fertility is decreased
among females with cystic fibrosis. In contrast to the severe
effects of two copies of the CF associated gene, individuals with a
single copy of the CF associated gene exhibit increased resistance
to cholera and to dehydration resulting from diarrhea--perhaps
explaining the relatively high frequency of the CF gene within the
population.
[0005] 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 (http://www.genet.sickkids.on.ca/cftr/app).
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% of the cases of cystic fibrosis and is associated
with a severe disease.
[0006] 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.
[0007] Compound 1 in salt form is disclosed in International PCT
Publication WO2007056341 and U.S. Pat. No. 7,741,321 as an inducer
of CFTR activity and thus as a useful treatment for CFTR-mediated
diseases such as cystic fibrosis. Compound 1 Form I, which is a
substantially crystalline and salt-free form, is disclosed in
International PCT Publication WO2009073757 and U.S. Pat. No.
8,507,534. Compound 2 is disclosed in International PCT Publication
WO2006002421 and U.S. Pat. No. 7,495,103 as an inducer of CFTR
activity and thus as useful treatment for CFTR-mediated diseases
such as cystic fibrosis. A solid dispersion comprising
substantially amorphous Compound 2 is disclosed in International
PCT Publication WO2010019239 and United States Published Patent
Application No. US20100074949. All above applications and patents
are incorporated in their entirety by reference herein.
[0008] Compounds which are CFTR potentiators, such as Compound 2,
and compounds which are CFTR correctors, such as Compound 1, have
been shown independently to have utility in the treatment of CFTR
related diseases, such as cystic fibrosis.
[0009] Accordingly, there is a need for novel treatments of CFTR
mediated diseases which involve CFTR corrector and potentiator
compounds.
[0010] Particularly, there is a need for combination therapies to
treat CFTR mediated diseases, such as cystic fibrosis, which
include CFTR potentiator and corrector compounds.
[0011] More particularly, there is a need for combination therapies
to treat CFTR mediated diseases, such as cystic fibrosis, which
include CFTR potentiator compounds, such as substantially amorphous
Compound 2, in combination with CFTR corrector compounds, such as
Compound 1 Form I.
[0012] Compound 1 as part of a combination with Compound 2 has been
granted a Breakthrough Therapy Designation from the Food and Drug
Administration (FDA) for the treatment of cystic fibrosis, one of
only two such grants at the time of the filing of this application
(the other being for Compound 2). This demonstrates a significant
unmet need for the effective treatment of the cause of cystic
fibrosis over symptomatic treatments. Additionally, a common
challenge for drugs approved by the FDA is the occasional lack of
drug availability for patients in need thereof. Accordingly, a
significant unmet need exists for the presently disclosed Compound
1 and Compound 2 formulations and processes for preparing them in a
continuous and controlled manner.
[0013] Additionally, patient compliance with treatment schedules
and dosage amounts is largely dependent on ease of drug
administration. A pharmaceutical composition comprising fixed
dosage amounts of a CFTR corrector and CFTR potentiator, wherein
the solid forms of said corrector and potentiator are stable, is a
significant breakthrough for the treatment of CFTR mediated
diseases such as cystic fibrosis.
SUMMARY
[0014] The invention features a pharmaceutical compositions
comprising 3-(6-(1-(2,2-difluorobenzo[d][1,3]
dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic
acid, Compound 1 Form I, which has the structure below:
##STR00001##
and a solid dispersion of substantially amorphous
N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide,
Compound 2, which has the structure below:
##STR00002##
methods of treatment, methods of manufacturing, methods of
administering, and kits thereof.
[0015] In one aspect, the present invention features a
pharmaceutical composition comprising:
[0016] a. Compound 1 Form I;
[0017] b. a solid dispersion comprising substantially amorphous
Compound 2;
[0018] c. a filler;
[0019] d. a disintegrant;
[0020] e. a surfactant; and
[0021] f. a binder;
referred to as PC-I.
[0022] In one embodiment, the pharmaceutical compositions of the
present invention comprise 30 to 55 percent by weight Compound 1
Form I, and 10 to 45 percent by weight solid dispersion comprising
substantially amorphous Compound 2.
[0023] In one embodiment, the filler is selected from cellulose,
modified cellulose, sodium carboxymethyl cellulose, ethyl cellulose
hydroxymethyl cellulose, hydroxypropylcellulose, cellulose acetate,
microcrystalline cellulose, dibasic calcium phosphate, sucrose,
lactose, corn starch, potato starch, or any combination thereof. In
another embodiment, the filler is microcrystalline cellulose, and
is present in an amount ranging from 10 to 20 percent by
weight.
[0024] In one embodiment, the disintegrant is selected from
agar-agar, algins, calcium carbonate, carboxmethylcellulose,
cellulose, hydroxypropylcellulose, low substituted
hydroxypropylcellulose, clays, croscarmellose sodium, crospovidone,
gums, magnesium aluminum silicate, methylcellulose, polacrilin
potassium, sodium alginate, sodium starch glycolate, maize starch,
potato starch, tapioca starch, or any combination thereof. In
another embodiment, the disintegrant is croscarmellose sodium, and
is present in an amount ranging from 1 to 3 percent by weight.
[0025] In one embodiment, the surfactant is selected from sodium
lauryl sulfate, sodium stearyl fumerate, polyoxyethylene 20
sorbitan mono-oleate, or any combination thereof. In another
embodiment, the surfactant is sodium lauryl sulfate, and is present
in an amount ranging from 0.5 to 2 percent by weight.
[0026] In one embodiment, the binder is selected from
polyvinylpyrrolidone, dibasic calcium phosphate, sucrose, corn
starch, modified cellulose, or any combination thereof. In another
embodiment, the binder is polyvinylpyrrolidone, and is present in
an amount ranging from 0 to 5 percent by weight.
[0027] In one embodiment, the present invention features a
pharmaceutical composition having the following formulation:
TABLE-US-00001 % by wgt. Compound 1 Form I 35-50 Solid dispersion
comprising 25-40 substantially amorphous Compound 2
Microcrystalline cellulose 10-20 Croscarmellose sodium 1-3 Sodium
lauryl sulfate 0.5-2 Polyvinylpyrrolidone 0-5
referred to as PC-II.
[0028] In another aspect, the present invention features a
pharmaceutical composition comprising:
[0029] a. Compound 1 Form I;
[0030] b. a solid dispersion comprising substantially amorphous
Compound 2;
[0031] c. a filler;
[0032] d. a disintegrant;
[0033] e. a surfactant;
[0034] f. a binder; and
[0035] g. a lubricant;
referred to as PC-III.
[0036] In one embodiment, the pharmaceutical compositions of
present invention comprise about 100 to 250 mg of Compound 1 Form
I, and about 100 to 150 mg of substantially amorphous Compound 2.
In another embodiment, the pharmaceutical compositions of the
present invention comprise about 200 mg of Compound 1 Form I, and
about 125 mg of substantially amorphous Compound 2. In another
embodiment, the pharmaceutical compositions of the present
invention comprise about 150 mg of Compound 1 Form I, and about 125
mg of substantially amorphous Compound 2.
[0037] In one embodiment, the pharmaceutical compositions of the
present invention comprise 25 to 50 percent by weight Compound 1
Form I, and 15 to 35 percent by weight a solid dispersion
comprising substantially amorphous Compound 2.
[0038] In one embodiment, the filler is selected from cellulose,
modified cellulose, sodium carboxymethyl cellulose, ethyl cellulose
hydroxymethyl cellulose, hydroxypropylcellulose, cellulose acetate,
microcrystalline cellulose, dibasic calcium phosphate, sucrose,
lactose, corn starch, potato starch, or any combination thereof. In
another embodiment, the filler is microcrystalline cellulose, and
is present in an amount ranging from 20 to 30 percent by
weight.
[0039] In one embodiment, the disintegrant is selected from
agar-agar, algins, calcium carbonate, carboxmethylcellulose,
cellulose, hydroxypropylcellulose, low substituted
hydroxypropylcellulose, clays, croscarmellose sodium, crospovidone,
gums, magnesium aluminum silicate, methylcellulose, polacrilin
potassium, sodium alginate, sodium starch glycolate, maize starch,
potato starch, tapioca starch, or any combination thereof. In
another embodiment, the disintegrant is croscarmellose sodium, and
is present in an amount ranging from 3 to 10 percent by weight.
[0040] In one embodiment, the surfactant is selected from sodium
lauryl sulfate, sodium stearyl fumerate, polyoxyethylene 20
sorbitan mono-oleate, or any combination thereof. In another
embodiment, the surfactant is sodium lauryl sulfate, and is present
in an amount ranging from 0.5 to 2 percent by weight.
[0041] In one embodiment, the binder is selected from
polyvinylpyrrolidone, dibasic calcium phosphate, sucrose, corn
starch, modified cellulose, or any combination thereof. In another
embodiment, the binder is polyvinylpyrrolidone, and is present in
an amount ranging from 0 to 5 percent by weight.
[0042] In one embodiment, the lubricant is selected from magnesium
stearate, calcium stearate, zinc stearate, sodium stearate, stearic
acid, aluminum stearate, leucine, glyceryl behenate, hydrogenated
vegetable oil or any combination thereof. In another embodiment,
the lubricant is magnesium stearate, and is present in an amount
ranging from 0.5 to 2 percent by weight.
[0043] In one embodiment, the present invention features a
pharmaceutical composition having the following formulation:
TABLE-US-00002 % by wgt. Compound 1 Form I 25-50 A solid dispersion
comprising 15-35 substantially amorphous Compound 2
Microcrystalline cellulose 20-30 Croscarmellose sodium 3-10 Sodium
lauryl sulfate 0.5-2 Polyvinylpyrrolidone 0-5 Magnesium stearate
0.5-2
referred to as PC-IV.
[0044] In one embodiment, the pharmaceutical compositions of the
present invention further comprise a colorant and optionally a wax.
In another embodiment, the colorant is present in an amount ranging
from 2 to 4 percent by weight. In another embodiment, the wax is
carnauba wax present in an amount ranging from 0 to 0.020 percent
by weight.
[0045] In one embodiment, the pharmaceutical compositions of the
present invention are solid oral pharmaceutical compositions. In
another embodiment, the solid oral pharmaceutical compositions are
a granular pharmaceutical composition or tablet.
[0046] In one embodiment, the granular pharmaceutical compositions
of the present invention have the following formulation:
TABLE-US-00003 % by wgt. Compound 1 Form I 43 Solid dispersion
comprising 34 substantially amorphous Compound 2 Microcrystalline
cellulose 17 Croscarmellose sodium 2 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3
referred to as PC-V.
[0047] In one embodiment, the granular pharmaceutical compositions
of the present invention have the following formulation:
TABLE-US-00004 % by wgt. Compound 1 Form I 38 Solid dispersion
comprising 40 substantially amorphous Compound 2 Microcrystalline
cellulose 16 Croscarmellose sodium 2 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3
referred to as PC-VI.
[0048] In one embodiment, the granular pharmaceutical compositions
of the present invention have the following formulation:
TABLE-US-00005 % by wgt. Compound 1 Form I 51 Solid dispersion
comprising 27 substantially amorphous Compound 2 Microcrystalline
cellulose 16 Croscarmellose sodium 2 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3
referred to as PC-VH.
[0049] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00006 % by wgt. Compound 1 Form I 35 Solid dispersion
comprising 28 substantially amorphous Compound 2 Microcrystalline
cellulose 26 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1
referred to as PC-VIII.
[0050] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00007 % by wgt. Compound 1 Form I 31 Solid dispersion
comprising 32 substantially amorphous Compound 2 Microcrystalline
cellulose 26 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1
referred to as PC-IX.
[0051] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00008 % by wgt. Compound 1 Form I 41 Solid dispersion
comprising 22 substantially amorphous Compound 2 Microcrystalline
cellulose 26 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1
referred to as PC-X.
[0052] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00009 mg Compound 1 Form I 200 Solid dispersion comprising
156 substantially amorphous Compound 2 Microcrystalline cellulose
150 Croscarmellose sodium 34 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 15 Magnesium stearate 6
referred to as PC-XI.
[0053] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00010 mg Compound 1 Form I 150 Solid dispersion comprising
156 substantially amorphous Compound 2 Microcrystalline cellulose
129 Croscarmellose sodium 30 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5
referred to as PC-XII.
[0054] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00011 mg Compound 1 Form I 200 Solid dispersion comprising
104 substantially amorphous Compound 2 Microcrystalline cellulose
128 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5
referred to as PC-XIII.
[0055] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00012 % by wgt. Compound 1 Form I 34 Solid dispersion
comprising 27 substantially amorphous Compound 2 Microcrystalline
cellulose 25 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1 Colorant 3
referred to as PC-XIV.
[0056] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00013 % by wgt. Compound 1 Form I 30 Solid dispersion
comprising 31 substantially amorphous Compound 2 Microcrystalline
cellulose 25 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1 Colorant 3
referred to as PC-XV.
[0057] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00014 % by wgt. Compound 1 Form I 40 Solid dispersion
comprising 21 substantially amorphous Compound 2 Microcrystalline
cellulose 25 Croscarmellose sodium 6 Sodium lauryl sulfate 1
Polyvinylpyrrolidone 3 Magnesium stearate 1 Colorant 3
referred to as PC-XVI.
[0058] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00015 mg Compound 1 Form I 200 Solid dispersion comprising
156 substantially amorphous Compound 2 Micro crystalline cellulose
150 Croscarmellose sodium 34 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 15 Magnesium stearate 6 Colorant 17
referred to as PC-XVII.
[0059] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00016 mg Compound 1 Form I 200 Substantially amorphous 125
Compound 2 Microcrystalline cellulose 150 Croscarmellose sodium 34
Sodium lauryl sulfate 4 Polyvinylpyrrolidone 15 Magnesium stearate
6 Colorant 17
referred to as PC-XVIII
[0060] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00017 mg Compound 1 Form I 150 Solid dispersion comprising
156 substantially amorphous Compound 2 Microcrystalline cellulose
129 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5 Colorant 15
referred to as PC-XIX.
[0061] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00018 mg Compound 1 Form I 200 Solid dispersion comprising
104 substantially amorphous Compound 2 Microcrystalline cellulose
128 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5 Colorant 14
referred to as PC-XX.
[0062] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00019 mg Compound 1 Form I 200 Solid dispersion comprising
83 substantially amorphous Compound 2 Microcrystalline cellulose
128 Croscarmellose sodium 29 Sodium lauryl sulfate 4
Polyvinylpyrrolidone 13 Magnesium stearate 5 Colorant 14
referred to as PC-XXI.
[0063] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00020 Component % by wgt. Compound 1 Form I 20-40 Solid
dispersion comprising 30-40 substantially amorphous Compound 2
Microcrystalline cellulose 20-30 Croscarmellose sodium 1-10
Polyvinylpyrrolidone 1-5 Sodium lauryl sulfate 0.1-1 Magnesium
stearate 0.5-1.5
referred to as PC-XXII.
[0064] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00021 Compound 1/Compound 2 100 mg/125 mg Component % in
Granule % in Tablet mg/Tablet Compound 1 Form I 30 25 100 Solid
dispersion 47 38 156 comprising substantially amorphous Compound 2
Microcrystalline 17 13 55 cellulose Croscarmellose 2 2 7 sodium
Polyvinylpyrrolidone 3 3 11 Sodium lauryl sulfate 1 1 3 Total
Granules 100 82 332 Croscarmellose 4 18 sodium Microcrystalline 13
53 cellulose Magnesium stearate 1 4 Total Tablet 100 407
referred to as PC-XXIII.
[0065] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00022 Compound 1/Compound 2 150 mg/125 mg Component % in
Granule % in Tablet mg/Tablet Compound 1 Form I 38 31 150 Solid
dispersion 40 32 156 comprising substantially amorphous Compound 2
Microcrystalline 16 13 65 cellulose Croscarmellose 2 2 8 sodium
Polyvinylpyrrolidone 3 3 13 Sodium lauryl sulfate 1 1 4 Total
Granules 100 82 396 Croscarmellose 4 22 sodium Microcrystalline 13
64 cellulose Magnesium stearate 1 5 Total Tablet 100 487
referred to as PC-XXIV.
[0066] In one embodiment, the tablets of the present invention have
the following formulation:
TABLE-US-00023 Compound 1/Compound 2 75 mg/125 mg Component % in
Granule % in Tablet mg/Tablet Compound 1 Form I 25 20 75 Solid
dispersion 52 43 156 comprising substantially amorphous Compound 2
Microcrystalline 17 13 49 cellulose Croscarmellose 2 2 6 sodium
Polyvinylpyrrolidone 3 3 10 Sodium lauryl sulfate 1 1 3 Total
Granules 100 82 299 Croscarmellose 4 17 sodium Microcrystalline 13
48 cellulose Magnesium stearate 1 4 Core Tablet 100 368 Pink Opadry
3 11 Film Coated Tablet 379
Referred to as PC-XXV.
[0067] In one aspect, the present invention features a method of
treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering to the
patient an effective amount of the pharmaceutical composition,
granular pharmaceutical composition, or tablet of the present
invention.
[0068] In embodiment, the present invention features a method of
treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering to the
patient an effective amount of the pharmaceutical composition,
granular pharmaceutical composition, or tablet of any one of
formulations PC-I through PC-XXV.
[0069] In one embodiment, the patient has a .DELTA.F508 CFTR
mutation. In another embodiment, the patient is homozygous in
.DELTA.F508. In another embodiment, the patient is heterozygous in
.DELTA.F508. In another embodiment, two tablets are administered to
the patient per day.
[0070] In one aspect, the present invention features a method of
preparing a granular pharmaceutical composition comprising wet
granulating the following components:
[0071] a. Compound 1 Form I;
[0072] b. a solid dispersion comprising substantially amorphous
Compound 2;
[0073] c. a filler;
[0074] d. a disintegrant;
[0075] e. a surfactant; and
[0076] f. a binder.
[0077] In one aspect the present invention features a method of
preparing a tablet comprising compressing:
[0078] i) a plurality of granular pharmaceutical compositions
comprising the following components: [0079] a. Compound 1 Form I;
[0080] b. a solid dispersion comprising substantially amorphous
Compound 2; [0081] c. a filler; [0082] d. a disintegrant; [0083] e.
a surfactant; and [0084] f. a binder;
[0085] ii) a disintegrant;
[0086] iii) a filler; and
[0087] iv) a lubricant.
[0088] In one aspect, the present invention features a kit
comprising pharmaceutical compositions, granular pharmaceutical
compositions, or tablets of the present invention, and a separate
therapeutic agent or pharmaceutical composition thereof.
[0089] In one embodiment, the pharmaceutical compositions, granular
pharmaceutical compositions, or tablets of the present invention,
and the separate therapeutic agent or pharmaceutical composition
thereof are in separate containers. In another embodiment, the
separate containers are bottles. In another embodiment, the
separate containers are vials. In another embodiment, the separate
containers are blister packs.
[0090] In another aspect, the invention provides a continuous or
semi-continuous process for making the pharmaceutical compositions
described herein by a twin screw wet granulation process comprising
the steps of screening and weighing Compound 1, Compound 2, and
excipients; mixing Compound 1, Compound 2, and excipients in a
blender and feeding the blend into a continuous granulator while
adding a granulation fluid comprising surfactant and a binder at a
suitable rate for a suitable amount of time and chopping the
mixture into granules; drying the granules; blending the granules
with extra-granular excipients for a suitable amount of time;
compressing the blend into tablets; coating the tablets; and,
optionally, printing a monogram on one or both tablet faces.
BRIEF DESCRIPTION OF DRAWINGS
[0091] FIG. 1 is an X-ray diffraction pattern calculated from a
single crystal structure of Compound 1 Form I.
[0092] FIG. 2 is an actual X-ray powder diffraction pattern of
Compound 1 Form I.
[0093] FIG. 3 is a graph depicting Compound 1 pH gradient
dissolution profiles for a tablet made by a high shear granulation
(HSG) process and a twin screw wet granulation (TSWG) process (LOD
stands for loss on drying, a measure to define the amount of water
in a powder/granule).
[0094] FIG. 4 is a graph depicting the stability of the
substantially amorphous form of Compound 2 in tablet formulation
PC-XVII at 50.degree. C. after pre-equilibrating at 60% relative
humidity by showing only a small amount of crystallinity over
time.
[0095] FIG. 5 is a graph depicting the stability of the
substantially amorphous form of Compound 2 in tablet formulation
PC-XVII at 60.degree. C. after pre-equilibrating at 60% relative
humidity by showing only a small amount of crystallinity over
time.
[0096] FIG. 6 is a graph depicting the stability of the
substantially amorphous form of Compound 2 in tablet formulation
PC-XX at 60.degree. C. after pre-equilibrating at 60% relative
humidity by showing only a small amount of crystallinity over
time.
[0097] FIG. 7 is a graph depicting the stability of the
substantially amorphous form of Compound 2 in tablet formulation
PC-XX at 50.degree. C. after pre-equilibrating at 60% relative
humidity by showing only a small amount of crystallinity over
time.
[0098] FIG. 8 is an .sup.1HNMR spectrum of Compound 1.
[0099] FIG. 9 is an .sup.1HNMR spectrum of Compound 1 HCl salt.
[0100] FIG. 10 is a differential scanning calorimetry (DSC) trace
of Compound 1 Form I.
[0101] FIG. 11 is a conformational picture of Compound 1 Form I
based on single crystal X-ray analysis.
[0102] FIG. 12 is a flow chart describing the preparation of a
tablet of Compound 1 and Compound 2 by continuous methods.
DETAILED DESCRIPTION
Definitions
[0103] As used herein, "CFTR" stands for cystic fibrosis
transmembrane conductance regulator.
[0104] As used herein, a ".DELTA.F508 mutation" or "F508-del
mutation" is a specific mutation within the CFTR protein. The
mutation is a deletion of the three nucleotides that comprise the
codon for amino acid phenylalanine at position 508, resulting in
CFTR protein that lacks this phenylalanine residue.
[0105] As used herein, a patient who is "homozygous" for a
particular mutation, e.g. .DELTA.F508, has the same mutation on
each allele.
[0106] As used herein, a patient who is "heterozygous" for a
particular mutation, e.g. .DELTA.F508, has this mutation on one
allele, and a different mutation on the other allele.
[0107] As used herein, the term "CFTR corrector" refers to a
compound that increases the amount of functional CFTR protein to
the cell surface, resulting in enhanced ion transport.
[0108] As used herein, the term "CFTR potentiator" refers to a
compound that increases the channel activity of CFTR protein
located at the cell surface, resulting in enhanced ion
transport.
[0109] As used herein, the term "active pharmaceutical ingredient"
or "API" refers to a biologically active compound.
[0110] The terms "solid form", "solid forms" and related terms,
when used herein refer to Compound 1 or Compound 2, in a particular
solid form e.g. crystals, amorphous states, and the like.
[0111] As used herein, the term "substantially amorphous" refers to
a solid material having little or no long range order in the
position of its molecules. For example, substantially amorphous
materials have less than about 15% crystallinity (e.g., less than
about 10% crystallinity or less than about 5% crystallinity). It is
also noted that the term `substantially amorphous` includes the
descriptor, `amorphous`, which refers to materials having no (0%)
crystallinity.
[0112] As used herein, the term "substantially crystalline" (as in
the phrase substantially crystalline Compound 1 Form I refers to a
solid material having predominantly long range order in the
position of its molecules. For example, substantially crystalline
materials have more than about 85% crystallinity (e.g., more than
about 90% crystallinity or more than about 95% crystallinity). It
is also noted that the term `substantially crystalline` includes
the descriptor, `crystalline`, which refers to materials having
100% crystallinity.
[0113] The term "crystalline" and related terms used herein, when
used to describe a substance, component, product, or form, means
that the substance, component or product is substantially
crystalline as determined by X-ray diffraction. (See, e.g.,
Remington: The Science and Practice of Pharmacy, 21st Ed.,
Lippincott Williams & Wilkins, Baltimore, Md. (2003); The
United States Pharmacopeia, 23.sup.rd ed., 1843-1844 (1995)).
[0114] As used herein, an "excipient" includes functional and
non-functional ingredients in a pharmaceutical composition.
[0115] As used herein, a "disintegrant" is an excipient that
hydrates a pharmaceutical composition and aids in tablet
dispersion. As used herein, a "diluent" or "filler" is an excipient
that adds bulkiness to a pharmaceutical composition.
[0116] As used herein, a "surfactant" is an excipient that imparts
pharmaceutical compositions with enhanced solubility and/or
wetability.
[0117] As used herein, a "binder" is an excipient that imparts a
pharmaceutical composition with enhanced cohesion or tensile
strength (e.g., hardness).
[0118] As used herein, a "glidant" is an excipient that imparts a
pharmaceutical compositions with enhanced flow properties.
[0119] As used herein, a "colorant" is an excipient that imparts a
pharmaceutical composition, e.g. a tablet, with a desired color.
Examples of colorants include commercially available pigments such
as FD&C Blue #1 Aluminum Lake, FD&C Blue #2, other FD&C
Blue colors, titanium dioxide, iron oxide, and/or combinations
thereof. In one embodiment, the tablet provided by the invention is
pink.
[0120] As used herein, a "lubricant" is an excipient that is added
to pharmaceutical compositions that are pressed into tablets. The
lubricant aids in compaction of granules into tablets and ejection
of a tablet of a pharmaceutical composition from a die press.
[0121] As used herein, "cubic centimeter" and "cc" are used
interchangeably to represent a unit of volume. Note that 1 cc=1
mL.
[0122] As used herein, "kiloPond" and "kP" are used interchangeably
and refer to the measure of force where a kP=approximately 9.8
Newtons.
[0123] As used herein, "friability" refers to the property of a
tablet to remain intact and hold its form despite an external force
of pressure. Friability can be quantified using the mathematical
expression presented in equation 1:
% friabiliy = 100 .times. ( W 0 - W f ) W 0 ( 1 ) ##EQU00001##
wherein W.sub.o is the original weight of the tablet and W.sub.f is
the final weight of the tablet after it is put through the
friabilator. Friability is measured using a standard USP testing
apparatus that tumbles experimental tablets for 100 or 400
revolutions. Some tablets of the invention have a friability of
less than 5.0%. In another embodiment, the friability is less than
2.0%. In another embodiment, the target friability is less than
1.0% after 400 revolutions.
[0124] As used herein, "mean particle diameter" is the average
particle diameter as measured using techniques such as laser light
scattering, image analysis, or sieve analysis. In one embodiment,
the granules used to prepare the pharmaceutical compositions
provided by the invention have a mean particle diameter of less
than 1.0 mm.
[0125] As used herein, "bulk density" is the mass of particles of
material divided by the total volume the particles occupy. The
total volume includes particle volume, inter-particle void volume
and internal pore volume. Bulk density is not an intrinsic property
of a material; it can change depending on how the material is
processed. In one embodiment, the granules used to prepare the
pharmaceutical compositions provided by the invention have a bulk
density of about 0.5-0.7 g/cc.
[0126] An "effective amount" or "therapeutically effective amount"
of a compound of the invention may vary according to factors such
as the disease state, age, and weight of the subject, and the
ability of the compound of the invention to elicit a desired
response in the subject. Dosage regimens may be adjusted to provide
the optimum therapeutic response. An effective amount is also one
in which any toxic or detrimental effects (e.g., side effects) of
the compound of the invention are outweighed by the therapeutically
beneficial effects.
[0127] As used herein, and unless otherwise specified, the terms
"therapeutically effective amount" and "effective amount" of a
compound mean an amount sufficient to provide a therapeutic benefit
in the treatment or management of a disease or disorder, or to
delay or minimize one or more symptoms associated with the disease
or disorder. A "therapeutically effective amount" and "effective
amount" of a compound mean an amount of therapeutic agent, alone or
in combination with one or more other agent(s), which provides a
therapeutic benefit in the treatment or management of the disease
or disorder. The terms "therapeutically effective amount" and
"effective amount" can encompass an amount that improves overall
therapy, reduces or avoids symptoms or causes of disease or
disorder, or enhances the therapeutic efficacy of another
therapeutic agent.
[0128] "Substantially pure" as used in the phrase "substantially
pure Compound 1 Form I" means greater than about 90% purity. In
another embodiment, substantially pure refers to greater than about
95% purity. In another embodiment, substantially pure refers to
greater than about 98% purity. In another embodiment, substantially
pure refers to greater than about 99% purity.
[0129] With respect to Compound 1 Form I, or a solid dispersion
comprising substantially amorphous Compound 2, the terms "about"
and "approximately", when used in connection with doses, amounts,
or weight percent of ingredients of a composition or a dosage form,
mean a dose, amount, or weight percent that is recognized by one of
ordinary skill in the art to provide a pharmacological effect
equivalent to that obtained from the specified dose, amount, or
weight percent. Specifically the term "about" or "approximately"
means an acceptable error for a particular value as determined by
one of ordinary skill in the art, which depends in part on how the
value is measured or determined. In certain embodiments, the term
"about" or "approximately" means within 1, 2, 3, or 4 standard
deviations. In certain embodiments, the term "about" or
"approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or
range.
Pharmaceutical Compositions
[0130] The invention provides pharmaceutical compositions
comprising Compound 1 Form I and a solid dispersion comprising
substantially amorphous Compound 2. In some embodiments of this
aspect, the amount of Compound 1 Form I that is present in the
pharmaceutical composition is 100 mg, 125 mg, 150 mg, 200 mg, 250
mg, 300 mg, or 400 mg. In some embodiments of this aspect, wgt.
percent of Compound 1 Form I present in the pharmaceutical
composition is from 10 to 75 percent. In these and other
embodiments, Compound 1 Form I is present as substantially pure
Compound 1 Form I. In some embodiments of this aspect, the amount
of substantially amorphous Compound 2 that is present in the
pharmaceutical composition is 100 mg, 125 mg, 150 mg, 200 mg, or
250 mg. In some embodiments of this aspect, wgt. percent of
substantially amorphous Compound 2 that is present in the
pharmaceutical composition is from 10 to 75 percent. In these and
other embodiments, substantially amorphous Compound 2 is present as
substantially pure and amorphous Compound 2. "Substantially pure"
means greater than ninety percent pure; preferably greater than 95
percent pure; more preferably greater than 99.5 percent pure.
[0131] Thus in one aspect, the invention provides a pharmaceutical
composition comprising:
[0132] a. Compound 1 Form I;
[0133] b. a solid dispersion of substantially amorphous Compound
2;
[0134] c. a filler;
[0135] d. a disintegrant;
[0136] e. a surfactant; and
[0137] f. a binder.
[0138] In one embodiment of this aspect, the pharmaceutical
composition comprises 25 mg of Compound 1 Form I. In another
embodiment of this aspect, the pharmaceutical composition comprises
50 mg of Compound 1 Form I. In another embodiment of this aspect,
the pharmaceutical composition comprises 100 mg of Compound 1 Form
I. In another embodiment of this aspect, the pharmaceutical
composition comprises 125 mg of Compound 1 Form I. In another
embodiment of this aspect, the pharmaceutical composition comprises
150 mg of Compound 1 Form I. In another embodiment of this aspect,
the pharmaceutical composition comprises 200 mg of Compound 1 Form
I. In another embodiment of this aspect, the pharmaceutical
composition comprises 250 mg of Compound 1 Form I. In another
embodiment of this aspect, the pharmaceutical composition comprises
400 mg of Compound 1 Form I.
[0139] In one embodiment of this aspect, the pharmaceutical
composition comprises 25 mg of substantially amorphous Compound 2.
In another embodiment of this aspect, the pharmaceutical
composition comprises 50 mg of substantially amorphous Compound 2.
In another embodiment of this aspect, the pharmaceutical
composition comprises 100 mg of substantially amorphous Compound 2.
In another embodiment of this aspect, the pharmaceutical
composition comprises 125 mg of substantially amorphous Compound 2.
In another embodiment of this aspect, the pharmaceutical
composition comprises 150 mg of substantially amorphous Compound 2.
In another embodiment of this aspect, the pharmaceutical
composition comprises 200 mg of substantially amorphous Compound 2.
In another embodiment of this aspect, the pharmaceutical
composition comprises 250 mg of substantially amorphous Compound
2.
[0140] In some embodiments, the pharmaceutical compositions
comprises Compound 1 Form I, wherein Compound 1 Form I is present
in an amount of at least 15 wt % (e.g., at least 20 wt %, at least
30 wt %, at least 40 wt %, at least 50 wt %, or at least 60 wt %)
by weight of the composition.
[0141] In some embodiments, the pharmaceutical compositions
comprises substantially amorphous Compound 2, wherein the
substantially amorphous Compound 2 is present in an amount of at
least 15 wt % (e.g., at least 20 wt %, at least 30 wt %, at least
40 wt %, at least 50 wt %, or at least 60 wt %) by weight of the
composition.
[0142] In some embodiments, the pharmaceutical composition
comprises Compound 1 Form I, a solid dispersion comprising
substantially amorphous Compound 2, a filler, a disintegrant, a
surfactant, and a binder. In this embodiment, the composition
comprises from about 25 wt % to about 55 wt % (e.g., about 30-50 wt
%) of Compound 1 Form I by weight of the composition, and more
typically, from 40 wt % to about 45 wt % of Compound 1 Form I by
weight of the composition. In this embodiment, the composition
comprises from about 15 wt % to about 40 wt % (e.g., about 20-35 wt
%) of substantially amorphous Compound 2 by weight of the
composition, and more typically, from 25 wt % to about 30 wt % of
substantially amorphous Compound 2 by weight of the
composition.
[0143] The concentration of Compound 1 Form I and substantially
amorphous Compound 2 in the composition depends on several factors
such as the amount of pharmaceutical composition needed to provide
a desired amount of Compound 1 Form I and substantially amorphous
Compound 2 and the desired dissolution profile of the
pharmaceutical composition.
[0144] In another embodiment, the pharmaceutical composition
comprises Compound 1 Form I, in which Compound 1 Form I in its
solid form has a mean particle diameter, measured by light
scattering (e.g., using a Malvern Mastersizer available from
Malvern Instruments in England) of 0.1 microns to 10 microns. In
another embodiment, the particle size of Compound 1 Form I is 1
micron to 5 microns. In another embodiment, Compound 1 Form I has a
particle size D50 of 2.0 microns.
[0145] As indicated, in addition to Compound 1 Form I and a solid
dispersion of substantially amorphous Compound 2, in some
embodiments of the invention, the pharmaceutical compositions which
are oral formulations also comprise one or more excipients such as
fillers, disintegrants, surfactants, diluents, binders, glidants,
lubricants, colorants, or fragrances and any combination
thereof.
[0146] Fillers suitable for the invention are compatible with the
ingredients of the pharmaceutical composition, i.e., they do not
substantially reduce the solubility, the hardness, the chemical
stability, the physical stability, or the biological activity of
the pharmaceutical composition. Exemplary fillers include:
celluloses, modified celluloses, (e.g. sodium carboxymethyl
cellulose, ethyl cellulose hydroxymethyl cellulose,
hydroxypropylcellulose), cellulose acetate, microcrystalline
cellulose, calcium phosphates, dibasic calcium phosphate, starches
(e.g. corn starch, potato starch), sugars (e.g., sorbitol) lactose,
sucrose, or the like), or any combination thereof.
[0147] Thus, in one embodiment, the pharmaceutical composition
comprises at least one filler in an amount of at least 5 wt %
(e.g., at least about 20 wt %, at least about 30 wt %, or at least
about 40 wt %) by weight of the composition. For example, the
pharmaceutical composition comprises from about 10 wt % to about 60
wt % (e.g., from about 20 wt % to about 55 wt %, from about 25 wt %
to about 50 wt %, or from about 27 wt % to about 45 wt %) of
filler, by weight of the composition. In another example, the
pharmaceutical composition comprises at least about 20 wt % (e.g.,
at least 30 wt % or at least 40 wt %) of microcrystalline
cellulose, for example MCC Avicel PH102, by weight of the
composition. In yet another example, the pharmaceutical composition
comprises from about 10 wt % to about 60 wt % (e.g., from about 20
wt % to about 55 wt % or from about 25 wt % to about 45 wt %) of
microcellulose, by weight of the composition.
[0148] Disintegrants suitable for the invention enhance the
dispersal of the pharmaceutical composition and are compatible with
the ingredients of the pharmaceutical composition, i.e., they do
not substantially reduce the chemical stability, the physical
stability, the hardness, or the biological activity of the
pharmaceutical composition. Exemplary disintegrants include
croscarmellose sodium, sodium starch glycolate, or a combination
thereof.
[0149] Thus, in one embodiment, the pharmaceutical composition
comprises disintegrant in an amount of about 10 wt % or less (e.g.,
about 7 wt % or less, about 6 wt % or less, or about 5 wt % or
less) by weight of the composition. For example, the pharmaceutical
composition comprises from about 1 wt % to about 10 wt % (e.g.,
from about 1.5 wt % to about 7.5 wt % or from about 2.5 wt % to
about 6 wt %) of disintegrant, by weight of the composition. In
another example, the pharmaceutical composition comprises about 10
wt % or less (e.g., 7 wt % or less, 6 wt % or less, or 5 wt % or
less) of croscarmellose sodium, by weight of the composition. In
yet another example, the pharmaceutical composition comprises from
about 1 wt % to about 10 wt % (e.g., from about 1.5 wt % to about
7.5 wt % or from about 2.5 wt % to about 6 wt %) of croscarmellose
sodium, by weight of the composition. In some examples, the
pharmaceutical composition comprises from about 0.1% to about 10 wt
% (e.g., from about 0.5 wt % to about 7.5 wt % or from about 1.5 wt
% to about 6 wt %) of disintegrant, by weight of the composition.
In still other examples, the pharmaceutical composition comprises
from about 0.5% to about 10 wt % (e.g., from about 1.5 wt % to
about 7.5 wt % or from about 2.5 wt % to about 6 wt %) of
disintegrant, by weight of the composition.
[0150] Surfactants suitable for the invention enhance the
wettability of the pharmaceutical composition and are compatible
with the ingredients of the pharmaceutical composition, i.e., they
do not substantially reduce the chemical stability, the physical
stability, the hardness, or the biological activity of the
pharmaceutical composition. Exemplary surfactants include sodium
lauryl sulfate (SLS), sodium stearyl fumarate (SSF),
polyoxyethylene 20 sorbitan mono-oleate (e.g., Tween.TM.), any
combination thereof, or the like.
[0151] Thus, in one embodiment, the pharmaceutical composition
comprises a surfactant in an amount of about 10 wt % or less (e.g.,
about 5 wt % or less, about 2 wt % or less, about 1 wt % or less,
about 0.8 wt % or less, or about 0.6 wt % or less) by weight of the
composition. For example, the pharmaceutical composition includes
from about 10 wt % to about 0.1 wt % (e.g., from about 5 wt % to
about 0.2 wt % or from about 2 wt % to about 0.3 wt %) of
surfactant, by weight of the composition. In another example, the
pharmaceutical composition comprises 10 wt % or less (e.g., about 5
wt % or less, about 2 wt % or less, about 1 wt % or less, about 0.8
wt % or less, or about 0.6 wt % or less) of sodium lauryl sulfate,
by weight of the composition. In yet another example, the
pharmaceutical composition comprises from about 10 wt % to about
0.1 wt % (e.g., from about 5 wt % to about 0.2 wt % or from about 2
wt % to about 0.3 wt %) of sodium lauryl sulfate, by weight of the
composition.
[0152] Binders suitable for the invention enhance the tablet
strength of the pharmaceutical composition and are compatible with
the ingredients of the pharmaceutical composition, i.e., they do
not substantially reduce the chemical stability, the physical
stability, or the biological activity of the pharmaceutical
composition. Exemplary binders include polyvinylpyrrolidone,
dibasic calcium phosphate, sucrose, corn (maize) starch, modified
cellulose (e.g., hydroxymethyl cellulose), or any combination
thereof.
[0153] Thus, in one embodiment, the pharmaceutical composition
comprises a binder in an amount of at least about 0.1 wt % (e.g.,
at least about 1 wt %, at least about 3 wt %, at least about 4 wt
%, or at least about 5 wt %) by weight of the composition. For
example, the pharmaceutical composition comprises from about 0.1 wt
% to about 10 wt % (e.g., from about 1 wt % to about 10 wt % or
from about 2 wt % to about 7 wt %) of binder, by weight of the
composition. In another example, the pharmaceutical composition
comprises at least about 0.1 wt % (e.g., at least about 1 wt %, at
least about 2 wt %, at least about 3 wt %, or at least about 4 wt
%) of polyvinylpyrrolidone, by weight of the composition. In yet
another example, the pharmaceutical composition comprises a glidant
in an amount ranging from about 0.1 wt % to about 10 wt % (e.g.,
from about 1 wt % to about 8 wt % or from about 2 wt % to about 5
wt %) of polyvinylpyrrolidone, by weight of the composition.
[0154] Diluents suitable for the invention may add necessary bulk
to a formulation to prepare tablets of the desired size and are
generally compatible with the ingredients of the pharmaceutical
composition, i.e., they do not substantially reduce the solubility,
the hardness, the chemical stability, the physical stability, or
the biological activity of the pharmaceutical composition.
Exemplary diluents include: sugars, for example, confectioner's
sugar, compressible sugar, dextrates, dextrin, dextrose, lactose,
mannitol, sorbitol, cellulose, and modified celluloses, for
example, powdered cellulose, talc, calcium phosphate, starch, or
any combination thereof.
[0155] Thus, in one embodiment, the pharmaceutical composition
comprises a diluent in an amount of 40 wt % or less (e.g., 35 wt %
or less, 30 wt % or less, or 25 wt % or less, or 20 wt % or less,
or 15 wt % or less, or 10 wt % or less) by weight of the
composition. For example, the pharmaceutical composition comprises
from about 40 wt % to about 1 wt % (e.g., from about 35 wt % to
about 5 wt % or from about 30 wt % to about 7 wt %, from about 25
wt % to about 10 wt %, from about 20 wt % to about 15 wt %) of
diluent, by weight of the composition. In another example, the
pharmaceutical composition comprises 40 wt % or less (e.g., 35 wt %
or less, 25 wt % or less, or 15 wt % or less) of mannitol, by
weight of the composition. In yet another example, the
pharmaceutical composition comprises from about 35 wt % to about 1
wt % (e.g., from about 30 wt % to about 5 wt % or from about 25 wt
% to about 10 wt %) of mannitol, by weight of the composition.
[0156] Glidants suitable for the invention enhance the flow
properties of the pharmaceutical composition and are compatible
with the ingredients of the pharmaceutical composition, i.e., they
do not substantially reduce the solubility, the hardness, the
chemical stability, the physical stability, or the biological
activity of the pharmaceutical composition. Exemplary glidants
include colloidal silicon dioxide, talc, or a combination
thereof.
[0157] Thus, in one embodiment, the pharmaceutical composition
comprises a glidant in an amount of 2 wt % or less (e.g., 1.75 wt
%, 1.25 wt % or less, or 1.00 wt % or less) by weight of the
composition. For example, the pharmaceutical composition comprises
from about 2 wt % to about 0.05 wt % (e.g., from about 1.5 wt % to
about 0.07 wt % or from about 1.0 wt % to about 0.09 wt %) of
glidant, by weight of the composition. In another example, the
pharmaceutical composition comprises 2 wt % or less (e.g., 1.75 wt
%, 1.25 wt % or less, or 1.00 wt % or less) of colloidal silicon
dioxide, by weight of the composition. In yet another example, the
pharmaceutical composition comprises from about 2 wt % to about
0.05 wt % (e.g., from about 1.5 wt % to about 0.07 wt % or from
about 1.0 wt % to about 0.09 wt %) of colloidal silicon dioxide, by
weight of the composition.
[0158] In some embodiments, the pharmaceutical composition can
include an oral solid pharmaceutical dosage form which can comprise
a lubricant that can prevent adhesion of a granulate-bead admixture
to a surface (e.g., a surface of a mixing bowl, a compression die
and/or punch). A lubricant can also reduce interparticle friction
within the granulate and improve the compression and ejection of
compressed pharmaceutical compositions from a die press. The
lubricant is also compatible with the ingredients of the
pharmaceutical composition, i.e., they do not substantially reduce
the solubility, the hardness, or the biological activity of the
pharmaceutical composition. Exemplary lubricants include magnesium
stearate, calcium stearate, zinc stearate, sodium stearate, stearic
acid, aluminum stearate, leucine, glyceryl behenate, hydrogenated
vegetable oil or any combination thereof. In one embodiment, the
pharmaceutical composition comprises a lubricant in an amount of 5
wt % or less (e.g., 4.75 wt %, 4.0 wt % or less, or 3.00 wt % or
less, or 2.0 wt % or less) by weight of the composition. For
example, the pharmaceutical composition comprises from about 5 wt %
to about 0.10 wt % (e.g., from about 4.5 wt % to about 0.5 wt % or
from about 3 wt % to about 1 wt %) of lubricant, by weight of the
composition. In another example, the pharmaceutical composition
comprises 5 wt % or less (e.g., 4.0 wt % or less, 3.0 wt % or less,
or 2.0 wt % or less, or 1.0 wt % or less) of magnesium stearate, by
weight of the composition. In yet another example, the
pharmaceutical composition comprises from about 5 wt % to about
0.10 wt % (e.g., from about 4.5 wt % to about 0.15 wt % or from
about 3.0 wt % to about 0.50 wt %) of magnesium stearate, by weight
of the composition.
[0159] Pharmaceutical compositions of the invention can optionally
comprise one or more colorants, flavors, and/or fragrances to
enhance the visual appeal, taste, and/or scent of the composition.
Suitable colorants, flavors, or fragrances are compatible with the
ingredients of the pharmaceutical composition, i.e., they do not
substantially reduce the solubility, the chemical stability, the
physical stability, the hardness, or the biological activity of the
pharmaceutical composition. In one embodiment, the pharmaceutical
composition comprises a colorant, a flavor, and/or a fragrance. In
one embodiment, the pharmaceutical compositions provided by the
invention are purple.
[0160] In some embodiments, the pharmaceutical composition includes
or can be made into tablets and the tablets can be coated with a
colorant and optionally labeled with a logo, other image and/or
text using a suitable ink. In still other embodiments, the
pharmaceutical composition includes or can be made into tablets and
the tablets can be coated with a colorant, waxed, and optionally
labeled with a logo, other image and/or text using a suitable ink.
Suitable colorants and inks are compatible with the ingredients of
the pharmaceutical composition, i.e., they do not substantially
reduce the solubility, the chemical stability, the physical
stability, the hardness, or the biological activity of the
pharmaceutical composition. The suitable colorants and inks can be
any color and are water based or solvent based. In one embodiment,
tablets made from the pharmaceutical composition are coated with a
colorant and then labeled with a logo, other image, and/or text
using a suitable ink. For example, tablets comprising
pharmaceutical composition as described herein can be coated with
about 3 wt % (e.g., less than about 6 wt % or less than about 4 wt
%) of film coating comprising a colorant. The colored tablets can
be labeled with a logo and text indicating the strength of the
active ingredient in the tablet using a suitable ink. In another
example, tablets comprising pharmaceutical composition as described
herein can be coated with about 3 wt % (e.g., less than about 6 wt
% or less than about 4 wt %) of a film coating comprising a
colorant.
[0161] In another embodiment, tablets made from the pharmaceutical
composition are coated with a colorant, waxed, and then labeled
with a logo, other image, and/or text using a suitable ink. For
example, tablets comprising pharmaceutical composition as described
herein can be coated with about 3 wt % (e.g., less than about 6 wt
% or less than about 4 wt %) of film coating comprising a colorant.
The colored tablets can be waxed with Carnauba wax powder weighed
out in the amount of about 0.01% w/w of the starting tablet core
weight. The waxed tablets can be labeled with a logo and text
indicating the strength of the active ingredient in the tablet
using a suitable ink. In another example, tablets comprising
pharmaceutical composition as described herein can be coated with
about 3 wt % (e.g., less than about 6 wt % or less than about 4 wt
%) of a film coating comprising a colorant The colored tablets can
be waxed with Carnauba wax powder weighed out in the amount of
about 0.01% w/w of the starting tablet core weight. The waxed
tablets can be labeled with a logo and text indicating the strength
of the active ingredient in the tablet using a pharmaceutical grade
ink such as a black ink (e.g., Opacode.RTM. S-1-17823, a solvent
based ink, commercially available from Colorcon, Inc. of West
Point, Pa.).
[0162] One exemplary pharmaceutical composition comprises from
about 15 wt % to about 70 wt % (e.g., from about 15 wt % to about
60 wt %, from about 15 wt % to about 50 wt %, or from about 20 wt %
to about 70 wt %, or from about 30 wt % to about 70 wt %) of
Compound 1 Form I, by weight of the composition; and from about 15
wt % to about 40 wt % (e.g., about 20-35 wt %) of substantially
amorphous Compound 2 by weight of the composition, and more
typically, from 25 wt % to about 30 wt % of substantially amorphous
Compound 2 by weight of the composition. The aforementioned
compositions can also include one or more pharmaceutically
acceptable excipients, for example, from about 20 wt % to about 50
wt % of a filler; from about 1 wt % to about 5 wt % of a
disintegrant; from about 2 wt % to about 0.3 wt % of a surfactant;
and from about 0.1 wt % to about 5 wt % of a binder.
[0163] Another exemplary pharmaceutical composition comprises from
about 15 wt % to about 70 wt % (e.g., from about 15 wt % to about
60 wt %, from about 15 wt % to about 50 wt %, or from about 15 wt %
to about 40 wt % or from about 20 wt % to about 70 wt %, or from
about 30 wt % to about 70 wt %, or from about 40 wt % to about 70
wt %, or from about 50 wt % to about 70 wt %) of Compound 1 Form I
by weight of the composition, from about 15 wt % to about 40 wt %
(e.g., about 20-35 wt %) of substantially amorphous Compound 2 by
weight of the composition, and more typically, from 25 wt % to
about 30 wt % of substantially amorphous Compound 2 by weight of
the composition, and one or more excipients, for example, from
about 20 wt % to about 50 wt % of a filler; from about 1 wt % to
about 5 wt % of a disintegrant; from about 2 wt % to about 0.3 wt %
of a surfactant; from about 0.1 wt % to about 5 wt % of a binder;
and from about 2 wt % to about 0.1 wt % of a lubricant.
[0164] Another exemplary pharmaceutical composition comprises from
about 15 wt % to about 70 wt % (e.g., from about 15 wt % to about
60 wt %, from about 15 wt % to about 50 wt %, or from about 15 wt %
to about 40 wt % or from about 20 wt % to about 70 wt %, or from
about 30 wt % to about 70 wt %, or from about 40 wt % to about 70
wt %, or from about 50 wt % to about 70 wt %) of Compound 1 Form I
by weight of the composition, from about 15 wt % to about 40 wt %
(e.g., about 20-35 wt %) of substantially amorphous Compound 2 by
weight of the composition, and more typically, from 25 wt % to
about 30 wt % of substantially amorphous Compound 2 by weight of
the composition, and one or more excipients, for example, from
about 20 wt % to about 50 wt % of a filler; from about 1 wt % to
about 5 wt % of a disintegrant; from about 2 wt % to about 0.3 wt %
of a surfactant; from about 0.1 wt % to about 5 wt % of a binder;
from about 2 wt % to about 0.1 wt % of a lubricant; from about 2 wt
% to about 4 wt % colorant; and about 0.005 wt % to about 0.015 wt
% wax.
[0165] In one embodiment, the invention is a granular
pharmaceutical composition comprising:
[0166] a. about 43 wt % of Compound 1 Form I by weight of the
composition;
[0167] b. about 34 wt % of a solid dispersion comprising
substantially amorphous Compound 2 by weight of the
composition;
[0168] c. about 17 wt % of microcrystalline cellulose by weight of
the composition;
[0169] d. about 2 wt % of croscarmellose sodium by weight of the
composition;
[0170] e. about 1 wt % of sodium lauryl sulfate by weight of the
composition; and
[0171] f. about 3 wt % of polyvinylpyrrolidone by weight of the
composition.
[0172] In one embodiment, the invention is a tablet comprising:
[0173] a. about 35 wt % of Compound 1 Form I by weight of the
composition;
[0174] b. about 28 wt % of a solid dispersion comprising
substantially amorphous Compound 2 by weight of the
composition;
[0175] c. about 26 wt % of microcrystalline cellulose by weight of
the composition;
[0176] d. about 6 wt % of croscarmellose sodium by weight of the
composition;
[0177] e. about 3 wt % of polyvinylpyrrolidone by weight of the
composition;
[0178] f. about 1 wt % of sodium lauryl sulfate by weight of the
composition; and
[0179] g. about 1 wt % of magnesium stearate by weight of the
composition.
[0180] In one embodiment, the invention is a tablet comprising:
[0181] a. about 34 wt % of Compound 1 Form I by weight of the
composition;
[0182] b. about 27 wt % of a solid dispersion comprising
substantially amorphous Compound 2 by weight of the
composition;
[0183] c. about 26 wt % of microcrystalline cellulose by weight of
the composition;
[0184] d. about 6 wt % of croscarmellose sodium by weight of the
composition;
[0185] e. about 2 wt % of polyvinylpyrrolidone by weight of the
composition
[0186] f. about 1 wt % of sodium lauryl sulfate by weight of the
composition;
[0187] g. about 1 wt % of magnesium stearate by weight of the
composition;
[0188] h. about 3 wt % of a colorant by weight of the composition;
and
[0189] i. about 0.010 wt % of a wax by weight of the
composition.
[0190] Another tablet of the invention comprises:
[0191] a. about 150 to 250 mg of Compound 1 Form I;
[0192] b. about 100 to 150 mg of substantially amorphous Compound
2;
[0193] c. about 125 to 175 mg of microcrystalline cellulose;
[0194] d. about 20 to 40 mg of croscarmellose sodium;
[0195] e. about 10 to 20 mg of polyvinylpyrrolidone;
[0196] f. about 2 to 6 mg of sodium lauryl sulfate; and
[0197] g. about 3 to 7 mg of magnesium stearate.
[0198] Another tablet of the invention comprises:
[0199] a. about 200 mg of Compound 1 Form I;
[0200] b. about 125 mg of substantially amorphous Compound 2;
[0201] c. about 150 mg of microcrystalline cellulose;
[0202] d. about 34 mg of croscarmellose sodium;
[0203] e. about 15 mg of polyvinylpyrrolidone;
[0204] f. about 4 mg of sodium lauryl sulfate; and
[0205] g. about 6 mg of magnesium stearate.
[0206] Another tablet of the invention comprises:
[0207] a. about 200 mg of Compound 1 Form I;
[0208] b. about 125 mg of substantially amorphous Compound 2;
[0209] c. about 150 mg of microcrystalline cellulose;
[0210] d. about 34 mg of croscarmellose sodium;
[0211] e. about 15 mg of polyvinylpyrrolidone;
[0212] f. about 4 mg of sodium lauryl sulfate;
[0213] g. about 6 mg of magnesium stearate;
[0214] h. about 17 mg of a colorant; and
[0215] i. about 0.06 mg of a wax.
[0216] In one embodiment, the invention is a granular
pharmaceutical composition comprising:
[0217] a. about 38 wt % of Compound 1 Form I by weight of the
composition;
[0218] b. about 40 wt % of a solid dispersion comprising
substantially amorphous Compound 2 by weight of the
composition;
[0219] c. about 16 wt % of microcrystalline cellulose by weight of
the composition;
[0220] d. about 2 wt % of croscarmellose sodium by weight of the
composition;
[0221] e. about 1 wt % of sodium lauryl sulfate by weight of the
composition; and
[0222] f. about 3 wt % of polyvinylpyrrolidone by weight of the
composition.
[0223] In one embodiment, the invention is a tablet comprising:
[0224] a. about 31 wt % of Compound 1 Form I by weight of the
composition;
[0225] b. about 32 wt % of a solid dispersion comprising
substantially amorphous Compound 2 by weight of the
composition;
[0226] c. about 26 wt % of microcrystalline cellulose by weight of
the composition;
[0227] d. about 6 wt % of croscarmellose sodium by weight of the
composition;
[0228] e. about 3 wt % of polyvinylpyrrolidone by weight of the
composition
[0229] f. about 1 wt % of sodium lauryl sulfate by weight of the
composition;
[0230] g. about 1 wt % of magnesium stearate by weight of the
composition; and
[0231] h. about 3 wt % of a colorant by weight of the
composition.
[0232] Another tablet of the invention comprises:
[0233] a. about 100 to 200 mg of Compound 1 Form I;
[0234] b. about 100 to 150 mg of substantially amorphous Compound
2;
[0235] c. about 100 to 150 mg of microcrystalline cellulose;
[0236] d. about 20 to 40 mg of croscarmellose sodium;
[0237] e. about 10 to 20 mg of polyvinylpyrrolidone;
[0238] f. about 2 to 6 mg of sodium lauryl sulfate; and
[0239] g. about 3 to 7 mg of magnesium stearate.
[0240] Another tablet of the invention comprises:
[0241] a. about 150 mg of Compound 1 Form I;
[0242] b. about 125 mg of substantially amorphous Compound 2;
[0243] c. about 129 mg of microcrystalline cellulose;
[0244] d. about 29 mg of croscarmellose sodium;
[0245] e. about 13 mg of polyvinylpyrrolidone;
[0246] f. about 4 mg of sodium lauryl sulfate;
[0247] g. about 5 mg of magnesium stearate; and
[0248] h. about 15 mg of a colorant.
[0249] The pharmaceutical compositions of the invention can be
processed into a tablet form, capsule form, pouch form, lozenge
form, or other solid form that is suited for oral administration.
Thus in some embodiments, the pharmaceutical compositions are in
tablet form.
[0250] Another aspect of the invention provides a pharmaceutical
formulation consisting of a tablet that includes Compound 1 Form I,
a solid dispersion comprising substantially amorphous Compound 2,
and excipients (e.g., a filler, a disintegrant, a surfactant, a
binder, a colorant, a lubricant, or any combination thereof), each
of which is described above and in the Examples below, wherein the
tablet has a dissolution of at least about 50% (e.g., at least
about 60%, at least about 70%, at least about 80%, at least about
90%, or at least about 99%) in about 30 minutes.
[0251] In one example, the pharmaceutical composition consists of a
tablet that includes Compound 1 Form I in an amount ranging from 25
mg to 400 mg, for example, 25 mg, or 50 mg, or 75 mg, or 100 mg, or
150 mg, 200 mg, 250 mg, 300 mg, or 400 mg, substantially amorphous
Compound 2 in an amount ranging from 25 mg to 250 mg, for example,
25 mg, or 50 mg, or 75 mg, or 100 mg, or 150 mg, 200 mg, 250 mg,
and one or more excipients (e.g., a filler, a disintegrant, a
surfactant, a binder, a colorant, a lubricant, or any combination
thereof) each of which is described above and in the Examples
below, wherein the tablet has a dissolution of from about 50% to
about 100% (e.g., from about 55% to about 95% or from about 60% to
about 90%) in about 30 minutes.
[0252] Dissolution can be measured with a standard USP Type II
apparatus that employs a dissolution media of 0.1% CTAB dissolved
in 900 mL of DI water, buffered at pH 6.8 with 50 mM potassium
phosphate monoasic, stirring at about 50-75 rpm at a temperature of
about 37.degree. C. A single experimental tablet is tested in each
test vessel of the apparatus. Dissolution can also be measured with
a standard USP Type II apparatus that employs a dissolution media
of 0.7% sodium lauryl sulfate dissolved in 900 mL of 50 mM sodium
phosphate buffer (pH 6.8), stirring at about 65 rpm at a
temperature of about 37.degree. C. A single experimental tablet is
tested in each test vessel of the apparatus. Dissolution can also
be measured with a standard USP Type II apparatus that employs a
dissolution media of 0.5% sodium lauryl sulfate dissolved in 900 mL
of 50 mM sodium phosphate buffer (pH 6.8), stirring at about 65 rpm
at a temperature of about 37.degree. C. A single experimental
tablet is tested in each test vessel of the apparatus.
Methods for Making Compound 1 Form I and A Solid Dispersion
Comprising Substantially Amorphous Compound 2
Compound 1
[0253] Compound 1 is used as the starting point for Compound 1 Form
I and can be prepared by coupling an acid chloride moiety with an
amine moiety according to Schemes 1-4.
##STR00003##
[0254] Scheme 1 depicts the preparation of
1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl
chloride, which is used in Scheme 3 to make the amide linkage of
Compound 1.
[0255] The starting material,
2,2-difluorobenzo[d][1,3]dioxole-5-carboxylic acid, is commercially
available from Saltigo (an affiliate of the Lanxess Corporation).
Reduction of the carboxylc acid moiety in
2,2-difluorobenzo[d][1,3]dioxole-5-carboxylic acid to the primary
alcohol, followed by conversion to the corresponding chloride using
thionyl chloride (SOCl.sub.2), provides
5-(chloromethyl)-2,2-difluorobenzo[d][1,3]dioxole, which is
subsequently converted to
2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile using sodium
cyanide. Treatment of
2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile with base and
1-bromo-2-chloroethane provides
1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile.
The nitrile moiety in
1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile is
converted to a carboxylic acid using base to give
1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic
acid, which is converted to the desired acid chloride using thionyl
chloride.
##STR00004##
[0256] Scheme 2 depicts an alternative synthesis of the requisite
acid chloride. 5-bromomethyl-2,2-difluoro-1,3-benzodioxole is
coupled with ethyl cyanoacetate in the presence of a palladium
catalyst to form the corresponding alpha cyano ethyl ester.
Saponification of the ester moiety to the carboxylic acid gives the
cyanoethyl compound. Alkylation of the cyanoethyl compound with
1-bromo-2-chloro ethane in the presence of base gives the
cyanocyclopropyl compound. Treatment of the cyanocyclopropyl
compound with base gives the carboxylate salt, which is converted
to the carboxylic acid by treatment with acid. Conversion of the
carboxylic acid to the acid chloride is then accomplished using a
chlorinating agent such as thionyl chloride or the like.
##STR00005##
[0257] Scheme 3 depicts the preparation of the requisite tert-butyl
3-(6-amino-3-methylpyridin-2-yl)benzoate, which is coupled with
1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl
chloride in Scheme 3 to give Compound 1. Palladium-catalyzed
coupling of 2-bromo-3-methylpyridine with
3-(tert-butoxycarbonyl)phenylboronic acid gives tert-butyl
3-(3-methylpyridin-2-yl)benzoate, which is subsequently converted
to the desired compound.
##STR00006##
[0258] Scheme 4 depicts the coupling of
1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl
chloride with tert-butyl 3-(6-amino-3-methylpyridin-2-yl)benzoate
using triethyl amine and 4-dimethylaminopyridine to initially
provide the tert-butyl ester of Compound 1.
Compound 1 Form I
[0259] Compound 1 Form I is prepared by dispersing or dissolving a
salt form, such as the HCl salt, of Compound 1 in an appropriate
solvent for an effective amount of time. Treatment of the
tert-butyl ester with an acid such as HCl, gives the HCL salt of
Compound 1, which is typically a crystalline solid. Compound 1 Form
I may also be prepared directly from the t-butyl ester precursor by
treatment with an appropriate acid, such as formic acid.
[0260] The HCl salt of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid can be used to make Form I by
dispersing or dissolving the HCl salt of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid in an appropriate solvent for an
effective amount of time. Other salts of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid may be used, such as, for example,
salts derived from other mineral or organic acids. The other salts
result from acid-mediated hydrolysis of the t-butyl ester moiety.
Salts derived from other acids may include, for example, nitric,
sulfuric, phosphoric, boric, acetic, benzoic and malonic. These
salt forms of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid may or may not be soluble,
depending upon the solvent used, but lack of solubility does not
hinder formation of Compound 1 Form I. For example, in one
embodiment, the appropriate solvent may be water or an
alcohol/water mixture such as 50% methanol/water mixture, even
though the HCl salt form of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid is only sparingly soluble in water.
In one embodiment, the appropriate solvent is water.
[0261] The effective amount of time for formation of Compound 1
Form I from the salt of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid can be any time between 2 to 24
hours or greater. It is recognized that the amount of time needed
is inversely proportional to the temperature. That is, the higher
the temperature the less time needed to affect dissociation of acid
to form Compound 1 Form I. When the solvent is water, stirring the
dispersion for approximately 24 hours at room temperature provides
Compound 1 Form I in an approximately 98% yield. If a solution of
the salt of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid is desired for process purposes, an
elevated temperature may be used. After stirring the solution for
an effective amount of time at the elevated temperature,
recrystallization upon cooling provides substantially pure Compound
1 Form I. In one embodiment, substantially pure refers to greater
than about 90% purity. In another embodiment, substantially pure
refers to greater than about 95% purity. In another embodiment,
substantially pure refers to greater than about 98% purity. In
another embodiment, substantially pure refers to greater than about
99% purity. The temperature selected depends in part on the solvent
used and is well within the determination capabilities of one of
ordinary skill in the art. In one embodiment, the temperature is
between room temperature and about 80.degree. C. In another
embodiment, the temperature is between room temperature and about
40.degree. C. In another embodiment, the temperature is between
about 40.degree. C. and about 60.degree. C. In another embodiment,
the temperature is between about 60.degree. C. and about 80.degree.
C.
[0262] Compound 1 Form I may also be formed directly from
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate (cf. Scheme 3), which is a
precursor to the salt of Compound 1. Thus,
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate is allowed to undergo reaction
with an appropriate acid, such as, for example, formic acid under
appropriate reaction conditions to give Compound 1 Form I.
[0263] Compound 1 Form I may be further purified by
recrystallization from an organic solvent. Examples of organic
solvents include, but are not limited to, toluene, cumene, anisol,
1-butanol, isopropyl acetate, butyl acetate, isobutyl acetate,
methyl t-butyl ether, methyl isobutyl ketone and 1-propanol-water
mixtures. The temperature may be as described above. For example,
Compound 1 Form I is dissolved in 1-butanol at 75.degree. C. until
it is completely dissolved. Cooling down the solution to 10.degree.
C. at a rate of 0.2.degree. C./min yields crystals of Compound 1
Form I which may be isolated by filtration.
[0264] In one embodiment, Compound 1 Form I is characterized by one
or more peaks at 15.2 to 15.6 degrees, 16.1 to 16.5 degrees, and
14.3 to 14.7 degrees in an X-ray powder diffraction obtained using
Cu K alpha radiation. In another embodiment, Compound 1 Form I is
characterized by one or more peaks at 15.4, 16.3, and 14.5 degrees.
In another embodiment, Compound 1 Form I is further characterized
by a peak at 14.6 to 15.0 degrees. In another embodiment, Compound
1 Form I is further characterized by a peak at 14.8 degrees. In
another embodiment, Compound 1 Form I is further characterized by a
peak at 17.6 to 18.0 degrees. In another embodiment, Compound 1
Form I is further characterized by a peak at 17.8 degrees. In
another embodiment, Compound 1 Form I is further characterized by a
peak at 16.4 to 16.8 degrees. In another embodiment, Compound 1
Form I is further characterized by a peak at 16.4 to 16.8 degrees.
In another embodiment, Compound 1 Form I is further characterized
by a peak at 16.6 degrees. In another embodiment, Compound 1 Form I
is further characterized by a peak at 7.6 to 8.0 degrees. In
another embodiment, Compound 1 Form I is further characterized by a
peak at 7.8 degrees. In another embodiment, Compound 1 Form I is
further characterized by a peak at 25.8 to 26.2 degrees. In another
embodiment, Compound 1 Form I is further characterized by a peak at
26.0 degrees. In another embodiment, Compound 1 Form I is further
characterized by a peak at 21.4 to 21.8 degrees. In another
embodiment, Compound 1 Form I is further characterized by a peak at
21.6 degrees. In another embodiment, Compound 1 Form I is further
characterized by a peak at 23.1 to 23.5 degrees. In another
embodiment, Compound 1 Form I is further characterized by a peak at
23.3 degrees. In some embodiments, Compound 1 Form I is
characterized by a diffraction pattern substantially similar to
that of FIG. 1. In some embodiments, Compound 1 Form I is
characterized by a diffraction pattern substantially similar to
that of FIG. 2.
[0265] In some embodiments, the particle size distribution of D90
is about 82 .mu.m or less for Compound 1 Form I. In some
embodiments, the particle size distribution of D50 is about 30
.mu.m or less for Compound 1 Form I.
Compound 2
[0266] Compound 2 is the starting point for the solid dispersion
comprising substantially amorphous Compound 2 and can be prepared
by coupling a 4-oxo-dihydroquinoline carboxylic acid moiety with an
amine moiety according to Schemes 5-7.
##STR00007##
##STR00008##
##STR00009##
Solid Dispersion Comprising Substantially Amorphous Compound 2
[0267] Starting from Compound 2 the amorphous form of Compound 2
may be prepared by spray dried methods. Spray drying is a process
that converts a liquid feed to a dried particulate form.
Optionally, a secondary drying process such as fluidized bed drying
or vacuum drying, may be used to reduce residual solvents to
pharmaceutically acceptable levels. Typically, spray drying
involves contacting a highly dispersed liquid suspension or
solution, and a sufficient volume of hot air to produce evaporation
and drying of the liquid droplets. The preparation to be spray
dried can be any solution, coarse suspension, slurry, colloidal
dispersion, or paste that may be atomized using the selected spray
drying apparatus. In a standard procedure, the preparation is
sprayed into a current of warm filtered air that evaporates the
solvent and conveys the dried product to a collector (e.g. a
cyclone). The spent air is then exhausted with the solvent, or
alternatively the spent air is sent to a condenser to capture and
potentially recycle the solvent. Commercially available types of
apparatus may be used to conduct the spray drying. For example,
commercial spray dryers are manufactured by Buchi Ltd. And Niro
(e.g., the PSD line of spray driers manufactured by Niro) (see, US
2004/0105820; US 2003/0144257).
[0268] Spray drying typically employs solid loads of material from
about 3% to about 30% by weight, (i.e., drug and excipients), for
example about 4% to about 20% by weight, preferably at least about
10%. In general, the upper limit of solid loads is governed by the
viscosity of (e.g., the ability to pump) the resulting solution and
the solubility of the components in the solution. Generally, the
viscosity of the solution can determine the size of the particle in
the resulting powder product.
[0269] Techniques and methods for spray drying may be found in
Perry's Chemical Engineering Handbook, 6.sup.th Ed., R. H. Perry,
D. W. Green & J. O. Maloney, eds.), McGraw-Hill book co.
(1984); and Marshall "Atomization and Spray-Drying" 50, Chem. Eng.
Prog. Monogr. Series 2 (1954). In general, the spray drying is
conducted with an inlet temperature of from about 60.degree. C. to
about 200.degree. C., for example, from about 95.degree. C. to
about 185.degree. C., from about 110.degree. C. to about
182.degree. C., from about 96.degree. C. to about 180.degree. C.,
e.g., about 145.degree. C. The spray drying is generally conducted
with an outlet temperature of from about 30.degree. C. to about
90.degree. C., for example from about 40.degree. C. to about
80.degree. C., about 45.degree. C. to about 80.degree. C. e.g.,
about 75.degree. C. The atomization flow rate is generally from
about 4 kg/h to about 12 kg/h, for example, from about 4.3 kg/h to
about 10.5 kg/h, e.g., about 6 kg/h or about 10.5 kg/h. The feed
flow rate is generally from about 3 kg/h to about 10 kg/h, for
example, from about 3.5 kg/h to about 9.0 kg/h, e.g., about 8 kg/h
or about 7.1 kg/h. The atomization ratio is generally from about
0.3 to 1.7, e.g., from about 0.5 to 1.5, e.g., about 0.8 or about
1.5.
[0270] Removal of the solvent may require a subsequent drying step,
such as tray drying, fluid bed drying (e.g., from about room
temperature to about 100.degree. C.), vacuum drying, microwave
drying, rotary drum drying or biconical vacuum drying (e.g., from
about room temperature to about 200.degree. C.).
[0271] In one embodiment, the spray dried dispersion is fluid bed
dried.
[0272] In one process, the solvent includes a volatile solvent, for
example a solvent having a boiling point of less than about
100.degree. C. In some embodiments, the solvent includes a mixture
of solvents, for example a mixture of volatile solvents or a
mixture of volatile and non-volatile solvents. Where mixtures of
solvents are used, the mixture can include one or more non-volatile
solvents, for example, where the non-volatile solvent is present in
the mixture at less than about 15%, e.g., less than about 12%, less
than about 10%, less than about 8%, less than about 5%, less than
about 3%, or less than about 2%.
[0273] Preferred solvents are those solvents where Compound 2 has a
solubility of at least about 10 mg/ml, (e.g., at least about 15
mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml,
50 mg/ml, or greater). More preferred solvents include those where
Compound 2 has a solubility of at least about 20 mg/ml.
[0274] Exemplary solvents that could be tested include acetone,
cyclohexane, dichloromethane, N,N-dimethylacetamide (DMA),
N,N-dimethylformamide (DMF), 1,3-dimethyl-2-imidazolidinone (DMI),
dimethyl sulfoxide (DMSO), dioxane, ethyl acetate, ethyl ether,
glacial acetic acid (HAc), methyl ethyl ketone (MEK),
N-methyl-2-pyrrolidinone (NMP), methyl tert-butyl ether (MTBE),
tetrahydrofuran (THF), pentane, acetonitrile, methanol, ethanol,
isopropyl alcohol, isopropyl acetate, and toluene. Exemplary
co-solvents include acetone/DMSO, acetone/DMF, acetone/water,
MEK/water, THF/water, dioxane/water. In a two solvent system, the
solvents can be present in of from about 0.1% to about 99.9%. In
some preferred embodiments, water is a co-solvent with acetone
where water is present from about 0.1% to about 15%, for example
about 9% to about 11%, e.g., about 10%. In some preferred
embodiments, water is a co-solvent with MEK where water is present
from about 0.1% to about 15%, for example about 9% to about 11%,
e.g., about 10%. In some embodiments the solvent solution include
three solvents. For example, acetone and water can be mixed with a
third solvent such as DMA, DMF, DMI, DMSO, or HAc. In instances
where substantially amorphous Compound 2 is a component of a solid
dispersion, preferred solvents dissolve both Compound 2 and the
polymer. Suitable solvents include those described above, for
example, MEK, acetone, water, methanol, and mixtures thereof.
[0275] The particle size and the temperature drying range may be
modified to prepare an optimal spray dry dispersion. As would be
appreciated by skilled practitioners, a small particle size would
lead to improved solvent removal. Applicants have found however,
that smaller particles can lead to fluffy particles that, under
some circumstances do not provide optimal spray dry dispersions for
downstream processing such as tableting. At higher temperatures,
crystallization or chemical degradation of substantially amorphous
Compound 2 may occur. At lower temperatures, a sufficient amount of
the solvent may not be removed. The methods herein provide an
optimal particle size and an optimal drying temperature.
[0276] In general, particle size is such that D10 (.mu.m) is less
than about 5, e.g., less than about 4.5, less than about 4.0, or
less than about 3.5, D50 (.mu.m) is generally less than about 17,
e.g., less than about 16, less than about 15, less than about 14,
less than about 13, and D90 (.mu.m) is generally less than about
175, e.g., less than about 170, less than about 170, less than
about 150, less than about 125, less than about 100, less than
about 90, less than about 80, less than about 70, less than about
60, or less than about less than about 50. In general bulk density
of the spray dried particles is from about 0.08 g/cc to about 0.20
g/cc, e.g., from about 0.10 to about 0.15 g/cc, e.g., about 0.11
g/cc or about 0.14 g/cc. Tap density of the spray dried particles
generally ranges from about 0.08 g/cc to about 0.20 g/cc, e.g.,
from about 0.10 to about 0.15 g/cc, e.g., about 0.11 g/cc or about
0.14 g/cc, for 10 taps; 0.10 g/cc to about 0.25 g/cc, e.g., from
about 0.11 to about 0.21 g/cc, e.g., about 0.15 g/cc, about 0.19
g/cc, or about 0.21 g/cc for 500 taps; 0.15 g/cc to about 0.27
g/cc, e.g., from about 0.18 to about 0.24 g/cc, e.g., about 0.18
g/cc, about 0.19 g/cc, about 0.20 g/cc, or about 0.24 g/cc for 1250
taps; and 0.15 g/cc to about 0.27 g/cc, e.g., from about 0.18 to
about 0.24 g/cc, e.g., about 0.18 g/cc, about 0.21 g/cc, about 0.23
g/cc, or about 0.24 g/cc for 2500 taps.
[0277] Polymers
[0278] Spray dried dispersions including amorphous Compound 2 and a
polymer (or solid state carrier) also are included herein. For
example, Compound 2 is present as an amorphous compound as a
component of a solid amorphous dispersion. The solid amorphous
dispersion, generally includes substantially amorphous Compound 2
and a polymer. Exemplary polymers include cellulosic polymers such
as HPMC or HPMCAS and pyrrolidone containing polymers such as
PVP/VA. In some embodiments, the solid amporphous dispersion
includes one or more additional exipients, such as a
surfactant.
[0279] In one embodiment, a polymer is able to dissolve in aqueous
media. The solubility of the polymers may be pH-independent or
pH-dependent. The latter include one or more enteric polymers. The
term "enteric polymer" refers to a polymer that is preferentially
soluble in the less acidic environment of the intestine relative to
the more acid environment of the stomach, for example, a polymer
that is insoluble in acidic aqueous media but soluble when the pH
is above 5-6. An appropriate polymer should be chemically and
biologically inert. In order to improve the physical stability of
the spray dry dispersions, the glass transition temperature
(T.sub.g) of the polymer should be as high as possible. For
example, preferred polymers have a glass transition temperature at
least equal to or greater than the glass transition temperature of
the drug (i.e., Compound 2). Other preferred polymers have a glass
transition temperature that is within about 10 to about 15.degree.
C. of the drug (i.e., Compound 2). Examples of suitable glass
transition temperatures of the polymers include at least about
90.degree. C., at least about 95.degree. C., at least about
100.degree. C., at least about 105.degree. C., at least about
110.degree. C., at least about 115.degree. C., at least about
120.degree. C., at least about 125.degree. C., at least about
130.degree. C., at least about 135.degree. C., at least about
140.degree. C., at least about 145.degree. C., at least about
150.degree. C., at least about 155.degree. C., at least about
160.degree. C., at least about 165.degree. C., at least about
170.degree. C., or at least about 175.degree. C. (as measured under
dry conditions). Without wishing to be bound by theory, it is
believed that the underlying mechanism is that a polymer with a
higher T.sub.g generally has lower molecular mobility at room
temperature, which can be a crucial factor in stabilizing the
physical stability of the amorphous spray dry dispersion.
[0280] Additionally, the hygroscopicity of the polymers should be
as low, e.g., less than about 10%. For the purpose of comparison in
this application, the hygroscopicity of a polymer or composition is
characterized at about 60% relative humidity. In some preferred
embodiments, the polymer has less than about 10% water absorption,
for example less than about 9%, less than about 8%, less than about
7%, less than about 6%, less than about 5%, less than about 4%,
less than about 3%, or less than about 2% water absorption. The
hygroscopicity can also affect the physical stability of the spray
dry dispersions. Generally, moisture adsorbed in the polymers can
greatly reduce the T.sub.g of the polymers as well as the resulting
spray dry dispersions, which will further reduce the physical
stability of the spray dry dispersions as described above.
[0281] In one embodiment, the polymer is one or more water-soluble
polymer(s) or partially water-soluble polymer(s). Water-soluble or
partially water-soluble polymers include but are not limited to,
cellulose derivatives (e.g., hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC)) or ethylcellulose;
polyvinylpyrrolidones (PVP); polyethylene glycols (PEG); polyvinyl
alcohols (PVA); acrylates, such as polymethacrylate (e.g.,
Eudragit.RTM. E); cyclodextrins (e.g., .beta.-cyclodextin) and
copolymers and derivatives thereof, including for example PVP-VA
(polyvinylpyrollidone-vinyl acetate).
[0282] In some embodiments, the polymer is
hydroxypropylmethylcellulose (HPMC), such as HPMCAS, HPMC E50,
HPMCE15, or HPMC60SHSO).
[0283] As discussed herein, the polymer can be a pH-dependent
enteric polymer. Such pH-dependent enteric polymers include, but
are not limited to, cellulose derivatives (e.g., cellulose acetate
phthalate (CAP)), hydroxypropyl methyl cellulose phthalates
(HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS),
carboxymethylcellulose (CMC) or a salt thereof (e.g., a sodium salt
such as (CMC-Na)); cellulose acetate trimellitate (CAT),
hydroxypropylcellulose acetate phthalate (HPCAP),
hydroxypropylmethyl-cellulose acetate phthalate (HPMCAP), and
methylcellulose acetate phthalate (MCAP), or polymethacrylates
(e.g., Eudragit.RTM. S). In some embodiments, the polymer is
hydroxypropyl methyl cellulose acetate succinate (HPMCAS). In some
embodiments, the polymer is hydroxypropyl methyl cellulose acetate
succinate HG grade (HPMCAS-HG).
[0284] In yet another embodiment, the polymer is a
polyvinylpyrrolidone co-polymer, for example,
avinylpyrrolidone/vinyl acetate co-polymer (PVP/VA).
[0285] In embodiments where Compound 2 forms a spray dry dispersion
with a polymer, for example with an HPMC, HPMCAS, or PVP/VA
polymer, the amount of polymer relative to the total weight of the
spray dry dispersion ranges from about 0.1% to 99% by weight.
Unless otherwise specified, percentages of drug, polymer and other
excitpients as described within a dispersion are given in weight
percentages. The amount of polymer is typically at least about 20%,
and preferably at least about 30%, for example, at least about 35%,
at least about 40%, at least about 45%, or about 50% (e.g., 49.5%).
The amount is typically about 99% or less, and preferably about 80%
or less, for example about 75% or less, about 70% or less, about
65% or less, about 60% or less, or about 55% or less. In one
embodiment, the polymer is in an amount of up to about 50% of the
total weight of the dispersion (and even more specifically, between
about 40% and 50%, such as about 49%, about 49.5%, or about 50%).
HPMC and HPMCAS are available in a variety of grades from ShinEtsu,
for example, HPMCAS is available in a number of varieties,
including AS-LF, AS-MF, AS-HF, AS-LG, AS-MG, AS-HG. Each of these
grades vary with the percent substitution of acetate and
succinate.
[0286] In some embodiments, substantially amorphous Compound 2 and
polymer are present in roughly equal amounts, for example each of
the polymer and the drug make up about half of the percentage
weight of the dispersion. For example, the polymer is present in
about 49.5% and the drug is present in about 50%.
[0287] In some embodiments, substantially amorphous Compound 2 and
the polymer combined represent 1% to 20% w/w total solid content of
the non-spray dry dispersion prior to spray drying. In some
embodiments, substantially amorphous Compound 2 and the polymer
combined represent 5% to 15% w/w total solid content of the
non-spray dry dispersion prior to spray drying. In some
embodiments, substantially amorphous Compound 2 and the polymer
combined represent about 11% w/w total solid content of the
non-spray dry dispersion prior to spray drying.
[0288] In some embodiments, the dispersion further includes other
minor ingredients, such as a surfactant (e.g., SLS). In some
embodiments, the surfactant is present in less than about 10% of
the dispersion, for example less than about 9%, less than about 8%,
less than about 7%, less than about 6%, less than about 5%, less
than about 4%, less than about 3%, less than about 2%, about 1%, or
about 0.5%.
[0289] In embodiments including a polymer, the polymer should be
present in an amount effective for stabilizing the spray dry
dispersion. Stabilizing includes inhibiting or preventing, the
crystallization of substantially amorphous Compound 2. Such
stabilizing would inhibit the conversion Compound 2 from amorphous
to crystalline form. For example, the polymer would prevent at
least a portion (e.g., about 5%, about 10%, about 15%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,
about 55%, about 60%, about 65%, about 70%, about 75%, or greater)
of Compound 2 from converting from an amorphous to a crystalline
form. Stabilization can be measured, for example, by measuring the
glass transition temperature of the spray dry dispersion, measuring
the rate of relaxation of the amorphous material, or by measuring
the solubility or bioavailability of Compound 2.
[0290] Suitable polymers for use in combination with Compound 2,
for example to form a spray dry dispersion such as an amorphous
spray dry dispersion, should have one or more of the following
properties:
[0291] The glass transition temperature of the polymer should have
a temperature of no less than about 10-15.degree. C. lower than the
glass transition temperature of substantially amorphous Compound 2.
Preferably, the glass transition temperature of the polymer is
greater than the glass transition temperature of substantially
amorphous Compound 2, and in general at least 50.degree. C. higher
than the desired storage temperature of the drug product. For
example, at least about 100.degree. C., at least about 105.degree.
C., at least about 105.degree. C., at least about 110.degree. C.,
at least about 120.degree. C., at least about 130.degree. C., at
least about 140.degree. C., at least about 150.degree. C., at least
about 160.degree. C., at least about 160.degree. C., or
greater.
[0292] The polymer should be relatively non-hygroscopic. For
example, the polymer should, when stored under standard conditions,
absorb less than about 10% water, for example, less than about 9%,
less than about 8%, less than about 7%, less than about 6%, or less
than about 5%, less than about 4%, or less than about 3% water.
Preferably the polymer will, when stored under standard conditions,
be substantially free of absorbed water.
[0293] The polymer should have similar or better solubility in
solvents suitable for spray drying processes relative to that of
Compound 2. In preferred embodiments, the polymer will dissolve in
one or more of the same solvents or solvent systems as Compound 2.
It is preferred that the polymer is soluble in at least one
non-hydroxy containing solvent such as methylene chloride, acetone,
or a combination thereof.
[0294] The polymer, when combined with substantially amorphous
Compound 2, for example in a spray dry dispersion or in a liquid
suspension, should increase the solubility of Compound 2 in aqueous
and physiologically relative media either relative to the
solubility of Compound 2 in the absence of polymer or relative to
the solubility of Compound 2 when combined with a reference
polymer. For example, the polymer could increase the solubility of
amorphous Compound 2 by reducing the amount of amorphous Compound 2
that converts to crystalline Compound 2, either from a solid
amorphous dispersion or from a liquid suspension.
[0295] The polymer should decrease the relaxation rate of the
amorphous substance.
[0296] The polymer should increase the physical and/or chemical
stability of substantially amorphous Compound 2.
[0297] The polymer should improve the manufacturability of
substantially amorphous Compound 2.
[0298] The polymer should improve one or more of the handling,
administration or storage properties of substantially amorphous
Compound 2.
[0299] The polymer should not interact unfavorably with other
pharmaceutical components, for example excipients.
[0300] The suitability of a candidate polymer (or other component)
can be tested using the spray drying methods (or other methods)
described herein to form an amorphous composition. The candidate
composition can be compared in terms of stability, resistance to
the formation of crystals, or other properties, and compared to a
reference preparation, e.g., a preparation of neat amorphous
Compound 2 or crystalline Compound 2. For example, a candidate
composition could be tested to determine whether it inhibits the
time to onset of solvent mediated crystallization, or the percent
conversion at a given time under controlled conditions, by at least
50%, 75%, 100%, or 110% as well as the reference preparation, or a
candidate composition could be tested to determine if it has
improved bioavailability or solubility relative to crystalline
Compound 2.
[0301] Surfactants
[0302] The spray dry dispersion may include a surfactant. A
surfactant or surfactant mixture would generally decrease the
interfacial tension between the spray dry dispersion and an aqueous
medium. An appropriate surfactant or surfactant mixture may also
enhance aqueous solubility and bioavailability of Compound 2 from a
spray dry dispersion. The surfactants for use in connection with
the present invention include, but are not limited to, sorbitan
fatty acid esters (e.g., Spans.RTM.), polyoxyethylene sorbitan
fatty acid esters (e.g., Tweens.RTM.), sodium lauryl sulfate (SLS),
sodium dodecylbenzene sulfonate (SDBS) dioctyl sodium
sulfosuccinate (Docusate), dioxycholic acid sodium salt (DOSS),
Sorbitan Monostearate, Sorbitan Tristearate, hexadecyltrimethyl
ammonium bromide (HTAB), Sodium N-lauroylsarcosine, Sodium Oleate,
Sodium Myristate, Sodium Stearate, Sodium Palmitate, Gelucire
44/14, ethylenediamine tetraacetic acid (EDTA), Vitamin E d-alpha
tocopheryl polyethylene glycol 1000 succinate (TPGS), Lecithin, MW
677-692, Glutanic acid monosodium monohydrate, Labrasol, PEG 8
caprylic/capric glycerides, Transcutol, diethylene glycol monoethyl
ether, Solutol HS-15, polyethylene glycol/hydroxystearate,
Taurocholic Acid, Pluronic F68, Pluronic F108, and Pluronic F127
(or any other polyoxyethylene-polyoxypropylene co-polymers
(Pluronics.RTM.) or saturated polyglycolized glycerides
(Gelucirs.RTM.)). Specific example of such surfactants that may be
used in connection with this invention include, but are not limited
to, Span 65, Span 25, Tween 20, Capryol 90, Pluronic F108, sodium
lauryl sulfate (SLS), Vitamin E TPGS, pluronics and copolymers. SLS
is generally preferred.
[0303] The amount of the surfactant (e.g., SLS) relative to the
total weight of the spray dry dispersion may be between 0.1-15%.
Preferably, it is from about 0.5% to about 10%, more preferably
from about 0.5 to about 5%, e.g., about 0.5 to 4%, about 0.5 to 3%,
about 0.5 to 2%, about 0.5 to 1%, or about 0.5%.
[0304] In certain embodiments, the amount of the surfactant
relative to the total weight of the spray dry dispersion is at
least about 0.1%, preferably about 0.5%. In these embodiments, the
surfactant would be present in an amount of no more than about 15%,
and preferably no more than about 12%, about 11%, about 10%, about
9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%,
about 2% or about 1%. An embodiment wherein the surfactant is in an
amount of about 0.5% by weight is preferred.
[0305] Candidate surfactants (or other components) can be tested
for suitability for use in the invention in a manner similar to
that described for testing polymers.
Methods for Making the Pharmaceutical Compositions
[0306] The pharmaceutical compositions of the invention can be
produced by, wet granulation, compacting or compressing an
admixture or composition, for example, a powder or granules, under
pressure to form a stable three-dimensional shape (e.g., a tablet).
As used herein, "tablet" includes compressed pharmaceutical dosage
unit forms of all shapes and sizes, whether coated or uncoated.
[0307] The term "tablet" as used herein refers to a physically
discrete unit of agent appropriate for the patient to be treated.
In general, a compacted mixture has a density greater than that of
the mixture prior to compaction. A dosage tablet of the invention
can have almost any shape including concave and/or convex faces,
rounded or angled corners, and a rounded to rectilinear shape. In
some embodiments, the compressed tablets of the invention comprise
a rounded tablet having flat faces. The tablets of the invention
can be prepared by any compaction and compression method known by
persons of ordinary skill in the art of forming compressed solid
pharmaceutical dosage forms. In particular embodiments, the
formulations provided herein may be prepared using conventional
methods known to those skilled in the field of pharmaceutical
formulation, as described, e.g., in pertinent textbooks. See, e.g.,
Remington: The Science and Practice of Pharmacy, 21st Ed.,
Lippincott Williams & Wilkins, Baltimore, Md. (2003); Ansel et
al., Pharmaceutical Dosage Forms And Drug Delivery Systems, 7th
Edition, Lippincott Williams & Wilkins, (1999); The Handbook of
Pharmaceutical Excipients, 4.sup.th edition, Rowe et al., Eds.,
American Pharmaceuticals Association (2003); Gibson, Pharmaceutical
Preformulation And Formulation, CRC Press (2001), these references
hereby incorporated herein by reference in their entirety.
Granulation and Compression
[0308] In some embodiments, the ingredients are weighed according
to the formula set herein. Next, all of the intragranular
ingredients are sifted and mixed well. The ingredients can be
lubricated with a suitable lubricant, for example, magnesium
stearate. The next step can comprise compaction/slugging of the
powder admixture and sized ingredients. Next, the compacted or
slugged blends are milled into granules and sifted to obtain the
desired size. Next, the granules can be further lubricated with,
for example, magnesium stearate. Next the granular composition of
the invention can be compressed on suitable punches into various
pharmaceutical formulations in accordance with the invention.
Optionally the tablets can be coated with a film, colorant or other
coating.
[0309] Another aspect of the invention provides a method for
producing a pharmaceutical composition comprising providing an
admixture of a composition comprising Compound 1 Form I, a solid
dispersion comprising substantially amorphous Compound 2 and one or
more excipients selected from: a filler, a diluent, a binder, a
surfactant, a lubricant, a disintegrant, and compressing the
composition into a tablet having a dissolution of at least about
50% in about 30 minutes.
[0310] In another embodiment, a wet granulation process is
performed to yield the pharmaceutical formulation of the invention
from an admixture of powdered and liquid ingredients. For example,
a pharmaceutical composition comprising an admixture of a
composition comprising Compound 1 Form I, a solid dispersion
comprising substantially amorphous Compound 2, and one or more
excipients selected from: a filler, a binder, a surfactant, or a
disintegrant, are weighed as per the formula set herein. Next, all
of the intragranular ingredients are sifted and mixed in a high
shear or low shear granulator using water or water with a
surfactant or water with a binder or water with a surfactant and a
binder to granulate the powder blend. A fluid other than water can
also be used with or without surfactant and/or binder to granulate
the powder blend. Next, the wet granules can optionally be milled
using a suitable mill. Next, water may optionally be removed from
the admixture by drying the ingredients in any suitable manner.
Next, the dried granules can optionally be milled to the required
size. Next, extra granular excipients can be added by blending (for
example a filler, a diluent, and a disintegrant). Next, the sized
granules can be further lubricated with magnesium stearate and a
disintegrant, for example, croscarmellose sodium. Next the granular
composition of the invention can be sifted for sufficient time to
obtain the correct size and then compressed on suitable punches
into various pharmaceutical formulations in accordance with the
invention. Optionally, the tablets can be coated with a film,
colorant or other coating. Surprisingly, wet granulation can be
carried out without substantial loss of the solid state forms of
Compound 1 Form I or substantially amorphous Compound 2.
[0311] In a particularly favored embodiment, the pharmaceutical
compositions of the present invention are prepared by a continuous
twin screw wet granulation (TSWG) process. Continuous manufacturing
delivers high quality and highly consistent product with on-line
monitoring and control. Continuous manufacturing also facilitates
quality by design development with a "data rich" design space and
an easier to understand impact of upstream variables on the
downstream process and final product quality. In addition, the
pharmaceutical compositions of the present invention can be
finalized early on commercial scale equipment which avoids scale-up
risks and formulation changes late in development. Finally,
continuous manufacturing has commercial manufacturing advantages
such as improved process control, reduced product handling, and
real time release efficiencies. The overall result is a more
robust, controllable, and scalable process that has fewer process
checks resulting in increased product quality and therefore greater
patient safety. These advantages address Janet Woodcock's (director
of the Center for Drug Evaluation and Research (CDER)) concerns
that chemistry, manufacturing, and controls (CMC) won't be able to
keep up with rapid clinical development of highly effective
therapies ("What we are seeing is that often the rate limiting step
is going to be manufacturing," Jul. 24, 2013 Friends of Cancer
hosted congressional briefing "Answering a Compelling Need
Expediting Life-Saving Treatments to Patients" to discuss the Food
and Drug Administration's Breakthrough Therapy Designation).
[0312] For example, high shear granulation (HSG), a common
granulation technique is well known for the risk of
over-granulation and poor process control. Scale-up of this process
is very challenging and involves significant risk. Changing from a
HSG process to a continuous TSWG process, allows scale-up using the
same equipment to produce different batch sizes, by running for a
longer time. This eliminates the scale-up risk commonly encountered
with other granulation processes. Additionally, it was found that
the TSWG process is more robust, being less sensitive to
over-granulation. As can be seen in FIG. 3 for a Compound 1 tablet,
the HSG process showed significant dissolution slow-down with
increasing water content, while the TSWG process did not show a
change for a similar range of water addition. Surprisingly, no
performance changes were found with the tablet formulations
comprising Compound 1 between 45-55 percent by weight and the
tablet formulations comprising Compound 1 between 60-70 percent by
weight using the twin screw wet granulation process. This was not
the case with the HSG process. Additionally, this continuous and
increased product quality process addresses a common complaint by
the FDA regarding the lack of drug availability for patients in
need thereof.
[0313] In one embodiment the continuous process starts with feeding
individual excipients, Compound 1, and Compound 2 into a continuous
in-line blender through loss-in-weight feeding. From this blender,
the material is continuously conveyed and processed through twin
screw wet granulation, drying, milling, extra-granular excipient
addition, blending, compression and film coating.
[0314] For example, in one embodiment, a tablet comprising Compound
1 and Compound 2 may be prepared continuously according to FIG.
12.
[0315] Each of the ingredients of this exemplary admixture is
described above and in the Examples below. Furthermore, the
admixture can comprise optional additives, such as, one or more
colorants, one or more flavors, and/or one or more fragrances as
described above and in the Examples below. In some embodiments, the
relative concentrations (e.g., wt %) of each of these ingredients
(and any optional additives) in the admixture are also presented
above and in the Examples below. The ingredients constituting the
admixture can be provided sequentially or in any combination of
additions; and, the ingredients or combination of ingredients can
be provided in any order. In one embodiment, the lubricant is the
last component added to the admixture.
[0316] In another embodiment, the admixture comprises a composition
of Compound 1 Form I, a solid dispersion of substantially amorphous
Compound 2, and any one or more of the excipients; a binder, a
surfactant, a diluent, a lubricant, a disintegrant, and a filler,
wherein each of these ingredients is provided in a powder form
(e.g., provided as particles having a mean or average diameter,
measured by light scattering, of 250 .mu.m or less (e.g., 150 .mu.m
or less, 100 .mu.m or less, 50 .mu.m or less, 45 .mu.m or less, 40
.mu.m or less, or 35 .mu.m or less)).
[0317] In another embodiment, compressing the admixture into a
tablet is accomplished by filling a form (e.g., a mold) with the
admixture and applying pressure to admixture. This can be
accomplished using a die press or other similar apparatus. In some
embodiments, the admixture of Compound 1 Form I, a solid dispersion
comprising substantially amorphous Compound 2, and excipients can
be first processed into granular form. The granules can then be
sized and compressed into tablets or formulated for encapsulation
according to known methods in the pharmaceutical art. It is also
noted that the application of pressure to the admixture in the form
can be repeated using the same pressure during each compression or
using different pressures during the compressions. In another
example, the admixture of powdered ingredients or granules can be
compressed using a die press that applies sufficient pressure to
form a tablet having a dissolution of about 50% or more at about 30
minutes (e.g., about 55% or more at about 30 minutes or about 60%
or more at about 30 minutes). For instance, the admixture is
compressed using a die press to produce a tablet hardness of at
least about 5 kP (at least about 5.5 kP, at least about 6 kP, at
least about 7 kP, at least about 10 kP, or at least 15 kP). In some
instances, the admixture is compressed to produce a tablet hardness
of between about 5 and 20 kP.
[0318] In some embodiments, tablets comprising a pharmaceutical
composition as described herein can be coated with about 3.0 wt %
of a film coating comprising a colorant by weight of the tablet. In
certain instances, the colorant suspension or solution used to coat
the tablets comprises about 20% w/w of solids by weight of the
colorant suspension or solution. In still further instances, the
coated tablets can be labeled with a logo, other image or text.
[0319] In another embodiment, the method for producing a
pharmaceutical composition comprises providing an admixture of a
solid forms, e.g. an admixture of powdered and/or liquid
ingredients, the admixture comprising Compound 1 Form I, a solid
dispersion comprising substantially amorphous Compound 2, and one
or more excipients selected from: a binder, a diluent, a
surfactant, a lubricant, a disintegrant, and a filler; mixing the
admixture until the admixture is substantially homogenous, and
compressing or compacting the admixture into a granular form. Then
the granular composition comprising Compound 1 Form I and a solid
dispersion comprising substantially amorphous Compound 2 can be
compressed into tablets or formulated into capsules as described
above or in the Examples below. Alternatively, methods for
producing a pharmaceutical composition comprises providing an
admixture of Compound 1 Form I, a solid dispersion comprising
substantially amorphous Compound 2, and one or more excipients,
e.g. a binder, a diluent, a surfactant, a lubricant, a
disintegrant, and a filler; mixing the admixture until the
admixture is substantially homogenous, and compressing/compacting
the admixture into a granular form using a high shear wet granule
compaction process as set forth in the Examples below.
Pharmaceutical formulations, for example a tablet as described
herein, can be made using the granules prepared incorporating
Compound 1 Form I and a solid dispersion comprising substantially
amorphous Compound 2 in addition to the selected excipients
described herein.
[0320] In some embodiments, the admixture is mixed by stirring,
blending, shaking, or the like using hand mixing, a mixer, a
blender, any combination thereof, or the like. When ingredients or
combinations of ingredients are added sequentially, mixing can
occur between successive additions, continuously throughout the
ingredient addition, after the addition of all of the ingredients
or combinations of ingredients, or any combination thereof. The
admixture is mixed until it has a substantially homogenous
composition.
[0321] In another embodiment, the present invention comprises jet
milling a pharmaceutical composition comprising Compound 1 Form I
and a solid dispersion comprising substantially amorphous Compound
2 in a suitable, conventional milling apparatus using air pressure
suitable to produce particles having a significant particle size
fraction between 0.1 microns and 50 microns. In another embodiment,
the particle size is between 0.1 microns and 20 microns. In another
embodiment, the particles size is between 0.1 microns and 10
microns. In another embodiment, the particle size is between 1.0
microns and 5 microns. In still another embodiment, the
pharmaceutical composition has a particle size D50 of 2.0
microns.
[0322] The formulations of the present invention provide a fixed
dosage of two APIs for the effective treatment of cystic fibrosis,
a combination that has received one of only two Breakthrough
Therapy Designation from the FDA, and does so with surprising
stability as measured by the small loss of the amorphous solid form
of Compound 2. FIG. 4 depicts the small amount of crystallinity of
Compound 2 over time in PC-XVII at 50.degree. C. after
pre-equilibration at 60% relative humidity. Even after close to
1000 hours under these conditions, less than 5% by weight of
Compound 2 has crystallized. FIG. 5 shows for PC-XVII that even at
the higher temperature of 60.degree. C. after pre-equilibrating at
60% relative humidity, at close to 1000 hours under these
conditions, less than 10% by weight of Compound 2 has crystallized.
FIGS. 6 and 7 show similar results for PC-XIX. The present
formulations, therefore, provide the convenience of a fixed dosage
of two breakthrough API's in a surprisingly stable pharmaceutical
composition. Such formulations increase patient compliance which
directly relates to the effective treatment of diseases.
[0323] Dosage forms prepared as above can be subjected to in vitro
dissolution evaluations according to Test 711 "Dissolution" in
United States Pharmacopoeia 29, United States Pharmacopeial
Convention, Inc., Rockville, Md., 2005 ("USP"), to determine the
rate at which the active substance is released from the dosage
forms. The content of active substance and the impurity levels are
conveniently measured by techniques such as high performance liquid
chromatography (HPLC).
[0324] In some embodiments, the invention includes use of packaging
materials such as containers and closures of high-density
polyethylene (HDPE), low-density polyethylene (LDPE) and or
polypropylene and/or glass, glassine foil, aluminum pouches, and
blisters or strips composed of aluminum or high-density polyvinyl
chloride (PVC), optionally including a desiccant, polyethylene
(PE), polyvinylidene dichloride (PVDC), PVC/PE/PVDC, and the like.
These package materials can be used to store the various
pharmaceutical compositions and formulations in a sterile fashion
after appropriate sterilization of the package and its contents
using chemical or physical sterilization techniques commonly
employed in the pharmaceutical arts.
Methods for Administering the Pharmaceutical Compositions
[0325] In one aspect, the pharmaceutical compositions of the
invention can be administered to a patient once daily or about
every twenty four hours. Alternatively, the pharmaceutical
compositions of the invention can be administered to a patient
twice daily. Alternatively, the pharmaceutical composition of the
invention can be administered about every twelve hours. These
pharmaceutical compositions are administered as oral formulations
containing about 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250
mg, 300 mg, or 400 mg of Compound 1 Form I; and about 25 mg, 50 mg,
100 mg, 125 mg, 150 mg, 200 mg, or 250 mg of substantially
amorphous Compound 2. In this aspect, in addition to Compound 1
Form I and substantially amorphous Compound 2, the pharmaceutical
compositions comprise a filler; a disintegrant; a surfactant; a
binder; and a lubricant (depending on whether the pharmaceutical
composition is a granule or a tablet). For instance, a dose of 400
mg of Compound 1 Form I, may comprise two tablets of the invention
each containing 200 mg of Compound 1 Form I. A dose of 250 mg of
substantially amorphous Compound 2, may comprise two tablets of the
invention each containing 125 mg of substantially amorphous
Compound 2.
[0326] It will also be appreciated that the compound and
pharmaceutically acceptable compositions and formulations of the
invention can be employed in combination therapies; that is,
Compound 1 Form I and a solid dispersion of substantially amorphous
Compound 2 and pharmaceutically acceptable compositions thereof can
be administered concurrently with, prior to, or subsequent to, one
or more other desired therapeutics or medical procedures.
[0327] In one embodiment, the additional therapeutic agent is
selected from a mucolytic agent, bronchodialator, an antibiotic, an
anti-infective agent, an anti-inflammatory agent, a compound that
induces CFTR activity other than Compound 1 Form I and
substantially amorphous Compound 2, or a nutritional agent.
[0328] In one embodiment, the additional agent 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)cyclopropanecarbox-
amide. In another embodiment, the additional agent is
4-(3-(1(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)
isoquinolin-1-yl)benzoic acid. In another embodiment, the
additional agent is selected from Table 1:
TABLE-US-00024 TABLE 1 ##STR00010## 1 ##STR00011## 2 ##STR00012## 3
##STR00013## 4 ##STR00014## 5 ##STR00015## 6 ##STR00016## 7
##STR00017## 8 ##STR00018## 9 ##STR00019## 10 ##STR00020## 11
##STR00021## 12 ##STR00022## 13 ##STR00023## 14
[0329] In another embodiment, the additional agent is any
combination of the above agents. For example, the combination may
comprise a pharmaceutical composition or tablet of the present
invention comprising Compound 1 Form I and a solid dispersion of
substantially amorphous Compound 2, and the additional therapeutic
agent 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)cyclopropanecarbox-
amide. In another example, the combination may comprise a
pharmaceutical composition or tablet of the present invention
comprising Compound 1 Form I and a solid dispersion of
substantially amorphous Compound 2, and the additional therapeutic
agent is
4-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)
isoquinolin-1-yl)benzoic acid. In another example, the combination
may comprise a pharmaceutical composition or tablet of the present
invention comprising Compound 1 Form I and a solid dispersion of
substantially amorphous Compound 2, and the additional therapeutic
agent is any one of the compounds from Table 1, i.e. compounds 1
through 14 of Table 1, or any combination thereof.
[0330] In another embodiment, the additional agent is selected from
Table 1:
TABLE-US-00025 TABLE 1 Compounds disclosed in U.S. Pat. No.
7,407,976 (Col 13, ln 35- col 66, ln 67; Compounds 1-100 in Table 1
at col 67, ln 1-col 127, ln 42) Compounds disclosed in U.S. Pat.
No. 7,645,789 (Col 16, ln 52- col 50, ln 22; Compounds 1-322 in
Table 1 at col 50, ln 24-col 167, ln 42) Compounds disclosed in
U.S. Pat. No. 7,659,268 (Col 16, ln 20- col 70, ln 52; Compounds
1-528 in Table 1 at col 70, ln 53-col 331, ln 34) Compounds
disclosed in U.S. Pat. No. 7,671,221 (Col 16, ln 12- col 54, ln 48;
Compounds 1-1216 in Table 1 at col 54, ln 49-col 699, ln 27)
Compounds disclosed in U.S. Pat. No. 7,691,902 (Col 16, ln 11- col
54, ln 29; Compounds 1-959 in Table 1 at col 54, ln 29-col 683, ln
44) Compounds disclosed in U.S. Pat. No. 7,741,321 (Col 16, ln 25-
col 72, ln 17; Compounds 1-422 in Table 1 at col 72, ln 20-col 279,
ln 15) Compounds disclosed in U.S. Pat. No. 7,754,739 (Col 16, ln
1- col 22, ln 47; Compounds 1-2 in Table 1 at col 18, ln 26-65)
Compounds disclosed in U.S. Pat. No. 7,776,905 (Col 16, ln 23- col
38, ln 40; Compounds 1-306 in Table 1 at col 38, ln 45-col 96, ln
40) Compounds disclosed in U.S. Pat. No. 7,973,169 (Col 9, ln 16-
col 40, ln 40; Compounds 1-289 in Table 1 at col. 40, ln 41-col
289, ln 39) Compounds disclosed in U.S. Pat. No. 7,977,322 (Col 6,
ln 26- col 37, ln 47; Compounds 1-498 in Table 1 at col 37, ln
50-col 141, ln 40) Compounds disclosed in U.S. Pat. No. 7,999,113
(Col 6, ln 13- col 10, ln 67; Compounds 1-13 in Table 1 at col 11,
ln 5-col 13, ln 65) Compounds disclosed in U.S. Pat. No. 8,227,615
(Col 6, ln 10- col 29, ln 66; Compounds 1-78 in Table 1 at col 30,
ln 1-col 46, ln 48) Compounds disclosed in U.S. Pat. No. 8,299,099
(Col 6, ln 10- col 34, ln 18; Compounds 1-47 in Table 1 at col 34,
ln 20-col 42, ln 35) Compounds disclosed in US Published
Application No. 2006- 0052358 (Paragraphs [0034]-[0056];
[0077]-[0240]; Compounds 1-320 in Table 1 at paragraph [0241])
Compounds disclosed in US Published Application No. 2009- 0143381
(Paragraphs [0102]-[0263]; Compounds 1-28 in Table 1 at paragraph
[0264]) Compounds disclosed in US Published Application No. 2009-
0170905 (Paragraphs [0012]-[0013]; [0030]-[0051]) Compounds
disclosed in US Published Application No. 2009- 0253736 (Paragraphs
[0031]-[0162]; Compounds 1-15 in Table 1 at paragraph [0163])
Compounds disclosed in US Published Application No. 2011- 0263654
(Paragraphs [0012]-[0013]; [0066]-[0141]) Compounds disclosed in US
Published Application No. 2011- 0251253 (Paragraphs [0012]-[0013];
[0054]-[0079]) Compounds disclosed in PCT application WO2008141119
(Paragraphs [0100]-[0339]; Compounds 1-117 in Table 1 at paragraph
[0340]) Compounds disclosed in U.S. application Ser. No. 11/047,361
Compounds disclosed in US Published Application No. 2013- 0116238
(Paragraphs [0028]-[0044]; [0117]-[0128]), or combinations
thereof.
[0331] In another embodiment, the additional agent is selected from
Table 2:
TABLE-US-00026 TABLE 2 Compounds disclosed in US Published
Application No. 2005- 0113423 (Paragraph [00146]; Compounds
IA-1-IA-136 and Compounds I-1-I-21 in Tables 1 and 2 at paragraphs
[0391]-[0392]) Compounds disclosed in US Published Application No.
2005-0059687 (Paragraphs [00100]-[00101]; Compounds 1-405 in Table
1 at paragraph [0169]) Compounds 1-108 disclosed in U.S. Pat. No.
7,598,412 (Col 22, ln 14-col 79, ln 20; Table 1) Compounds 1-485
disclosed in U.S. Pat. No. 7,495,103 (Col 51, ln 1-col 63, ln 43;
Table 1) Compounds 1-718 disclosed in U.S. Pat. No. 8,354,427 (Col
51, ln 3-col 71, ln 46; Table 1) Compounds 1-233 disclosed in US
Published Application No. 2007-0105833 (Paragraph [00145]; Table 1)
Compounds 1-26 disclosed in U.S. Pat. No. 8,242,149 (Col 46, ln
47-col 57, ln 37; Table 1) Compounds 1-18 disclosed in U.S. Pat.
No. 8,314,256 (Col 21, ln 1-col 26, ln 19) Compounds 1-14 disclosed
in U.S. Pat. No. 8,399,479 (Col 36, ln 20-col 38, ln 40; Table 1)
Compounds 1-18 disclosed in U.S. Pat. No. 8,188,283 (Col 38, ln
43-col 43, ln 36; Table 1) Compounds 1-16 disclosed in US Published
Application No. 2010-0249180 (Paragraph [0173]; Table 1) Compounds
1-19 disclosed in US Published Application No. 2011-0008259
(Paragraph [0172]; Table 1) Compounds 1-129 disclosed in U.S. Pat.
No. 8,367,660 (Col 57, ln 31-col 81, ln 24; Table 1)
[0332] In one embodiment, the additional therapeutic agent is an
antibiotic. Exemplary antibiotics useful herein include tobramycin,
including tobramycin inhaled powder (TIP), azithromycin, cayston,
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.
[0333] In another embodiment, the additional agent is a mucolyte.
Exemplary mucolytes useful herein includes Pulmozyme.RTM..
[0334] In another embodiment, the additional agent is a
bronchodialator. Exemplary bronchodialtors include albuterol,
metaprotenerol sulfate, pirbuterol acetate, salmeterol, or
tetrabuline sulfate.
[0335] 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).
[0336] 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.
[0337] In another embodiment, the additional agent is a compound
that augments or induces CFTR activity other than Compound 1 Form I
or a solid dispersion comprising substantially amorphous Compound
2, i.e., an agent that has the effect of inducing or augmenting
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), and cobiprostone
(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxoo-
ctahydrocyclopenta[b]pyran-5-yl}heptanoic acid).
[0338] 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.
[0339] 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 augmenters or inducers 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.
[0340] In another embodiment, the additional agent is a compound
selected from
3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic
acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
5-amino-6'-methyl-3-trifluoromethyl-[2,3]bipyridinyl-6-carboxylic
acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-cyclopropyl-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(tri-
fluoromethyl)picolinamide;
3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propyl)-
-5-(trifluoro methyl)picolinamide;
3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic
acid ((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic
acid((S-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid
((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-(2,4-dichloro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic
acid ((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-(2,4-dichloro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic
acid ((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic
acid (2-hydroxy-2-methyl-propyl)-amide;
3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid
((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid
((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
(S)-3-amino-6-ethoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trif-
luoro methyl)picolinamide;
3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid
((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid
((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic
acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid
((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;
3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid
((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide, or
pharmaceutically acceptable salts thereof. In another embodiment,
the additional agent is a compound disclosed in U.S. Pat. No.
8,247,436 and International PCT Publication WO 2011113894,
incorporated herein in their entirety by reference.
[0341] In another embodiment, the additional agent may be an
epithelial sodium channel (ENac) modulator disclosed in PCT
publications WO2012035158, WO2009074575, WO2011028740,
WO2009150137, WO2011079087, or WO2008135557, incorporated herein in
their entirety by reference.
[0342] In other embodiments, the additional agent is a compound
disclosed in WO 2004028480, WO 2004110352, WO 2005094374, WO
2005120497, or WO 2006101740, incorporated herein in their entirety
by reference. In another embodiment, the additional agent is a
benzo[c]quinolizinium derivative that exhibits CFTR inducing or
augmenting activity or a benzopyran derivative that exhibits CFTR
inducing or augmenting activity. 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, incorporated herein in their entirety by reference.
In another embodiment, the additional agent is a compound disclosed
in WO2004080972, WO2004111014, WO2005035514, WO2005049018,
WO2006099256, WO2006127588, or WO2007044560, incorporated herein in
their entirety by reference.
[0343] In one embodiment, 400 mg of Compound 1 Form I and 250 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of one or more tablets of the invention.
For example, administration of 400 mg of Compound 1 Form I and 250
mg of substantially amorphous Compound 2 may be achieved by
administering two tablets each containing 200 mg of Compound 1 Form
I, and 125 mg of substantially amorphous Compound 2. The duration
of administration may continue until amelioration of the disease is
achieved or until a subject's physician advises, e.g. duration of
administration may be less than a week, 1 week, 2 weeks, 3 weeks,
four weeks (28 days), or a month or longer. In one embodiment, two
tablets each comprising 200 mg of Compound 1 Form I, and 125 mg of
substantially amorphous Compound 2 may be administered to the
patient per day. In a further embodiment, the two tablets may be
administered at the same time or at different times during the day.
In a further embodiment, one tablet is administered every 12
hours.
[0344] In one embodiment, 400 mg of Compound 1 Form I and 500 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of two tablets each containing 200 mg of
Compound 1 Form I, and 250 mg of substantially amorphous Compound
2. In one embodiment a tablet is administered once every 12 hours.
In another embodiment, the dosage amount may also be achieved by
administering two tablets, each containing 100 mg of Compound 1
Form I and 125 mg of substantially amorphous Compound 2, every 12
hours. In another embodiment, the dosage amounts may also be
achieved by administering Compound 1 Form I and substantially
amorphous Compound 2 in separate tablets. For example, the dosage
amounts may be achieved by administering two tablets containing 200
mg of Compound 1 Form I, and four tablets containing 125 mg of
substantially amorphous Compound 2 or two tablets containing 150 mg
of substantially amorphous Compound 2 and two tablets containing
100 mg of substantially amorphous Compound 2. The duration of
administration may continue until amelioration of the disease is
achieved or until a subject's physician advises, e.g. duration of
administration may be less than a week, 1 week, 2 weeks, 3 weeks,
four weeks (28 days), or a month or longer. In one embodiment, two
tablets comprising 200 mg of Compound 1 Form I, and four tablets
comprising 125 mg of substantially amorphous Compound 2 may be
administered to the patient per day. In one embodiment, two tablets
comprising 200 mg of Compound 1 Form I may be administered to the
patient per day, and two tablets comprising 150 mg and 100 mg of
substantially amorphous Compound 2 may be administered to the
patient twice per day. In a further embodiment, the two tablets may
be administered at the same time or at different times during the
day. In a further embodiment, one tablet comprising 200 mg of
Compound 1 is administered every 12 hours, and two tablets
comprising 150 mg and 100 mg of substantially amorphous Compound 2
are administered every 12 hours.
[0345] In one embodiment, 300 mg of Compound 1 Form 1 and 250 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of one or more tablets of the invention.
For example, administration of 300 mg of Compound 1 Form I and 250
mg of substantially amorphous Compound 2 may be achieved by
administering two tablets each containing 150 mg of Compound 1 Form
I, and 125 mg of substantially amorphous Compound 2. The duration
of administration may continue until amelioration of the disease is
achieved or until a subject's physician advises, e.g. duration of
administration may be less than a week, 1 week, 2 weeks, 3 weeks,
four weeks (28 days), or a month or longer. In one embodiment, two
tablets each comprising 150 mg of Compound 1 Form I, and 125 mg of
substantially amorphous Compound 2 may be administered to the
patient per day. In a further embodiment, the two tablets may be
administered at the same time or at different times during the day.
In a further embodiment, one tablet is administered every 12
hours.
[0346] In one embodiment, 600 mg of Compound 1 Form I and 500 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of one or more tablets of the invention.
For example, administration of 600 mg of Compound 1 Form I and 500
mg of substantially amorphous Compound 2 may be achieved by
administering two tablets, each containing 150 mg of Compound 1
Form I, and 125 mg of substantially amorphous Compound 2, every 12
hours. The duration of administration may continue until
amelioration of the disease is achieved or until a subject's
physician advises, e.g. duration of administration may be less than
a week, 1 week, 2 weeks, 3 weeks, four weeks (28 days), or a month
or longer. In one embodiment, four tablets each comprising 150 mg
of Compound 1 Form I, and 125 mg of substantially amorphous
Compound 2 may be administered to the patient per day. In a further
embodiment, the four tablets may be administered at the same time
or at different times during the day. In a further embodiment, two
tablet is administered every 12 hours.
[0347] In one embodiment, 800 mg of Compound 1 Form I and 500 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of one or more tablets of the invention.
For example, administration of 800 mg of Compound 1 Form I and 500
mg of substantially amorphous Compound 2 may be achieved by
administering four tablets each containing 200 mg of Compound 1
Form I, and 125 mg of substantially amorphous Compound 2. The
duration of administration may continue until amelioration of the
disease is achieved or until a subject's physician advises, e.g.
duration of administration may be less than a week, 1 week, 2
weeks, 3 weeks, four weeks (28 days), or a month or longer. In one
embodiment, four tablets each comprising 200 mg of Compound 1 Form
I, and 125 mg of substantially amorphous Compound 2 may be
administered to the patient per day. In a further embodiment, the
four tablets may be administered at the same time or at different
times during the day. In a further embodiment, two tablets are
administered per dosing occasion, and there are two dosing
occasions per day. In a further embodiment, 800 mg of Compound 1
and 500 mg of Compound 2 are administered to the patient by
administering two tablets each comprising 200 mg of Compound 1 and
125 mg of Compound 2 twice a day (BID). In a further embodiment,
800 mg of Compound 1 and 500 mg of Compound 2 are administered to
the patient by administering two tablets each comprising 200 mg of
Compound 1 and 125 mg of Compound 2 every 12 hours (q12h).
[0348] In one embodiment, 600 mg of Compound 1 Form I and 250 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of one or more tablets of the invention.
For example, administration of 600 mg of Compound 1 Form I and 250
mg of substantially amorphous Compound 2 may be achieved by
administering three tablets each containing 200 mg of Compound 1
Form I, and 83.3 mg of substantially amorphous Compound 2. The
duration of administration may continue until amelioration of the
disease is achieved or until a subject's physician advises, e.g.
duration of administration may be less than a week, 1 week, 2
weeks, 3 weeks, four weeks (28 days), or a month or longer. In one
embodiment, three tablets each comprising 200 mg of Compound 1 Form
I, and 83.3 mg of substantially amorphous Compound 2 may be
administered to the patient per day. In a further embodiment, the
three tablets may be administered at the same time or at different
times during the day. In a further embodiment, three tablets are
administered at the same time.
[0349] In one embodiment, 600 mg of Compound 1 Form I and 500 mg of
substantially amorphous Compound 2 may be administered to a subject
in need thereof. In these embodiments, the dosage amounts may be
achieved by administration of one or more tablets of the invention.
For example, administration of 600 mg of Compound 1 Form I and 500
mg of substantially amorphous Compound 2 may be achieved by
administering three tablets each containing 200 mg of Compound 1
Form I, and 83.3 mg of substantially amorphous Compound 2, followed
by two additional tablets each comprising 125 mg of Compound 2. The
duration of administration may continue until amelioration of the
disease is achieved or until a subject's physician advises, e.g.
duration of administration may be less than a week, 1 week, 2
weeks, 3 weeks, four weeks (28 days), or a month or longer. In one
embodiment, 600 mg of Compound 1 may be administered daily (qd) and
250 mg of Compound 2 administered twice a day (bid) by
administering three tablets each comprising 200 mg of Compound 1
Form I, and 83.3 mg of substantially amorphous Compound 2 daily
(qd) and two tablets each comprising 125 mg of Compound 2 every 12
hours (q12h). In one embodiment, 600 mg of Compound 1 may be
administered daily (qd) and 250 mg of Compound 2 administered every
12 hours (q12h) by administering three tablets each comprising 200
mg of Compound 1 Form I, and 83.3 mg of substantially amorphous
Compound 2 daily (qd) and two tablets each comprising 125 mg of
Compound 2 every 12 hours (q12h).
[0350] These combinations are useful for treating the diseases
described herein including cystic fibrosis. These combinations are
also useful in the kits described herein. In another aspect, the
present invention features a kit comprising a pharmaceutical
composition or tablet of the present invention comprising Compound
1 Form I and a solid dispersion comprising substantially amorphous
Compound 2, and a separate additional therapeutic agent or
pharmaceutical composition thereof. In another embodiment, the
pharmaceutical composition or tablet of the present invention,
separate additional therapeutic agent or pharmaceutical composition
thereof are in separate containers. In another embodiment, the
separate containers are bottles. In another embodiment, the
separate containers are vials. In another embodiment, the separate
containers are blister packs.
[0351] 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.
Therapeutic Uses of the Composition
[0352] In one aspect, the invention also provides a method of
treating, lessening the severity of, or symptomatically treating a
disease in a patient, the method comprising administering an
effective amount of the pharmaceutical composition or tablet of the
invention to the patient, preferably a mammal, wherein the disease
is selected from cystic fibrosis, asthma, smoke induced COPD,
chronic bronchitis, rhinosinusitis, constipation, pancreatitis,
pancreatic insufficiency, male infertility caused by congenital
bilateral absence of the vas deferens (CBAVD), mild pulmonary
disease, idiopathic pancreatitis, allergic bronchopulmonary
aspergillosis (ABPA), liver disease, hereditary emphysema,
hereditary hemochromatosis, coagulation-fibrinolysis deficiencies,
such as protein C deficiency, Type 1 hereditary angioedema, lipid
processing deficiencies, such as familial hypercholesterolemia,
Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, congenital hyperthyroidism,
osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT
deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic
DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,
neurodegenerative diseases such as Alzheimer's disease, Parkinson's
disease, amyotrophic lateral sclerosis, progressive supranuclear
plasy, Pick's disease, several polyglutamine neurological disorders
such as Huntington's, spinocerebullar ataxia type I, spinal and
bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic
dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease (due to prion protein
processing defect), Fabry disease, Straussler-Scheinker syndrome,
COPD, dry-eye disease, or Sjogren's disease, osteoporosis,
osteopenia, bone healing and bone growth (including bone repair,
bone regeneration, reducing bone resorption and increasing bone
deposition), Gorham's Syndrome, chloride channelopathies such as
myotonia congenita (Thomson and Becker forms), Bartter's syndrome
type III, Dent's disease, hyperekplexia, epilepsy, lysosomal
storage disease, Angelman syndrome, and Primary Ciliary Dyskinesia
(PCD), a term for inherited disorders of the structure and/or
function of cilia, including PCD with situs inversus (also known as
Kartagener syndrome), PCD without situs inversus and ciliary
aplasia.
[0353] In one aspect, the invention also provides a method of
treating, lessening the severity of, or symptomatically treating a
disease in a patient comprising administering an effective amount
of the pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the disease is selected from
generalized epilepsy with ferbrile seizures plus (GEFS+), general
epilepsy with ferbile and aferbrile seizures, myotonia,
paramyotonia congenital, potassium-aggravated myotonia,
hyperkalemic periodic paralysis, LQTS, LQTS/Brugada syndrome,
autosomal-dominant LQTS with deafness, autosomal-recessive LQTS,
LQTS with dysmorphic features, congenital and acquired LQTS,
Timothy syndrome, persistent hyperinsulinemic hypolglycemia of
infancy, dilated cardiomyopathy, autosomal-dominant LQTS, Dent
disease, Osteopetrosis, Bartter syndrome type III, central core
disease, malignant hyperthermia, and catecholaminergic polymorphic
tachycardia.
[0354] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation N1303K, .DELTA.I507, or R560T.
[0355] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation G551D. In another embodiment, the patient
is homozygous in G551D. In another embodiment, the patient is
heterozygous in G551D wherein the other CFTR genetic mutation is
any one of .DELTA.F508, G542X, N1303K, W1282X, R117H, R553X,
1717-1G->A, 621+1G->T, 2789+5G->A, 3849+10 kbC->T,
R1162X, G85E, 3120+1G->A, .DELTA.I507, 1898+1G->A, 3659delC,
R347P, R560T, R334W, A455E, 2184delA, or 711+1G->T.
[0356] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation .DELTA.F508. In another embodiment, the
patient is homozygous in .DELTA.F508. In another embodiment, the
patient is heterozygous in .DELTA.F508 wherein the other CFTR
genetic mutation is any one of G551D, G542X, N1303K, W1282X, R117H,
R553X, 1717-1G->A, 621+1G->T, 2789+5G->A, 3849+10
kbC->T, R1162X, G85E, 3120+1G->A, .DELTA.I507, 1898+1G->A,
3659delC, R347P, R560T, R334W, A455E, 2184delA, or
711+1G->T.
[0357] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V,
G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E,
D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H,
1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A,
711+1G->T, 2622+1G->A, 405+1G->A, 406-1G->A,
4005+1G->A, 1812-1G->A, 1525-1G->A, 712-1G->T,
1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+1G->T,
3850-1G->A, 2789+5G->A, 3849+10 kbC->T, 3272-26A->G,
711+5G->A, 3120G->A, 1811+1.6 kbA->G, 711+3A->G,
1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A,
1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A,
1898+1G->T, 4005+2T->C and 621+3A->G.
[0358] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V and
G1069R. In one embodiment of this aspect, the invention provides a
method of treating CFTR comprising administering Compound 1 to a
patient possessing a human CFTR mutation selected from G178R,
G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R and S1251N. In
one aspect, the present invention is directed to a method of
treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from E193K, F1052V and
G1069R. In some embodiments of this aspect, the method produces a
greater than 10-fold increase in chloride transport relative to
baseline chloride transport.
[0359] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from R117C, D110H, R347H,
R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,
F1074L, D110E, D1270N and D1152H. In one embodiment of this aspect,
the method produces an increase in chloride transport which is
greater or equal to 10% above the baseline chloride transport.
[0360] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 1717-1G->A,
621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T,
2622+1G->A, 405+1G->A, 406-1G->A, 4005+1G->A,
1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A,
1341+1G->A, 3121-1G->A, 4374+1G->T, 3850-1G->A,
2789+5G->A, 3849+10 kbC->T, 3272-26A->G, 711+5G->A,
3120G->A, 1811+1.6 kbA->G, 711+3A->G, 1898+3A->G,
1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C,
1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T,
4005+2T->C and 621+3A->G. In one aspect, the present
invention is directed to a method of treating, lessening the
severity of, or symptomatically treating cystic fibrosis in a
patient comprising administering an effective amount of the
pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the patient possesses the
CFTR genetic mutation is selected from 1717-1G->A, 1811+1.6
kbA->G, 2789+5G->A, 3272-26A->G and 3849+10 kbC->T. In
one aspect, the present invention is directed to a method of
treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 2789+5G->A and
3272-26A->G.
[0361] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V,
G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E,
D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H,
1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A,
711+1G->T, 2622+1G->A, 405+1G->A, 406-1G->A,
4005+1G->A, 1812-1G->A, 1525-1G->A, 712-1G->T,
1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+1G->T,
3850-1G->A, 2789+5G->A, 3849+10 kbC->T, 3272-26A->G,
711+5G->A, 3120G->A, 1811+1.6 kbA->G, 711+3A->G,
1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A,
1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A,
1898+1G->T, 4005+2T->C and 621+3A->G, and a human CFTR
mutation selected from .DELTA.F508, R117H, and G551D.
[0362] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V and
G1069R, and a human CFTR mutation selected from .DELTA.F508, R117H,
and G551D. In one aspect, the present invention is directed to a
method of treating, lessening the severity of, or symptomatically
treating cystic fibrosis in a patient comprising administering an
effective amount of the pharmaceutical composition or tablet of the
invention to the patient, preferably a mammal, wherein the patient
possesses the CFTR genetic mutation is selected from G178R, G551S,
G970R, G1244E, S1255P, G1349D, S549N, S549R and S1251N, and a human
CFTR mutation selected from .DELTA.F508, R117H, and G551D. In one
aspect, the present invention is directed to a method of treating,
lessening the severity of, or symptomatically treating cystic
fibrosis in a patient comprising administering an effective amount
of the pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the patient possesses the
CFTR genetic mutation is selected from E193K, F1052V and G1069R,
and a human CFTR mutation selected from .DELTA.F508, R117H, and
G551D. In some embodiments of this aspect, the method produces a
greater than 10-fold increase in chloride transport relative to
baseline chloride transport.
[0363] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from R117C, D110H, R347H,
R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,
F1074L, D110E, D1270N and D1152H, and a human CFTR mutation
selected from .DELTA.F508, R117H, and G551D. In one embodiment of
this aspect, the method produces an increase in chloride transport
which is greater or equal to 10% above the baseline chloride
transport.
[0364] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 1717-1G->A,
621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T,
2622+1G->A, 405+1G->A, 406-1G->A, 4005+1G->A,
1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A,
1341+1G->A, 3121-1G->A, 4374+1G->T, 3850-1G->A,
2789+5G->A, 3849+10 kbC->T, 3272-26A->G, 711+5G->A,
3120G->A, 1811+1.6 kbA->G, 711+3A->G, 1898+3A->G,
1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C,
1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T,
4005+2T->C and 621+3A->G, and a human CFTR mutation selected
from .DELTA.F508, R117H, and G551D. In one aspect, the present
invention is directed to a method of treating, lessening the
severity of, or symptomatically treating cystic fibrosis in a
patient comprising administering an effective amount of the
pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the patient possesses the
CFTR genetic mutation is selected from 1717-1G->A, 1811+1.6
kbA->G, 2789+5G->A, 3272-26A->G and 3849+10 kbC->T, and
a human CFTR mutation selected from .DELTA.F508, R117H, and G551D.
In one aspect, the present invention is directed to a method of
treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 2789+5G->A and
3272-26A->G, and a human CFTR mutation selected from
.DELTA.F508, R117H.
[0365] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V,
G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E,
D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H,
1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A,
711+1G->T, 2622+1G->A, 405+1G->A, 406-1G->A,
4005+1G->A, 1812-1G->A, 1525-1G->A, 712-1G->T,
1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+1G->T,
3850-1G->A, 2789+5G->A, 3849+10 kbC->T, 3272-26A->G,
711+5G->A, 3120G->A, 1811+1.6 kbA->G, 711+3A->G,
1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A,
1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A,
1898+1G->T, 4005+2T->C and 621+3A->G, and a human CFTR
mutation selected from .DELTA.F508, R117H, and G551D.
[0366] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V and
G1069R. In one aspect, the present invention is directed to a
method of treating, lessening the severity of, or symptomatically
treating cystic fibrosis in a patient comprising administering an
effective amount of the pharmaceutical composition or tablet of the
invention to the patient, preferably a mammal, wherein the patient
possesses the CFTR genetic mutation is selected from G178R, G551S,
G970R, G1244E, S1255P, G1349D, S549N, S549R and S1251N. In one
aspect, the present invention is directed to a method of treating,
lessening the severity of, or symptomatically treating cystic
fibrosis in a patient comprising administering an effective amount
of the pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the patient possesses the
CFTR genetic mutation is selected from E193K, F1052V and G1069R. In
some embodiments of this aspect, the method produces a greater than
10-fold increase in chloride transport relative to baseline
chloride transport.
[0367] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from R117C, D110H, R347H,
R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,
F1074L, D110E, D1270N and D1152H. In one embodiment of this aspect,
the method produces an increase in chloride transport which is
greater or equal to 10% above the baseline chloride transport.
[0368] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 1717-1G->A,
621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T,
2622+1G->A, 405+1G->A, 406-1G->A, 4005+1G->A,
1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A,
1341+1G->A, 3121-1G->A, 4374+1G->T, 3850-1G->A,
2789+5G->A, 3849+10 kbC->T, 3272-26A->G, 711+5G->A,
3120G->A, 1811+1.6 kbA->G, 711+3A->G, 1898+3A->G,
1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C,
1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T,
4005+2T->C and 621+3A->G. In one aspect, the present
invention is directed to a method of treating, lessening the
severity of, or symptomatically treating cystic fibrosis in a
patient comprising administering an effective amount of the
pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the patient possesses the
CFTR genetic mutation is selected from 1717-1G->A, 1811+1.6
kbA->G, 2789+5G->A, 3272-26A->G and 3849+10 kbC->T. In
one aspect, the present invention is directed to a method of
treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 2789+5G->A and
3272-26A->G.
[0369] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V,
G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E,
D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H,
1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A,
711+1G->T, 2622+1G->A, 405+1G->A, 406-1G->A,
4005+1G->A, 1812-1G->A, 1525-1G->A, 712-1G->T,
1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+1G->T,
3850-1G->A, 2789+5G->A, 3849+10 kbC->T, 3272-26A->G,
711+5G->A, 3120G->A, 1811+1.6 kbA->G, 711+3A->G,
1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A,
1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A,
1898+1G->T, 4005+2T->C and 621+3A->G, and a human CFTR
mutation selected from .DELTA.F508, R117H, and G551D, and one or
more human CFTR mutations selected from .DELTA.F508, R117H, and
G551D.
[0370] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from G178R, G551S, G970R,
G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V and
G1069R, and one or more human CFTR mutations selected from
.DELTA.F508, R117H, and G551D. In one aspect, the present invention
is directed to a method of treating, lessening the severity of, or
symptomatically treating cystic fibrosis in a patient comprising
administering an effective amount of the pharmaceutical composition
or tablet of the invention to the patient, preferably a mammal,
wherein the patient possesses the CFTR genetic mutation is selected
from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R and
S1251N, and one or more human CFTR mutations selected from
.DELTA.F508, R117H, and G551D. In one aspect, the present invention
is directed to a method of treating, lessening the severity of, or
symptomatically treating cystic fibrosis in a patient comprising
administering an effective amount of the pharmaceutical composition
or tablet of the invention to the patient, preferably a mammal,
wherein the patient possesses the CFTR genetic mutation is selected
from E193K, F1052V and G1069R, and one or more human CFTR mutations
selected from .DELTA.F508, R117H, and G551D. In some embodiments of
this aspect, the method produces a greater than 10-fold increase in
chloride transport relative to baseline chloride transport.
[0371] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from R117C, D110H, R347H,
R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,
F1074L, D110E, D1270N and D1152H, and one or more human CFTR
mutations selected from .DELTA.F508, R117H, and G551D. In one
embodiment of this aspect, the method produces an increase in
chloride transport which is greater or equal to 10% above the
baseline chloride transport.
[0372] In one aspect, the present invention is directed to a method
of treating, lessening the severity of, or symptomatically treating
cystic fibrosis in a patient comprising administering an effective
amount of the pharmaceutical composition or tablet of the invention
to the patient, preferably a mammal, wherein the patient possesses
the CFTR genetic mutation is selected from 1717-1G->A,
621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T,
2622+1G->A, 405+1G->A, 406-1G->A, 4005+1G->A,
1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A,
1341+1G->A, 3121-1G->A, 4374+1G->T, 3850-1G->A,
2789+5G->A, 3849+10 kbC->T, 3272-26A->G, 711+5G->A,
3120G->A, 1811+1.6 kbA->G, 711+3A->G, 1898+3A->G,
1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C,
1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T,
4005+2T->C and 621+3A->G, and one or more human CFTR
mutations selected from .DELTA.F508, R117H, and G551D. In one
aspect, the present invention is directed to a method of treating,
lessening the severity of, or symptomatically treating cystic
fibrosis in a patient comprising administering an effective amount
of the pharmaceutical composition or tablet of the invention to the
patient, preferably a mammal, wherein the patient possesses the
CFTR genetic mutation is selected from 1717-1G->A, 1811+1.6
kbA->G, 2789+5G->A, 3272-26A->G and 3849+10 kbC->T, and
one or more human CFTR mutations selected from .DELTA.F508, R117H,
and G551D. In one aspect, the present invention is directed to a
method of treating, lessening the severity of, or symptomatically
treating cystic fibrosis in a patient comprising administering an
effective amount of the pharmaceutical composition or tablet of the
invention to the patient, preferably a mammal, wherein the patient
possesses the CFTR genetic mutation is selected from 2789+5G->A
and 3272-26A->G, and one or more human CFTR mutations selected
from .DELTA.F508, R117H, and G551D.
[0373] In certain embodiments, the pharmaceutically acceptable
composition or tablet of the present invention comprising Compound
1 Form I and a solid dispersion of substantially amorphous Compound
2 are useful for treating, lessening the severity of, or
symptomatically treating cystic fibrosis in patients who exhibit
residual CFTR activity in the apical membrane of respiratory and
non-respiratory epithelia. The presence of residual CFTR activity
at the epithelial surface can be readily detected using methods
known in the art, e.g., standard electrophysiological, biochemical,
or histochemical techniques. Such methods identify CFTR activity
using in vivo or ex vivo electrophysiological techniques,
measurement of sweat or salivary Cl.sup.- 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, as well as
other mutations such as the G551D mutation, or the R117H mutation.
In certain embodiments, the pharmaceutically acceptable
compositions or tablets comprising Compound 1 Form I and a solid
dispersion comprising substantially amorphous Compound 2 are useful
for treating, lessening the severity of, or symptomatically
treating cystic fibrosis in patients who exhibit little to no
residual CFTR activity. In certain embodiments, the
pharmaceutically acceptable compositions or tablets comprising
Compound 1 Form I and a solid dispersion comprising substantially
amorphous Compound 2 are useful for treating, lessening the
severity of, or symptomatically treating cystic fibrosis in
patients who exhibit little to no residual CFTR activity in the
apical membrane of respiratory epithelia.
[0374] In another embodiment, the compounds and compositions of the
present invention are useful for treating or lessening the severity
of cystic fibrosis in patients who have residual CFTR activity
induced or augmented using pharmacological methods. In another
embodiment, the compounds and compositions of the present invention
are useful for treating or lessening the severity of cystic
fibrosis in patients who have residual CFTR activity induced or
augmented using or gene therapy. Such methods increase the amount
of CFTR present at the cell surface, thereby inducing a hitherto
absent CFTR activity in a patient or augmenting the existing level
of residual CFTR activity in a patient.
[0375] In one embodiment, pharmaceutical compositions and tablets
of the present invention comprising Compound 1 Form I and a solid
dispersion comprising substantially amorphous Compound 2, as
described herein, are useful for treating or lessening the severity
of cystic fibrosis in patients within certain genotypes exhibiting
residual CFTR activity, e.g., Class I mutations (not synthesized),
class II mutation (misfolding), class III mutations (impaired
regulation or gating), class IV mutations (altered conductance), or
class V mutations (reduced synthesis).
[0376] In one embodiment, pharmaceutical compositions and tablets
of the present invention comprising Compound 1 Form I and a solid
dispersion comprising substantially amorphous Compound 2, as
described herein, are useful for treating, lessening the severity
of, or symptomatically treating 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 sufficiency.
[0377] In one embodiment, pharmaceutical compositions and tablets
of the present invention comprising Compound 1 Form I and a solid
dispersion comprising substantially amorphous Compound 2, as
described herein, are useful for treating, lessening the severity
of, or symptomatically treating patients diagnosed with pancreatic
sufficiency, idiopathic pancreatitis and congenital bilateral
absence of the vas deferens, or mild lung disease wherein the
patient exhibits residual CFTR activity.
[0378] In one embodiment, pharmaceutical compositions and tablets
of the present invention comprising Compound 1 Form I and a solid
dispersion comprising substantially amorphous Compound 2, as
described herein, are useful for treating, lessening the severity
of, or symptomatically treating patients diagnosed with pancreatic
sufficiency, idiopathic pancreatitis and congenital bilateral
absence of the vas deferens, or mild lung disease wherein the
patient has wild type CFTR.
[0379] In addition to cystic fibrosis, modulation of CFTR activity
may be beneficial for other diseases not directly caused by
mutations in CFTR, such as secretory diseases and other protein
folding diseases mediated by CFTR. These include, but are not
limited to, chronic obstructive pulmonary disease (COPD), dry eye
disease, and Sjogren's Syndrome. COPD is characterized by airflow
limitation that is progressive and not fully reversible. The
airflow limitation is due to mucus hypersecretion, emphysema, and
bronchiolitis. Activators of mutant or wild-type CFTR offer a
potential treatment of mucus hypersecretion and impaired
mucociliary clearance that is common in COPD. Specifically,
increasing anion secretion across CFTR may facilitate fluid
transport into the airway surface liquid to hydrate the mucus and
optimized periciliary fluid viscosity. This would lead to enhanced
mucociliary clearance and a reduction in the symptoms associated
with COPD. Dry eye disease is characterized by a decrease in tear
aqueous production and abnormal tear film lipid, protein and mucin
profiles. There are many causes of dry eye, some of which include
age, Lasik eye surgery, arthritis, medications, chemical/thermal
burns, allergies, and diseases, such as cystic fibrosis and
Sjogrens's syndrome. Increasing anion secretion via CFTR would
enhance fluid transport from the corneal endothelial cells and
secretory glands surrounding the eye to increase corneal hydration.
This would help to alleviate the symptoms associated with dry eye
disease. Sjogrens's syndrome is an autoimmune disease in which the
immune system attacks moisture-producing glands throughout the
body, including the eye, mouth, skin, respiratory tissue, liver,
vagina, and gut. Symptoms, include, dry eye, mouth, and vagina, as
well as lung disease. The disease is also associated with
rheumatoid arthritis, systemic lupus, systemic sclerosis, and
polymypositis/dermatomyositis. Defective protein trafficking is
believed to cause the disease, for which treatment options are
limited. Augmenters or inducers of CFTR activity may hydrate the
various organs afflicted by the disease and help to elevate the
associated symptoms.
[0380] In one embodiment, the invention relates to a method of
augmenting or inducing anion channel activity in vitro or in vivo,
comprising contacting the channel with any one of pharmaceutical
compositions PC-I to PC-XXV. In another embodiment, the anion
channel is a chloride channel or a bicarbonate channel. In another
embodiment, the anion channel is a chloride channel.
[0381] 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 invention will be decided by the attending
physician within the scope of sound medical judgment. The specific
effective dose level for any particular patient or organism will
depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed, and like factors well known in the
medical arts. The term "patient", as used herein, means an animal,
preferably a mammal, and most preferably a human.
[0382] 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.
EXAMPLES
XRPD (X-Ray Powder Diffraction)
[0383] The X-Ray diffraction (XRD) data of Compound 1 Form I were
collected on a Bruker D8 DISCOVER powder diffractometer with
HI-STAR 2-dimensional detector and a flat graphite monochromator.
Cu sealed tube with K.alpha. radiation was used at 40 kV, 35 mA.
The samples were placed on zero-background silicon wafers at
25.degree. C. For each sample, two data frames were collected at
120 seconds each at 2 different .theta..sub.2 angles: 8.degree. and
26.degree.. The data were integrated with GADDS software and merged
with DIFFRACTP.sup.plusEVA software. Uncertainties for the reported
peak positions are .+-.0.2 degrees.
[0384] Differential Scanning calorimetry (DSC)
[0385] The Differential scanning calorimetry (DSC) data of Compound
1 Form I were collected using a DSC Q100 V9.6 Build 290 (TA
Instruments, New Castle, Del.). Temperature was calibrated with
indium and heat capacity was calibrated with sapphire. Samples of
3-6 mg were weighed into aluminum pans that were crimped using lids
with 1 pin hole. The samples were scanned from 25.degree. C. to
350.degree. C. at a heating rate of 1.0.degree. C./min and with a
nitrogen gas purge of 50 ml/min. Data were collected by Thermal
Advantage Q Series.TM. version 2.2.0.248 software and analyzed by
Universal Analysis software version 4.1D (TA Instruments, New
Castle, Del.). The reported numbers represent single analyses.
[0386] Compound 1 Form I Single Crystal Structure Determination
[0387] Diffraction data were acquired on Bruker Apex II
diffractometer equipped with sealed tube Cu K-alpha source and an
Apex II CCD detector. The structure was solved and refined using
SHELX program (Sheldrick, G. M., Acta Cryst., (2008) A64, 112-122).
Based on systematic absences and intensities statistics the
structure was solved and refined in P2.sub.1/n space group.
[0388] 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.
[0389] 2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was
purchased from Saltigo (an affiliate of the Lanxess
Corporation).
Preparation of Compound 1
Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol
##STR00024##
[0391] Commercially available
2,2-difluoro-1,3-benzodioxole-5-carboxylic acid (1.0 eq) was
slurried in toluene (10 vol). Vitride.RTM. (2 eq) was added via
addition funnel at a rate to maintain the temperature at
15-25.degree. C. At the end of the addition, the temperature was
increased to 40.degree. C. for 2 hours (h), then 10% (w/w) aqueous
(aq) NaOH (4.0 eq) was carefully added via addition funnel,
maintaining the temperature at 40-50.degree. C. After stirring for
an additional 30 minutes (min), the layers were allowed to separate
at 40.degree. C. The organic phase was 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 was used directly
in the next step.
Preparation of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole
##STR00025##
[0393] (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) was
dissolved in MTBE (5 vol). A catalytic amount of
4-(N,N-dimethyl)aminopyridine (DMAP) (1 mol %) was added and
SOCl.sub.2 (1.2 eq) was added via addition funnel. The SOCl.sub.2
was added at a rate to maintain the temperature in the reactor at
15-25.degree. C. The temperature was increased to 30.degree. C. for
1 h, and then was cooled to 20.degree. C. Water (4 vol) was added
via addition funnel while maintaining the temperature at less than
30.degree. C. After stirring for an additional 30 min, the layers
were allowed to separate. The organic layer was stirred and 10%
(w/v) aq NaOH (4.4 vol) was added. After stirring for 15 to 20 min,
the layers were allowed to separate. The organic phase was then
dried (Na.sub.2SO.sub.4), filtered, and concentrated to afford
crude 5-chloromethyl-2,2-difluoro-1,3-benzodioxole that was used
directly in the next step.
Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile
##STR00026##
[0395] A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole
(1 eq) in DMSO (1.25 vol) was added to a slurry of NaCN (1.4 eq) in
DMSO (3 vol), while maintaining the temperature between
30-40.degree. C. The mixture was stirred for 1 h, and then water (6
vol) was added, followed by methyl tert-butyl ether (MTBE) (4 vol).
After stirring for 30 min, the layers were separated. The aqueous
layer was extracted with MTBE (1.8 vol). The combined organic
layers were 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 was
used directly in the next step.
Synthesis of
(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile
##STR00027##
[0397] 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
##STR00028##
[0399] 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).
Preparation of
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile
##STR00029##
[0401] A mixture of
(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (1.0 eq), 50 wt %
aqueous KOH (5.0 eq) 1-bromo-2-chloroethane (1.5 eq), and
Oct.sub.4NBr (0.02 eq) was heated at 70.degree. C. for 1 h. The
reaction mixture was cooled, then worked up with MTBE and water.
The organic phase was washed with water and brine. The solvent was
removed to afford
(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile.
Preparation of
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic
acid
##STR00030##
[0403] (2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile
was hydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at
80.degree. C. overnight. The mixture was cooled to room temperature
and the ethanol was evaporated under vacuum. The residue was taken
up in water and MTBE, 1 M HCl was added, and the layers were
separated. The MTBE layer was then treated with dicyclohexylamine
(DCHA) (0.97 equiv). The slurry was cooled to 0.degree. C.,
filtered and washed with heptane to give the corresponding DCHA
salt. The salt was taken into MTBE and 10% citric acid and stirred
until all the solids had dissolved. The layers were separated and
the MTBE layer was washed with water and brine. A solvent swap to
heptane followed by filtration gave
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid
after drying in a vacuum oven at 50.degree. C. overnight.
Preparation of
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonyl
chloride
##STR00031##
[0405] 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic
acid (1.2 eq) is slurried in toluene (2.5 vol) and the mixture was
heated to 60.degree. C. SOCl.sub.2 (1.4 eq) was added via addition
funnel. The toluene and SOCl.sub.2 were distilled from the reaction
mixture after 30 minutes. Additional toluene (2.5 vol) was added
and the resulting mixture was distilled again, leaving the product
acid chloride as an oil, which was used without further
purification.
Preparation of tert-butyl-3-(3-methylpyridin-2-yl)benzoate
##STR00032##
[0407] 2-Bromo-3-methylpyridine (1.0 eq) was dissolved in toluene
(12 vol). K.sub.2CO.sub.3 (4.8 eq) was added, followed by water
(3.5 vol). The resulting mixture was heated to 65.degree. C. under
a stream of N.sub.2 for 1 hour. 3-(t-Butoxycarbonyl)phenylboronic
acid (1.05 eq) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (0.015 eq)
were then added and the mixture was heated to 80.degree. C. After 2
hours, the heat was turned off, water was added (3.5 vol), and the
layers were allowed to separate. The organic phase was then washed
with water (3.5 vol) and extracted with 10% aqueous methanesulfonic
acid (2 eq MsOH, 7.7 vol). The aqueous phase was made basic with
50% aqueous NaOH (2 eq) and extracted with EtOAc (8 vol). The
organic layer was concentrated to afford crude
tert-butyl-3-(3-methylpyridin-2-yl)benzoate (82%) that was used
directly in the next step.
Preparation of
2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide
##STR00033##
[0409] tent-Butyl-3-(3-methylpyridin-2-yl)benzoate (1.0 eq) was
dissolved in EtOAc (6 vol). Water (0.3 vol) was added, followed by
urea-hydrogen peroxide (3 eq). Phthalic anhydride (3 eq) was then
added portionwise to the mixture as a solid at a rate to maintain
the temperature in the reactor below 45.degree. C. After completion
of the phthalic anhydride addition, the mixture was heated to
45.degree. C. After stirring for an additional 4 hours, the heat
was turned off. 10% w/w aqueous Na.sub.2SO.sub.3 (1.5 eq) was added
via addition funnel. After completion of Na.sub.2SO.sub.3 addition,
the mixture was stirred for an additional 30 min and the layers
separated. The organic layer was stirred and 10% wt/wt aqueous.
Na.sub.2CO.sub.3 (2 eq) was added. After stirring for 30 minutes,
the layers were allowed to separate. The organic phase was washed
13% w/v aq NaCl. The organic phase was then filtered and
concentrated to afford crude
2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide (95%)
that was used directly in the next step.
Preparation of
tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate
##STR00034##
[0411] A solution of
2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide (1 eq)
and pyridine (4 eq) in acetonitrile (8 vol) was heated to
70.degree. C. A solution of methanesulfonic anhydride (1.5 eq) in
MeCN (2 vol) was added over 50 min via addition funnel while
maintaining the temperature at less than 75.degree. C. The mixture
was stirred for an additional 0.5 hours after complete addition.
The mixture was then allowed to cool to ambient. Ethanolamine (10
eq) was added via addition funnel. After stirring for 2 hours,
water (6 vol) was added and the mixture was cooled to 10.degree. C.
After stirring for 3 hours, the solid was collected by filtration
and washed with water (3 vol), 2:1 acetonitrile/water (3 vol), and
acetonitrile (2.times.1.5 vol). The solid was dried to constant
weight (<1% difference) in a vacuum oven at 50.degree. C. with a
slight N.sub.2 bleed to afford
tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate as a red-yellow
solid (53% yield).
Preparation of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-cyclopropanecarboxamido)-3-
-methylpyridin-2-yl)-t-butylbenzoate
##STR00035##
[0413] The crude acid chloride described above was dissolved in
toluene (2.5 vol based on acid chloride) and added via addition
funnel to a mixture of
tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate (1 eq), DMAP,
(0.02 eq), and triethylamine (3.0 eq) in toluene (4 vol based on
tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate). After 2
hours, water (4 vol based on
tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate) was added to
the reaction mixture. After stirring for 30 minutes, the layers
were separated. The organic phase was then filtered and
concentrated to afford a thick oil of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate (quantitative crude yield).
Acetonitrile (3 vol based on crude product) was added and distilled
until crystallization occurs. Water (2 vol based on crude product)
was added and the mixture stirred for 2 h. The solid was collected
by filtration, washed with 1:1 (by volume) acetonitrile/water
(2.times.1 volumes based on crude product), and partially dried on
the filter under vacuum. The solid was dried to a constant weight
(<1% difference) in a vacuum oven at 60.degree. C. with a slight
N.sub.2 bleed to afford
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate as a brown solid.
Preparation of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid.HCL salt
##STR00036##
[0415] To a slurry of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate (1.0 eq) in MeCN (3.0 vol) was
added water (0.83 vol) followed by concentrated aqueous HCl (0.83
vol). The mixture was heated to 45.+-.5.degree. C. After stirring
for 24 to 48 h, the reaction was complete, and the mixture was
allowed to cool to ambient. Water (1.33 vol) was added and the
mixture stirred. The solid was collected by filtration, washed with
water (2.times.0.3 vol), and partially dried on the filter under
vacuum. The solid was dried to a constant weight (<1%
difference) in a vacuum oven at 60.degree. C. with a slight N.sub.2
bleed to afford
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid.HCl as an off-white solid.
[0416] An .sup.1HNMR spectrum of Compound 1 is shown in FIG. 8 and
FIG. 9 depicts an .sup.1HNMR spectrum of Compound 1 as an HCl
salt.
[0417] Table 2 below recites the .sup.1H NMR data for Compound
I.
TABLE-US-00027 TABLE 2 Compound LC/MS LC/RT No M + 1 minutes NMR 1
453.3 1.93 .sup.1HNMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93
(m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H),
7.41-7.33 (m, 2H), 2.24 (s, 3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m,
2H).
[0418] Preparation of Compound 1 Form I
[0419] Preparation of Compound 1 Form I, Method A.
##STR00037##
[0420] A slurry of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)benzoic acid.HCl (1 eq) in water (10 vol) was
stirred at ambient temperature. A sample was taken after stirring
for 24 h. The sample was filtered and the solid was washed with
water (2 times). The solid sample was submitted for DSC analysis.
When DSC analysis indicated complete conversion to Form I, the
solid was collected by filtration, washed with water (2.times.1.0
vol), and partially dried on a filter under vacuum. The solid was
then dried to a constant weight (<1% difference) in a vacuum
oven at 60.degree. C. with a slight N.sub.2 bleed to afford
Compound 1 Form I as an off-white solid (98% yield). .sup.1H NMR
(400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m,
1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24
(s, 3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).
[0421] Preparation of Compound 1 Form I, Method B.
##STR00038##
[0422] A solution of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate (1.0 eq) in formic acid (3.0
vol) was heated with stirring to 70.+-.10.degree. C., for 8 h. The
reaction was deemed complete when no more than 1.0% AUC by
chromatographic methods of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate) remained. The mixture was
allowed to cool to ambient. The solution was added to water (6
vol), heated at 50.degree. C., and the mixture was stirred. The
mixture was then heated to 70.+-.10.degree. C. until the level of
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3--
methylpyridin-2-yl)-t-butylbenzoate was no more than 0.8% (AUC).
The solid was collected by filtration, washed with water (2.times.3
vol), and partially dried on the filter under vacuum. The solid was
dried to a constant weight (<1% difference) in a vacuum oven at
60.degree. C. with a slight N.sub.2 bleed to afford Compound 1 Form
I as an off-white solid.
[0423] The DSC trace of Compound 1 Form I is shown in FIG. 10.
Melting for Compound 1 Form I occurs at about 204.degree. C.
[0424] An X-ray diffraction pattern was calculated from a single
crystal structure of Compound 1 Form I and is shown in FIG. 1.
Table 3 lists the calculated peaks for FIG. 1.
TABLE-US-00028 TABLE 3 2.theta. Angle Relative Peak Rank [degrees]
Intensity [%] 11 14.41 48.2 8 14.64 58.8 1 15.23 100.0 2 16.11 94.7
3 17.67 81.9 7 19.32 61.3 4 21.67 76.5 5 23.40 68.7 9 23.99 50.8 6
26.10 67.4 10 28.54 50.1
[0425] An actual X-ray powder diffraction pattern of Compound 1
Form I is shown in FIG. 2. Table 4 lists the actual peaks for FIG.
2.
TABLE-US-00029 TABLE 4 2.theta. Angle Relative Peak Rank [degrees]
Intensity [%] 7 7.83 37.7 3 14.51 74.9 4 14.78 73.5 1 15.39 100.0 2
16.26 75.6 6 16.62 42.6 5 17.81 70.9 9 21.59 36.6 10 23.32 34.8 11
24.93 26.4 8 25.99 36.9
[0426] Colorless crystals of Compound 1 Form I were obtained by
cooling a concentrated 1-butanol solution from 75.degree. C. to
10.degree. C. at a rate of 0.2.degree. C./min. A crystal with
dimensions of 0.50.times.0.08.times.0.03 mm was selected, cleaned
with mineral oil, mounted on a MicroMount and centered on a Bruker
APEX II system. Three batches of 40 frames separated in reciprocal
space were obtained to provide an orientation matrix and initial
cell parameters. Final cell parameters were obtained and refined
based on the full data set.
[0427] A diffraction data set of reciprocal space was obtained to a
resolution of 0.82 .ANG. using 0.5.degree. steps using 30 s
exposure for each frame. Data were collected at 100 (2) K.
Integration of intensities and refinement of cell parameters were
accomplished using APEXII software. Observation of the crystal
after data collection showed no signs of decomposition.
[0428] A conformational picture of Compound 1 Form I based on
single crystal X-ray analysis is shown in FIG. 11. Compound 1 Form
I is monoclinic, P.sub.2l/n, with the following unit cell
dimensions: a=4.9626(7) .ANG., b=12.299(2) .ANG., c=33.075 (4)
.ANG., .beta.=93.938(9).degree., V=2014.0 .ANG..sup.3, Z=4. Density
of Compound 1 Form I calculated from structural data is 1.492
g/cm.sup.3 at 100 K.
Preparation of Compound 2
Synthesis of 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)
##STR00039##
[0430] Procedure for the preparation of ethyl
4-oxo-1,4-dihydroquinoline-3-carboxylate (25)
##STR00040##
[0431] Compound 23 (4.77 g, 47.7 mmol) was added dropwise to
compound 22 (10 g, 46.3 mmol) with subsurface N.sub.2 flow to drive
out ethanol below 30.degree. C. for 0.5 hours. The solution was
then heated to 100-110.degree. C. and stirred for 2.5 hours. After
cooling the mixture to below 60.degree. C., diphenyl ether was
added. The resulting solution was added dropwise to diphenyl ether
that had been heated to 228-232.degree. C. for 1.5 hours with
subsurface N.sub.2 flow to drive out ethanol. The mixture was
stirred at 228-232.degree. C. for another 2 hours, cooled to below
100.degree. C. and then heptane was added to precipitate the
product. The resulting slurry was stirred at 30.degree. C. for 0.5
hours. The solids were then filtrated, and the cake was washed with
heptane and dried in vacuo to give compound 25 as brown solid.
.sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta. 12.25 (s), .delta. 8.49
(d), .delta. 8.10 (m), .delta. 7.64 (m), .delta. 7.55 (m), .delta.
7.34 (m), .delta. 4.16 (q), .delta. 1.23 (t).
Procedure for the preparation of
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)
##STR00041##
[0432] Method 1
[0433] Compound 25 (1.0 eq) was suspended in a solution of HCl
(10.0 eq) and H.sub.2O (11.6 vol). The slurry was heated to
85-90.degree. C., although alternative temperatures are also
suitable for this hydrolysis step. For example, the hydrolysis can
alternatively be performed at a temperature of from about 75 to
about 100.degree. C. In some instances, the hydrolysis is performed
at a temperature of from about 80 to about 95.degree. C. In others,
the hydrolysis step is performed at a temperature of from about 82
to about 93.degree. C. (e.g., from about 82.5 to about 92.5.degree.
C. or from about 86 to about 89.degree. C.). After stirring at
85-90.degree. C. for approximately 6.5 hours, the reaction was
sampled for reaction completion. Stirring may be performed under
any of the temperatures suited for the hydrolysis. The solution was
then cooled to 20-25.degree. C. and filtered. The reactor/cake was
rinsed with H.sub.2O (2 vol.times.2). The cake was then washed with
2 vol H.sub.2O until the pH>3.0. The cake was then dried under
vacuum at 60.degree. C. to give compound 26.
Method 2
[0434] Compound 25 (11.3 g, 52 mmol) was added to a mixture of 10%
NaOH (aq) (10 mL) and ethanol (100 mL). The solution was heated to
reflux for 16 hours, cooled to 20-25.degree. C. and then the pH was
adjusted to 2-3 with 8% HCl. The mixture was then stirred for 0.5
hours and filtered. The cake was washed with water (50 mL) and then
dried in vacuo to give compound 26 as a brown solid. .sup.1H NMR
(DMSO-d.sub.6; 400 MHz) .delta. 15.33 (s), .delta. 13.39 (s),
.delta. 8.87 (s), .delta. 8.26 (m), .delta. 7.87 (m), .delta. 7.80
(m), .delta. 7.56 (m).
Total synthesis of
N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide (Compound 2)
##STR00042## ##STR00043##
[0435] Procedure for the preparation of 2,4-di-tert-butylphenyl
methyl carbonate (30)
##STR00044##
[0436] Method 1
[0437] To a solution of 2,4-di-tert-butyl phenol, 29, (10 g, 48.5
mmol) in diethyl ether (100 mL) and triethylamine (10.1 mL, 72.8
mmol), was added methyl chloroformate (7.46 mL, 97 mmol) dropwise
at 0.degree. C. The mixture was then allowed to warm to room
temperature and stir for an additional 2 hours. An additional 5 mL
triethylamine and 3.7 mL methyl chloroformate was then added and
the reaction stirred overnight. The reaction was then filtered, the
filtrate was cooled to 0.degree. C., and an additional 5 mL
triethylamine and 3.7 mL methyl chloroformate was then added and
the reaction was allowed to warm to room temperature and then stir
for an addition 1 hours. At this stage, the reaction was almost
complete and was worked up by filtering, then washing with water
(2.times.), followed by brine. The solution was then concentrated
to produce a yellow oil and purified using column chromatography to
give compound 30. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.35
(d, J=2.4 Hz, 1H), 7.29 (dd, J=8.4, 2.4 Hz, 1H), 7.06 (d, J=8.4 Hz,
1H), 3.85 (s, 3H), 1.30 (s, 9H), 1.29 (s, 9H).
Method 2
[0438] To a reactor vessel charged with 4-dimethylaminopyridine
(DMAP, 3.16 g, 25.7 mmol) and 2,4-ditert-butyl phenol (compound 29,
103.5 g, 501.6 mmol) was added methylene chloride (415 g, 313 mL)
and the solution was agitated until all solids dissolved.
Triethylamine (76 g, 751 mmol) was then added and the solution was
cooled to 0-5.degree. C. Methyl chloroformate (52 g, 550.3 mmol)
was then added dropwise over 2.5-4 hours, while keeping the
solution temperature between 0-5.degree. C. The reaction mixture
was then slowly heated to 23-28.degree. C. and stirred for 20
hours. The reaction was then cooled to 10-15.degree. C. and charged
with 150 mL water. The mixture was stirred at 15-20.degree. C. for
35-45 minutes and the aqueous layer was then separated and
extracted with 150 mL methylene chloride. The organic layers were
combined and neutralized with 2.5% HCl (aq) at a temperature of
5-20.degree. C. to give a final pH of 5-6. The organic layer was
then washed with water and concentrated in vacuo at a temperature
below 20.degree. C. to 150 mL to give compound 30 in methylene
chloride.
Procedure for the preparation of 5-nitro-2,4-di-tert-butylphenyl
methyl carbonate (31)
##STR00045##
[0439] Method 1
[0440] To a stirred solution of compound 30 (6.77 g, 25.6 mmol) was
added 6 mL of a 1:1 mixture of sulfuric acid and nitric acid at
0.degree. C. dropwise. The mixture was allowed to warm to room
temperature and stirred for 1 hour. The product was purified using
liquid chromatography (ISCO, 120 g, 0-7% EtOAc/Hexanes, 38 min)
producing about an 8:1-10:1 mixture of regioisomers of compound 31
as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.63
(s, 1H), 7.56 (s, 1H), 3.87 (s, 3H), 1.36 (s, 9H), 1.32 (s, 9H).
HPLC ret. time 3.92 min 10-99% CH.sub.3CN, 5 min run; ESI-MS 310
m/z (MH).sup.+.
Method 2
[0441] To compound 30 (100 g, 378 mmol) was added DCM (540 g, 408
mL). The mixture was stirred until all solids dissolved, and then
cooled to -5-0.degree. C. Concentrated sulfuric acid (163 g) was
then added dropwise, while maintaining the initial temperature of
the reaction, and the mixture was stirred for 4.5 hours. Nitric
acid (62 g) was then added dropwise over 2-4 hours while
maintaining the initial temperature of the reaction, and was then
stirred at this temperature for an additional 4.5 hours. The
reaction mixture was then slowly added to cold water, maintaining a
temperature below 5.degree. C. The quenched reaction was then
heated to 25.degree. C. and the aqueous layer was removed and
extracted with methylene chloride. The combined organic layers were
washed with water, dried using Na.sub.2SO.sub.4, and concentrated
to 124-155 mL. Hexane (48 g) was added and the resulting mixture
was again concentrated to 124-155 mL. More hexane (160 g) was
subsequently added to the mixture. The mixture was then stirred at
23-27.degree. C. for 15.5 hours, and was then filtered. To the
filter cake was added hexane (115 g), the resulting mixture was
heated to reflux and stirred for 2-2.5 hours. The mixture was then
cooled to 3-7.degree. C., stirred for an additional 1-1.5 hours,
and filtered to give compound 31 as a pale yellow solid.
Procedure for the preparation of 5-amino-2,4-di-tert-butylphenyl
methyl carbonate (32)
##STR00046##
[0443] 2,4-Di-tert-butyl-5-nitrophenyl methyl carbonate (1.00 eq)
was charged to a suitable hydrogenation reactor, followed by 5%
Pd/C (2.50 wt % dry basis, Johnson-Matthey Type 37). MeOH (15.0
vol) was charged to the reactor, and the system was closed. The
system was purged with N.sub.2 (g), and was then pressurized to 2.0
Bar with H.sub.2 (g). The reaction was performed at a reaction
temperature of 25.degree. C.+/-5.degree. C. When complete, the
reaction was filtered, and the reactor/cake was washed with MeOH
(4.00 vol). The resulting filtrate was distilled under vacuum at no
more than 50.degree. C. to 8.00 vol. Water (2.00 vol) was added at
45.degree. C.+/-5.degree. C. The resultant slurry was cooled to
0.degree. C.+/-5. The slurry was held at 0.degree. C.+/-5.degree.
C. for no less than 1 hour, and filtered. The cake was washed once
with 0.degree. C.+/-5.degree. C. MeOH/H.sub.2O (8:2) (2.00 vol).
The cake was dried under vacuum (-0.90 bar and -0.86 bar) at
35.degree. C.-40.degree. C. to give compound 32. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 7.05 (s, 1H), 6.39 (s, 1H), 4.80 (s,
2H), 3.82 (s, 3H), 1.33 (s, 9H), 1.23 (s, 9H).
[0444] Once the reaction was complete, the resulting mixture was
diluted with from about 5 to 10 volumes of MeOH (e.g., from about 6
to about 9 volumes of MeOH, from about 7 to about 8.5 volumes of
MeOH, from about 7.5 to about 8 volumes of MeOH, or about 7.7
volumes of MeOH), heated to a temperature of about 35.+-.5.degree.
C., filtered, washed, and dried, as described above.
Preparation of
N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide (Compound 2)
##STR00047##
[0446] 4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq)
and 5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq)
were charged to a reactor. 2-MeTHF (4.0 vol, relative to the acid)
was added followed by T3P 50% solution in 2-MeTHF (1.7 eq). The T3P
charged vessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq)
was then added, and the resulting suspension was heated to
47.5+/-5.0.degree. C. and held at this temperature for 8 hours. A
sample was taken and checked for completion by HPLC. Once complete,
the resulting mixture was cooled to 25.0.degree. C.+/-2.5.degree.
C. 2-MeTHF was added (12.5 vol) to dilute the mixture. The reaction
mixture was washed with water (10.0 vol) 2 times. 2-MeTHF was added
to bring the total volume of reaction to 40.0 vol (-16.5 vol
charged). To this solution was added NaOMe/MeOH (1.7 equiv) to
perform the methanolysis. The reaction was stirred for no less than
1.0 hour, and checked for completion by HPLC. Once complete, the
reaction was quenched with 1 N HCl (10.0 vol), and washed with 0.1
N HCl (10.0 vol). The organic solution was polish filtered to
remove any particulates and placed in a second reactor. The
filtered solution was concentrated at no more than 35.degree. C.
(jacket temperature) and no less than 8.0.degree. C. (internal
reaction temperature) under reduced pressure to 20 vol. CH.sub.3CN
was added to 40 vol and the solution concentrated at no more than
35.degree. C. (jacket temperature) and no less than 8.0.degree. C.
(internal reaction temperature) to 20 vol. The addition of
CH.sub.3CN and concentration cycle was repeated 2 more times for a
total of 3 additions of CH.sub.3CN and 4 concentrations to 20 vol.
After the final concentration to 20 vol, 16.0 vol of CH.sub.3CN was
added followed by 4.0 vol of H.sub.2O to make a final concentration
of 40 vol of 10% H.sub.2O/CH.sub.3CN relative to the starting acid.
This slurry was heated to 78.0.degree. C.+/-5.0.degree. C.
(reflux). The slurry was then stirred for no less than 5 hours. The
slurry was cooled to 0.0.degree. C.+/-5.degree. C. over 5 hours,
and filtered. The cake was washed with 0.0.degree. C.+/-5.0.degree.
C. CH.sub.3CN (5 vol) 4 times. The resulting solid (Compound 2) was
dried in a vacuum oven at 50.0.degree. C.+/-5.0.degree. C. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 12.8 (s, 1H), 11.8 (s, 1H), 9.2
(s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H), 7.8
(d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).
Alternative Preparation of
N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide (Compound 2)
##STR00048##
[0448] 4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq)
and 5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq)
were charged to a reactor. 2-MeTHF (4.0 vol, relative to the acid)
was added followed by T3P.RTM. 50% solution in 2-MeTHF (1.7 eq).
The T3P charged vessel was washed with 2-MeTHF (0.6 vol). Pyridine
(2.0 eq) was then added, and the resulting suspension was heated to
47.5+/-5.0.degree. C. and held at this temperature for 8 hours. A
sample was taken and checked for completion by HPLC. Once complete,
the resulting mixture was cooled to 20.degree. C.+/-5.degree. C.
2-MeTHF was added (12.5 vol) to dilute the mixture. The reaction
mixture was washed with water (10.0 vol) 2 times and 2-MeTHF (16.5
vol) was charged to the reactor. This solution was charged with 30%
w/w NaOMe/MeOH (1.7 equiv) to perform the methanolysis. The
reaction was stirred at 25.0.degree. C.+/-5.0.degree. C. for no
less than 1.0 hour, and checked for completion by HPLC. Once
complete, the reaction was quenched with 1.2 N HCl/H.sub.2O (10.0
vol), and washed with 0.1 N HCl/H.sub.2O (10.0 vol). The organic
solution was polish filtered to remove any particulates and placed
in a second reactor.
[0449] The filtered solution was concentrated at no more than
35.degree. C. (jacket temperature) and no less than 8.0.degree. C.
(internal reaction temperature) under reduced pressure to 20 vol.
CH.sub.3CN was added to 40 vol and the solution concentrated at no
more than 35.degree. C. (jacket temperature) and no less than
8.0.degree. C. (internal reaction temperature) to 20 vol. The
addition of CH.sub.3CN and concentration cycle was repeated 2 more
times for a total of 3 additions of CH.sub.3CN and 4 concentrations
to 20 vol. After the final concentration to 20 vol, 16.0 vol of
CH.sub.3CN was charged followed by 4.0 vol of H.sub.2O to make a
final concentration of 40 vol of 10% H.sub.2O/CH.sub.3CN relative
to the starting acid. This slurry was heated to 78.0.degree.
C.+/-5.0.degree. C. (reflux). The slurry was then stirred for no
less than 5 hours. The slurry was cooled to 20 to 25.degree. C.
over 5 hours, and filtered. The cake was washed with CH.sub.3CN (5
vol) heated to 20 to 25.degree. C. 4 times. The resulting solid
(Compound 2) was dried in a vacuum oven at 50.0.degree.
C.+/-5.0.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
12.8 (s, 1H), 11.8 (s, 1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H),
7.2 (s, 1H), 7.9 (t, 1H), 7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H),
1.4 (s, 9H), 1.4 (s, 9H).
Procedure for the recrystallization of
N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide (Compound 2)
##STR00049##
[0451] Compound 2 (1.0 eq) was charged to a reactor. 2-MeTHF (20.0
vol) was added followed by 0.1N HCl (5.0 vol). The biphasic
solution was stirred and separated and the top organic phase was
washed twice more with 0.1N HCl (5.0 vol). The organic solution was
polish filtered to remove any particulates and placed in a second
reactor. The filtered solution was concentrated at no more than
35.degree. C. (jacket temperature) and no more than 8.0.degree. C.
(internal reaction temperature) under reduced pressure to 10 vol.
Isopropyl acetate (IPAc) (10 vol) was added and the solution
concentrated at no more than 35.degree. C. (jacket temperature) and
no more than 8.0.degree. C. (internal reaction temperature) to 10
vol. The addition of IPAc and concentration was repeated 2 more
times for a total of 3 additions of IPAc and 4 concentrations to 10
vol. After the final concentration, 10 vol of IPAc was charged and
the slurry was heated to reflux and maintained at this temperature
for 5 hours. The slurry was cooled to 0.0.degree. C.+/-5.degree. C.
over 5 hours and filtered. The cake was washed with IPAc (5 vol)
once. The resulting solid was dried in a vacuum oven at
50.0.degree. C.+/-5.0.degree. C.
[0452] Preparation of a Solid Dispersion Comprising Substantially
Amorphous Compound 2
[0453] A solvent system of MEK and DI water, formulated according
to the ratio 90 wt % MEK/10 wt % DI water, was heated to a
temperature of 20-30.degree. C. in a reactor, equipped with a
magnetic stirrer and thermal circuit. Into this solvent system,
hypromellose acetate succinate polymer (HPMCAS)(HG grade), SLS, and
Compound 2 were added according to the ratio 19.5 wt % hypromellose
acetate succinate/0.5 wt % SLS/80 wt % Compound 2. The resulting
mixture contained 10.5 wt % solids. The actual amounts of
ingredients and solvents used to generate this mixture are recited
in Table 5, below:
TABLE-US-00030 TABLE 5 Solid Spray Dispersion Ingredients for
Intermediate F. Units Batch Compound 2 Kg 70.0 HPMCAS Kg 17.1 SLS
Kg 0.438 Total Solids Kg 87.5 MEK Kg 671 Water Kg 74.6 Total
Solvents Kg 746 Total Spray Solution Weight Kg 833
[0454] The mixture temperature was adjusted to a range of
20-45.degree. C. and mixed until it was substantially homogenous
and all components were substantially dissolved.
[0455] A spray drier, Niro PSD4 Commercial Spray Dryer, fitted with
pressure nozzle (Spray Systems Maximum Passage series SK-MFP having
orifice/core size 54/21) equipped with anti-bearding cap, was used
under normal spray drying mode, following the dry spray process
parameters recited in Table 6, below.
TABLE-US-00031 TABLE 6 Dry spray process parameters used to
generate Intermediate F. Parameter Value Feed Pressure 20 bar Feed
Flow Rate 92-100 Kg/hr Inlet Temperature 93-99.degree. C. Outlet
Temperature 53-57.degree. C. Vacuum Dryer Temperature 80.degree. C.
for 2 hours then 110.degree. C. (+/-5.degree. C.) Vacuum Drying
Time 20-24 hours
[0456] A high efficiency cyclone separated the wet product from the
spray gas and solvent vapors. The wet product contained 8.5-9.7%
MEK and 0.56-0.83% Water and had a mean particle size of 17-19 um
and a bulk density of 0.27-0.33 g/cc. The wet product was
transferred to a 4000L stainless steel double cone vacuum dryer for
drying to reduce residual solvents to a level of less than about
5000 ppm and to generate dry spray dry dispersion of amorphous
Compound 2, containing <0.03% MEK and 0.3% Water.
Tablet Formation from a Fully Continuous Wet Granulation
Process
[0457] Equipment/Process
[0458] Equipment
[0459] Fully Continuous Development and Launch Rig (DLR) or similar
type of equipment. Screening
[0460] Compound 1 Form I, the solid dispersion comprising
substantially amorphous Compound 2, and excipients may be dispensed
in separate intermediate bin containers (IBCs). These materials may
be screened using a "bin-to-bin" screening operation. Appropriate
screen sizes are mesh 20, mesh 40, or mesh 60.
[0461] Blending
[0462] The IBCs containing the screened Compound 1 Form I, the
solid dispersion comprising substantially amorphous Compound 2, and
excipients may be docked to the a feeder system, which can feed the
materials in a controlled manner, e.g. using volumetric or
gravimetric loss in weight feeders, into a continuous blender. The
feed rates of the individual components is defined by the
formulation composition and the overall line rate. The line rate
may be 8 kg/hr to 30 kg/hr. The continuous blender can have
different blade configurations to allow appropriate blending and
the rotational speed of these blades may be between 80 RPM and 300
RPM.
[0463] Wet Granulation
[0464] A granulation solution may be prepared by dissolving 48 g
sodium lauryl sulfate and 159 g polyvinylpyrrolidone in 1,626 g
water in a stainless steel container, using an overhead stirrer
with a stirring speed of 700 RPM. The granulation solution may be
placed in a container from which the solution may be pumped into
the twin screw granulator using a peristaltic pump with a mass flow
meter and control, using a flow rate that is appropriate for the
process. The blend may be granulated using a twin screw granulator
such as the granulator that is part of the DLR. The blend may be
added to the twin screw granulator using a Loss in Weight feeder,
such as the K-Tron feeder on the DLR, with a feed rate of 8 kg/hr
to 24 kg/hr. The twin screw granulator may be operated with a
barrel temperature of 25 degrees Celsius and a screw speed of 200
to 950 RPM. The granulation process may be performed for three
minutes for small batch sizes or several hours for large batch
sizes.
[0465] Drying
[0466] The wet granules may be fed directly into a fluid bed dryer,
such as the segmented fluid bed dryer on the DLR. The drying
end-point may be chosen at a product temperature during discharge
ranging from 40 to 55 degrees Celsius at which point the water
content of the granules may be 2.1% w/w ("Loss on Drying, LOD") or
less. The drying time may be 12 minutes, or shorter or longer, to
reach the desired drying endpoint.
[0467] Milling
[0468] The dried granules may be milled to reduce the size of the
granules. A cone mill such as the integrated Quadro U10 CoMil may
be used for this.
[0469] Blending
[0470] The granules may be blended with extra-granular excipients
such as fillers and lubricant using loss in weight feeders and a
continuous blender. The blending speed may be 80-300 RPM.
[0471] Compression
[0472] The compression blend may be compressed into tablets using a
single station or rotary tablet press, such as the Courtoy Modul P
press, which is part of the DLR system, using appropriately sized
tooling. The weight of the tablets for a dose of 200 mg of Compound
1 Form 1 and 125 mg of substantially amorphous Compound 2 may be
about 500 or 600 mg.
[0473] Film Coating
[0474] Tablets may be film coated using the innovative Omega film
coater, which is part of the DLR system. This coater enables fast
film coating of sub-batches of 1 to 4 kg to allow continuous
manufacturing.
[0475] Printing
[0476] Film coated tablets may be printed with a monogram on one or
both tablet faces with, for example, an Ackley ramp printer.
[0477] Tablet Formation from Twin Screw Wet Granulation Process
[0478] Equipment/Process
[0479] Equipment
[0480] Twin Screw Wet Granulators: ConsiGma-1, ConsiGma-25 or
Leistritz nano.
[0481] Screening/Weighing
[0482] Compound 1 Form I, the solid dispersion comprising
substantially amorphous Compound 2, and excipients may be screened
prior to or after weigh-out. Appropriate screen sizes are mesh 20,
mesh 40, or mesh 60. Compound 1 Form I and/or the solid dispersion
comprising substantially amorphous Compound 2 may be pre-blended
with one or more of the excipients to simplify screening.
[0483] Blending
[0484] Compound 1 Form I, the solid dispersion comprising
substantially amorphous Compound 2, and excipients may be added to
the blender in different order. The blending may be performed in a
Turbula blender, a v-shell blender, or a bin blender. The
components may be blended for 10 minutes.
[0485] Wet Granulation
[0486] A granulation solution may be prepared by dissolving 48 g
sodium lauryl sulfate and 159 g polyvinylpyrrolidone in 1,626 g
water in a stainless steel container, using an overhead stirrer
with a stirring speed of 700 RPM. The blend may be granulated using
a twin screw granulator such as the ConsiGma-1. The granulation
solution may be added to the twin screw granulator using a
peristaltic pump, such as the pump on the ConsiGma-1, with a feed
rate of 67 g/min. The blend may be added to the twin screw
granulator using a Loss in Weight feeder, such as the Brabender
feeder on the ConsiGma-1, with a feed rate of 10 kg/hr. The twin
screw granulator may be operated with a barrel temperature of 25
degrees Celsius and a screw speed of 400 RPM. The granulation
process may be performed for four minutes. The granulation process
may be performed for a shorter or longer duration of time to
produce a smaller or larger amount of wet granules.
[0487] Drying
[0488] The wet granules may be fed directly into a fluid bed dryer,
such as the drying chamber on the ConsiGma-1 or the segmented fluid
bed dryer on the CTL-25. The drying end-point may be chosen at a
product temperature of 43 degrees Celsius at which point the water
content of the granules may be 1.6% w/w ("Loss on Drying, LOD").
The drying time may be 12 minutes, or shorter or longer, to reach
the desired drying endpoint. The drying may be performed with an
air flow of 59 m.sup.3/min and inlet temperature of 60 degrees
Celsius. Alternatively, the wet granules coming from the twin screw
granulator may be collected into a bin or container for a certain
period of time after which the wet granules are transferred to a
separate stand-alone fluid bed dryer, such as the Vector Multi
15.
[0489] Milling
[0490] The dried granules may be milled to reduce the size of the
granules. A cone mill such as the Quadro 194 CoMil may be used for
this.
[0491] Blending
[0492] The granules may be blended with extra-granular excipients
such as fillers and lubricant using a V-shell blender or a bin
blender. The blending time may be 5, 3 or 1 minute(s).
[0493] Compression
[0494] The compression blend may be compressed into tablets using a
single station or rotary tablet press, such as the Courtoy Modul P
press, using 0.55.degree..times.0.33.degree. oval shaped tooling.
The weight of the tablets for a dose of 200 mg of Compound 1 Form I
and 125 mg of substantially amorphous Compound 2 may be about 500
or 600 mg.
[0495] Film Coating
[0496] Tablets may be film coated using a pan coater, such as, for
example a Thomas Engineering Compu-Lab coater. A trace amount of
Carnauba wax may be added to improve tablet appearance and process
ability.
[0497] Printing
[0498] Film coated tablets may be printed with a monogram on one or
both tablet faces with, for example, a Hartnett Delta printer.
[0499] Tablet Formation from Continuous Twin Screw Wet Granulation
Process
[0500] Equipment/Process
[0501] Equipment
[0502] Granulator: ConsiGma or Leistritz or Thermo Fisher twin
screw granulator.
[0503] Screening/Weighing
[0504] Compound 1 and excipients may be screened prior to or after
weigh-out. Possible screen sizes are mesh 20, mesh 40, or mesh 60.
Compound 1 may be pre-blended with one or more of the excipients to
simplify screening.
[0505] Blending
[0506] Compound 1 and excipients may be added to the blender in
different order. The blending may be performed in a Turbula
blender, a v-shell blender, a bin blender, or a continuous blender.
The components may be blended for 10 minutes for batch blenders or
continuously for a continuous blender.
[0507] Granulation Operation
[0508] Granulation Fluid--SLS and binder are added to purified
water and mixed until dissolved. A suitable ratio is 2.5% w/w SLS
and 10.0% w/w PVP K30 in water.
[0509] Granulation--The blend containing Compound 1 and excipients
may be dosed into the twin screw granulator using a Loss in Weight
feeder at a rate of 10 kg/hr. The granulation fluid may be added
using a peristaltic pump at a rate of 3.5 kg/hr. The granulator may
be run at a speed of 400 RPM. A notable advantage of the present
twin screw wet granulation process is using a granulation fluid
that comprises both a surfactant and the binder for better
granulation through increased wettability. In one embodiment, the
surfactant is SLS. Another notable advantage is that because the
process is continuous and at any moment in time only a limited
amount of material is processed, the process can be well controlled
and results in a high quality product.
[0510] Milling
[0511] The granules may be reduced in size using a screen mill or a
cone mill, either before drying or after drying, or both.
[0512] Drying
[0513] The granules may be dried using a vacuum oven, tray dryer,
bi-conical dryer, or fluid bed drier.
[0514] Blending
[0515] The granules may be blended with extra-granular excipients.
The granules have been blended using a 300 liter bin blender for 60
revolutions.
[0516] Compression
[0517] The compression blend has been compressed into tablets using
a Courtoy Modul P rotary press
[0518] Film Coating
[0519] Tablets may be film coated using a pan coater, such as, for
example an O'Hara Labcoat.
[0520] Printing
[0521] Film coated tablets may be printed with a monogram on one or
both tablet faces with, for example, a Hartnett Delta printer.
Assays
[0522] Protocol 1
[0523] Assays for Detecting and Measuring .DELTA.F508-CFTR
Potentiation Properties of Compounds
[0524] Membrane Potential Optical Methods for Assaying
.DELTA.F508-CFTR Modulation Properties of Compounds
[0525] The assay utilizes fluorescent voltage sensing dyes to
measure changes in membrane potential using a fluorescent plate
reader (e.g., FLIPR III, Molecular Devices, Inc.) as a readout for
increase in functional .DELTA.F508-CFTR in NIH 3T3 cells. The
driving force for the response is the creation of a chloride ion
gradient in conjunction with channel activation by a single liquid
addition step after the cells have previously been treated with
compounds and subsequently loaded with a voltage sensing dye.
Identification of Potentiator Compounds
[0526] To identify potentiators of .DELTA.F508-CFTR, a
double-addition HTS assay format was developed. This HTS assay
utilizes fluorescent voltage sensing dyes to measure changes in
membrane potential on the FLIPR III as a measurement for increase
in gating (conductance) of .DELTA.F508 CFTR in
temperature-corrected .DELTA.F508 CFTR NIH 3T3 cells. The driving
force for the response is a Cl.sup.- ion gradient in conjunction
with channel activation with forskolin in a single liquid addition
step using a fluorescent plate reader such as FLIPR III after the
cells have previously been treated with potentiator compounds (or
DMSO vehicle control) and subsequently loaded with a redistribution
dye.
[0527] Solutions
[0528] 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.
[0529] Chloride-free bath solution: Chloride salts in Bath Solution
#1 (above) are substituted with gluconate salts.
Cell Culture
[0530] 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 .about.20,000/well
in 384-well matrigel-coated plates and cultured for 2 hrs at
37.degree. C. before culturing at 27.degree. C. for 24 hrs. for the
potentiator assay. For the correction assays, the cells are
cultured at 27.degree. C. or 37.degree. C. with and without
compounds for 16-24 hours.
[0531] Electrophysiological Assays for assaying .DELTA.F508-CFTR
modulation properties of compounds.
Using Chamber Assay
[0532] Using chamber experiments were performed on polarized airway
epithelial cells expressing .DELTA.F508-CFTR to further
characterize the .DELTA.F508-CFTR augmenters or inducers identified
in the optical assays. Non-CF and CF airway epithelia were isolated
from bronchial tissue, cultured as previously described (Galietta,
L. J. V., Lantero, S., Gazzolo, A., Sacco, O., Romano, L., Rossi,
G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev. Biol. 34,
478-481), and plated onto Costar.RTM. Snapwell.TM. filters that
were precoated with NIH3T3-conditioned media. After four days the
apical media was removed and the cells were grown at an air liquid
interface for >14 days prior to use. This resulted in a
monolayer of fully differentiated columnar cells that were
ciliated, features that are characteristic of airway epithelia.
Non-CF HBE were isolated from non-smokers that did not have any
known lung disease. CF-HBE were isolated from patients homozygous
for .DELTA.F508.
[0533] HBE grown on Costar.RTM. Snapwell.TM. cell culture inserts
were mounted in an Using chamber (Physiologic Instruments, Inc.,
San Diego, Calif.), and the transepithelial resistance and
short-circuit current in the presence of a basolateral to apical
Cl.sup.- gradient (I.sub.SC) were measured using a voltage-clamp
system (Department of Bioengineering, University of Iowa, Iowa).
Briefly, HBE were examined under voltage-clamp recording conditions
(V.sub.hold=0 mV) at 37.degree. C. The basolateral solution
contained (in mM) 145 NaCl, 0.83 K.sub.2HPO.sub.4, 3.3
KH.sub.2PO.sub.4, 1.2 MgCl.sub.2, 1.2 CaCl.sub.2, 10 Glucose, 10
HEPES (pH adjusted to 7.35 with NaOH) and the apical solution
contained (in mM) 145 NaGluconate, 1.2 MgCl.sub.2, 1.2 CaCl.sub.2,
10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).
Identification of Potentiator Compounds
[0534] Typical protocol utilized a basolateral to apical membrane
Cl.sup.- concentration gradient. To set up this gradient, normal
ringers was used on the basolateral membrane, whereas apical NaCl
was replaced by equimolar sodium gluconate (titrated to pH 7.4 with
NaOH) to give a large Cl.sup.- concentration gradient across the
epithelium. Forskolin (10 .mu.M) and all test compounds were added
to the apical side of the cell culture inserts. The efficacy of the
putative .DELTA.F508-CFTR potentiators was compared to that of the
known potentiator, genistein.
Patch-Clamp Recordings
[0535] Total Cl.sup.- current in .DELTA.F508-NIH3T3 cells was
monitored using the perforated-patch recording configuration as
previously described (Rae, J., Cooper, K., Gates, P., & Watsky,
M. (1991) J. Neurosci. Methods 37, 15-26). Voltage-clamp recordings
were performed at 22.degree. C. using an Axopatch 200B patch-clamp
amplifier (Axon Instruments Inc., Foster City, Calif.). The pipette
solution contained (in mM) 150 N-methyl-D-glucamine (NMDG)-Cl, 2
MgCl.sub.2, 2 CaCl.sub.2, EGTA, 10 HEPES, and 240 .mu.g/mL
amphotericin-B (pH adjusted to 7.35 with HCl). The extracellular
medium contained (in mM) 150 NMDG-Cl, 2 MgCl.sub.2, 2 CaCl.sub.2,
10 HEPES (pH adjusted to 7.35 with HCl). Pulse generation, data
acquisition, and analysis were performed using a PC equipped with a
Digidata 1320 A/D interface in conjunction with Clampex 8 (Axon
Instruments Inc.). To activate .DELTA.F508-CFTR, 10 .mu.M forskolin
and 20 .mu.M genistein were added to the bath and the
current-voltage relation was monitored every 30 sec.
Identification of Potentiator Compounds
[0536] The ability of .DELTA.F508-CFTR potentiators to increase the
macroscopic .DELTA.F508-CFTR Cl.sup.- current (I.sub..DELTA.F508)
in NIH3T3 cells stably expressing .DELTA.F508-CFTR was also
investigated using perforated-patch-recording techniques. The
potentiators identified from the optical assays evoked a
dose-dependent increase in I.DELTA..sub.F508 with similar potency
and efficacy observed in the optical assays. In all cells examined,
the reversal potential before and during potentiator application
was around -30 mV, which is the calculated E.sub.Cl (-28 mV).
Cell Culture
[0537] 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.
Single-Channel Recordings
[0538] Gating activity of wt-CFTR and temperature-corrected
.DELTA.F508-CFTR expressed in NIH3T3 cells was observed using
excised inside-out membrane patch recordings as previously
described (Dalemans, W., Barbry, P., Champigny, G., Jallat, S.,
Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J-P.,
Lazdunski, M. (1991) Nature 354, 526-528) using an Axopatch 200B
patch-clamp amplifier (Axon Instruments Inc.). The pipette
contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl.sub.2, 2
MgCl.sub.2, and 10 HEPES (pH adjusted to 7.35 with Tris base). The
bath contained (in mM): 150 NMDG-Cl, 2 MgCl.sub.2, 5 EGTA, 10 TES,
and 14 Tris base (pH adjusted to 7.35 with HCl). After excision,
both wt- and .DELTA.F508-CFTR were activated by adding 1 mM Mg-ATP,
75 nM of the catalytic subunit of cAMP-dependent protein kinase
(PKA; Promega Corp. Madison, Wis.), and 10 mM NaF to inhibit
protein phosphatases, which prevented current rundown. The pipette
potential was maintained at 80 mV. Channel activity was analyzed
from membrane patches containing .ltoreq.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.
Cell Culture
[0539] 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.
[0540] Protocol 2
[0541] Assays for Detecting and Measuring .DELTA.F508-CFTR
Correction Properties of Compounds
[0542] Membrane potential optical methods for assaying
.DELTA.F508-CFTR modulation properties of compounds.
[0543] 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).
[0544] 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.
Identification of Correction Compounds
[0545] 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.
Identification of Potentiator Compounds
[0546] 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
Cl.sup.--free medium containing 2-10 .mu.M forskolin was added to
activate .DELTA.F508-CFTR. The extracellular Cl.sup.- 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.
Solutions
[0547] 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. [0548] Chloride-free bath
solution: Chloride salts in Bath Solution #1 (above) are
substituted with gluconate salts. [0549] CC2-DMPE: Prepared as a 10
mM stock solution in DMSO and stored at -20.degree. C. [0550]
DiSBAC.sub.2(3): Prepared as a 10 mM stock in DMSO and stored at
-20.degree. C.
Cell Culture
[0551] 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.
[0552] Electrophysiological Assays for assaying .DELTA.F508-CFTR
modulation properties of compounds
Using Chamber Assay
[0553] Using chamber experiments were performed on polarized
epithelial cells expressing .DELTA.F508-CFTR to further
characterize the .DELTA.F508-CFTR augmenters or inducers identified
in the optical assays. FRT.sup..DELTA.F508-CFTR epithelial cells
grown on Costar Snapwell cell culture inserts were mounted in an
Using 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 KS.sup..about.2/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.).
Identification of Correction Compounds
[0554] 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).
[0555] 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.
Identification of Potentiator Compounds
[0556] 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.
Solutions
[0557] 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.
[0558] Apical solution (in mM): Same as basolateral solution with
NaCl replaced with Na Gluconate (135).
Cell Culture
[0559] Fisher rat epithelial (FRT) cells expressing
.DELTA.F508-CFTR (FRT.sup..DELTA.F508-CFTR) were used for Using
chamber experiments for the putative .DELTA.F508-CFTR augmenters or
inducers 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.
Whole-Cell Recordings
[0560] 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) 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.
Identification of Correction Compounds
[0561] 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.
Identification of Potentiator Compounds
[0562] 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).
Solutions
[0563] 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). [0564] 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).
Cell Culture
[0565] 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.
Single-Channel Recordings
[0566] 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.
[0567] 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.
Solutions
[0568] 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). [0569] 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).
Cell Culture
[0570] 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.
[0571] Compound 1 and Compound 2 of the invention are useful as
augmenters or inducers of CFTR activity. Table 5 below illustrates
the EC50 and relative efficacy of Compound 1 and Compound 2. In
Table 5 below, the following meanings apply. EC50: "+++" means
<10 uM; "++" means between 10 uM to 25 uM; "+" means between 25
uM to 60 uM. % Efficacy: "+" means <25%; "++" means between 25%
to 100%; "+++" means >100%.
TABLE-US-00032 TABLE 5 EC50 Cmpd. No. (.mu.M) % Activity 1 +++ +++
2 +++ ++
Other Embodiments
[0572] All publications and patents referred to in this disclosure
are incorporated herein by reference to the same extent as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Should the
meaning of the terms in any of the patents or publications
incorporated by reference conflict with the meaning of the terms
used in this disclosure, the meaning of the terms in this
disclosure are intended to be controlling. Furthermore, the
foregoing discussion discloses and describes merely exemplary
embodiments of the invention. One skilled in the art will readily
recognize from such discussion and from the accompanying drawings
and claims, that various changes, modifications and variations can
be made therein without departing from the spirit and scope of the
invention as defined in the following claims.
* * * * *
References