Pharmaceutical compositions for treating CFTR-mediated diseases
阅读说明:本技术 用于治疗cftr介导的疾病的药物组合物 (Pharmaceutical compositions for treating CFTR-mediated diseases ) 是由 M·J·沃维耶斯 R·卡尔卡里 M·D·摩尔 于 2013-11-01 设计创作,主要内容包括:公开了包含3-(6-(1-(2,2-二氟苯并[d][1,3]间二氧杂环戊烯-5-基)环丙烷甲酰氨基)-3-甲基吡啶-2-基)苯甲酸(化合物1)形式I和包含基本上无定形的N-(5-羟基-2,4-二叔丁基-苯基)-4-氧代-1H-喹啉-3-甲酰胺(化合物2)的固体分散体的药物组合物;治疗CFTR介导的疾病例如囊性纤维化、减轻其严重性或对症治疗它们的方法;其施用方法和药盒。(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-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (compound 2) are disclosed; methods of treating, lessening the severity of, or symptomatically treating CFTR mediated diseases such as cystic fibrosis; methods of administration and kits thereof.)
1. A pharmaceutical composition comprising: 200mg of form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (Compound 1) and a solid dispersion comprising substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (Compound 2);
wherein the pharmaceutical composition comprises 125mg of substantially amorphous compound 2.
2. A tablet, comprising: form I of a fixed dose of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (compound 1) and a solid dispersion comprising substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (compound 2);
wherein the tablet comprises 25-50% by weight of compound 1 form I and 15-35% by weight of a solid dispersion comprising substantially amorphous compound 2.
3. A pharmaceutical composition comprising:
(a) about 100mg of form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (Compound 1) and about 125mg of substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (Compound 2);
(b) about 150mg of form I of compound 1 and about 200mg of substantially amorphous compound 2; or
(c) About 75mg of form I of compound 1 and about 100mg of substantially amorphous compound 2.
Use of form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (Compound 1) and substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (Compound 2) in the manufacture of a medicament for the treatment of cystic fibrosis in a patient, wherein the medicament is administered to the patient in an amount of 800mg of form I of Compound 1 and 500mg of substantially amorphous Compound 2 per day.
Use of form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (Compound 1) and substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (Compound 2) in the manufacture of a medicament for the treatment of cystic fibrosis in a patient, wherein the medicament is administered to the patient in an amount of 400mg of form I of Compound 1 and 500mg of substantially amorphous Compound 2 per day.
6. A method of preparing a pharmaceutical composition comprising wet granulating:
form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (Compound 1);
b. a solid dispersion comprising substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (compound 2);
c. a filler;
d. a disintegrant;
e. a surfactant; and
f. and (3) an adhesive.
7. A process for preparing a tablet comprising compressing:
i) a multiparticulate pharmaceutical composition comprising:
form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (Compound 1);
b. a solid dispersion comprising substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (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.
8. A continuous process for preparing tablets comprising:
a) mixing form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (compound 1), a solid dispersion comprising substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (compound 2), a filler, and a disintegrant in a blender to form a blend;
b) preparing a granulation liquid with water, a binder and a surfactant;
c) feeding the blend from step a) into a continuous twin screw granulator while adding the granulation liquid from step b) to produce granules;
d) drying the granules obtained from step c) and grinding them;
e) blending the milled granules from step d) with a filler, a disintegrant, and a lubricant to form a blend; and
f) compressing the blend from step e) into tablets.
Technical Field
The present invention relates to pharmaceutical compositions, methods of treatment, methods of preparation, methods of administration, and kits thereof comprising form I of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid (compound 1) and a solid dispersion comprising substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide (compound 2).
Background
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 advances in the treatment of CF, there is still no cure.
In patients with CF, mutations in the endogenously expressed CFTR in respiratory epithelial cells cause reduced apical anion secretion, resulting in an imbalance in ion and fluid transport. The resulting decrease in anion transport promotes increased mucus accumulation in the lung, and concomitant microbial infection, which ultimately leads to death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency, which if left untreated, can lead to death. In addition, most men with cystic fibrosis are unable to give birth, while women with cystic fibrosis have a reduced fertility. In contrast to the severe effects of two copies of the CF-associated gene, individuals with a single copy of the CF-associated gene show increased resistance to cholera and dehydration due to diarrhea-perhaps explaining the relatively high frequency of the CF gene in the population.
Sequence analysis of the CFTR gene of the CF chromosome has revealed a number of causative mutations (Cutting, G.R. et al (1990) Nature 346; 366-. To date, over 1000 pathogenic mutations in the CF gene have been identified (http;/www.genet.sickkids.on.ca/cftr/app). The most common mutation is the deletion of phenylalanine at position 508 of the amino acid sequence of CFTR, commonly referred to as Δ F508-CFTR. This mutation occurs in approximately 70% of cystic fibrosis cases and is associated with severe disease.
Deletion of residue 508 in Δ F508-CFTR prevents proper folding of the nascent protein. This results in the inability of the mutein to exit the ER and be transported to the plasma membrane. As a result, the number of channels present in the membrane is much less than that observed in cells expressing wild-type CFTR. In addition to impaired transport, this mutation leads to defective channel gating. Together, the reduced number of channels in the membrane and defective gating lead to reduced anion transport through the epithelium, resulting in defective ion and fluid transport. (Quinton, P.M, (1990), FASEB J.4; 2709-. However, studies have shown that the reduction in the number of Δ F508-CFTR in the membrane is functional, although it is less than wild-type CFTR. (Dalemans et al (1991), Nature Lond.354; 526-. In addition to af 508-CFTR, other disease-causing mutations in CFTR that result in defective transport, synthesis, and/or channel gating can be up-or down-regulated to alter anion secretion, and alter disease progression and/or severity.
Compounds that are potentiators of CFTR (e.g., compound 2) and compounds that are correctors of CFTR (e.g., compound 1) have independently been demonstrated to have utility in the treatment of CFTR-related diseases (e.g., cystic fibrosis).
Thus, there is a need for new therapeutic approaches to CFTR mediated diseases involving CFTR corrector and potentiator compounds.
In particular, there is a need for combination therapies comprising CFTR potentiators and corrector compounds for the treatment of CFTR mediated diseases (e.g., cystic fibrosis).
More specifically, there is a need for combination therapies comprising a CFTR potentiator compound (e.g., substantially amorphous compound 2) in combination with a CFTR corrector compound (e.g.,
In addition, patients with compliance with treatment regimens and dosages rely primarily on ease of administration of the drug. Pharmaceutical compositions comprising fixed doses of a CFTR corrector and a CFTR potentiator, wherein the solid forms of the corrector and potentiator are stable, are significant breakthroughs for the treatment of CFTR mediated diseases, such as cystic fibrosis.
Disclosure of Invention
The invention is characterized in that: a pharmaceutical composition comprising: 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid,
substantially amorphous N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide in solid dispersion,
a
methods of treatment of the pharmaceutical compositions; a preparation method; a method of administration; and a kit thereof.
In one aspect, the invention features a pharmaceutical composition comprising:
a.
b. a solid dispersion comprising substantially
c. a filler;
d. a disintegrant;
e. a surfactant; and
f. a binder;
referred to as PC-I.
In one embodiment, the pharmaceutical composition of the invention comprises 30 to 55 weight percent of
In one embodiment, the filler is selected from cellulose, modified cellulose, sodium carboxymethylcellulose, ethylcellulose, sodium hydroxymethylcellulose, 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 of 10-20 weight percent.
In one embodiment, the disintegrant is selected from the group consisting of agar, algae, calcium carbonate, carboxymethyl cellulose, hydroxypropyl cellulose, low substituted hydroxypropyl cellulose, clay, croscarmellose sodium, crospovidone, gum, magnesium aluminum silicate, methyl cellulose, polacrilin potassium, sodium alginate, sodium starch glycolate, corn starch, potato starch, tapioca starch, or any combination thereof. In another embodiment, the disintegrant is croscarmellose sodium and is present in an amount of 1-3 weight percent.
In one embodiment, the surfactant is selected from sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene sorbitan monooleate, or any combination thereof. In another embodiment, the surfactant is sodium lauryl sulfate and is present in an amount of 0.5 to 2 weight percent.
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 of 0 to 5 weight percent.
In one embodiment, the invention features a pharmaceutical composition of the following formulation:
referred to as PC-II.
In another aspect, the invention features a pharmaceutical composition that includes:
a.
b. a solid dispersion comprising substantially
c. a filler;
d. a disintegrant;
e. a surfactant;
f. a binder; and
g. a lubricant;
referred to as PC-III.
In one embodiment, the pharmaceutical composition of the invention comprises about 100-250 mg of
In one embodiment, the pharmaceutical composition of the invention comprises 25-50 weight percent of
In one embodiment, the filler is selected from cellulose, modified cellulose, sodium carboxymethyl cellulose, ethyl cellulose sodium hydroxymethyl cellulose, hydroxypropyl cellulose, 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 of 20 to 30 weight percent.
In one embodiment, the disintegrant is selected from the group consisting of agar, algae, calcium carbonate, carboxymethyl cellulose, hydroxypropyl cellulose, low substituted hydroxypropyl cellulose, clay, croscarmellose sodium, crospovidone, gum, magnesium aluminum silicate, methyl cellulose, polacrilin potassium, sodium alginate, sodium starch glycolate, corn starch, potato starch, tapioca starch, or any combination thereof. In another embodiment, the disintegrant is croscarmellose sodium and is present in an amount of 3 to 10 weight percent.
In one embodiment, the surfactant is selected from sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene sorbitan monooleate, or any combination thereof. In another embodiment, the surfactant is sodium lauryl sulfate and is present in an amount of 0.5 to 2 weight percent.
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 of 0 to 5 weight percent.
In one embodiment, the lubricant is selected from magnesium stearate, calcium stearate, zinc stearate, sodium stearate, 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 of 0.5-2 weight percent.
In one embodiment, the invention features a pharmaceutical composition of the following formulation:
referred to as PC-IV.
In one embodiment, the pharmaceutical composition of the present invention further comprises a colorant and optionally a wax. In another embodiment, the colorant of the present invention is present in an amount of 2 to 4 weight percent. In another embodiment, the wax is carnauba wax present in an amount of 0 to 0.020 weight percent.
In one embodiment, the pharmaceutical composition of the present invention is a solid oral pharmaceutical composition. In another embodiment, the solid oral pharmaceutical composition is a granular pharmaceutical composition or a tablet.
In one embodiment, the particulate composition of the present invention has the following formulation:
referred to as PC-V.
In one embodiment, the particulate composition of the present invention has the following formulation:
referred to as PC-VI.
In one embodiment, the particulate composition of the present invention has the following formulation:
referred to as PC-VII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-VIII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-IX.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-X.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XI.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XIII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XIV.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XV.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XVI.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XVII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XVIII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XIX.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XX.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XXI.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XXII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XXIII.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XXIV.
In one embodiment, the tablet of the present invention has the following formulation:
referred to as PC-XXV.
In one aspect, the invention features a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition, particulate pharmaceutical composition, or tablet of the invention.
In embodiments, the invention features a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition, granular pharmaceutical composition, or tablet of any of the formulations PC-I-PC-XXV.
In one embodiment, the patient has the Δ F508CFTR mutation. In another embodiment, the patient is homozygous for Δ F508. In another embodiment, the patient is heterozygous for Δ F508. In another embodiment, two tablets are administered to the patient daily.
In one aspect, the invention features a method of making a granular pharmaceutical composition comprising wet granulated:
a.
b. a solid dispersion comprising substantially
c. a filler;
d. a disintegrant;
e. a surfactant; and
f. and (3) an adhesive.
In one aspect, the invention features a method of making a tablet, comprising compressing:
i) a multiparticulate pharmaceutical composition comprising:
a.
b. a solid dispersion comprising substantially
c. a filler;
d. a disintegrant;
e. a surfactant; and
f. a binder;
ii) a disintegrant;
iii) a filler; and
iv) a lubricant.
In one aspect, the invention features a kit comprising a pharmaceutical composition, granular pharmaceutical composition, or tablet of the invention and a therapeutic agent alone or a pharmaceutical composition thereof.
In one embodiment, the pharmaceutical composition, granular pharmaceutical composition or tablet of the invention and the therapeutic agent alone or a pharmaceutical composition thereof are in a disclosed container. In another embodiment, the disclosed container is a bottle. In another embodiment, the disclosed container is a vial. In another embodiment, the disclosed container is a blister pack.
In another aspect, the present invention provides a continuous or semi-continuous process for preparing a pharmaceutical composition as described herein by a twin screw wet granulation process comprising the steps of: screening and weighing the
Drawings
Figure 1 is an X-ray diffraction pattern calculated from the single crystal structure of
Figure 2 is an actual X-ray powder diffraction pattern of
Fig. 3 is a schematic diagram depicting compound 1pH gradient dissolution profiles (LOD denotes loss on drying, i.e. a measure defining the amount of water in the powder/granulate) for tablets prepared by the High Shear Granulation (HSG) process and the Twin Screw Wet Granulation (TSWG) process.
Figure 4 is a schematic diagram depicting the stability of
Figure 5 is a schematic diagram depicting the stability of
Figure 6 is a schematic diagram depicting the stability of
Figure 7 is a schematic diagram depicting the stability of
FIG. 8 is a drawing of
FIG. 9 is of compound 1HCl salt1HNMR spectroscopy.
Figure 10 is a Differential Scanning Calorimetry (DSC) profile of trace amounts of
Figure 11 is a conformational diagram of
Detailed Description
Definition of
As used herein, "CFTR" refers to the cystic fibrosis transmembrane conductance regulator.
As used herein, the "Δ F508 mutation" or "F508-del mutation" is a specific mutation within the CFTR protein. The mutation is a deletion of three nucleotides of the codon containing the amino acid phenylalanine at position 508, resulting in a CFTR protein that does not contain this particular phenylalanine residue.
As used herein, a patient "homozygous" for a particular mutation (e.g., af 508) has the same mutation on each allele.
As used herein, a patient who is "heterozygous" for a particular mutation (e.g., af 508) has that mutation on one allele and a different mutation on the other allele.
The term "CFTR corrector" as used herein refers to a compound that increases the amount of cell surface functional CFTR protein, resulting in enhanced ion transport.
The term "CFTR potentiator" as used herein refers to a compound that increases the channel activity of the CFTR protein located on the cell surface, resulting in enhanced ion transport.
The term "active pharmaceutical ingredient" or "API" as used herein refers to a biologically active compound.
The term "solid form" and related terms when used herein to refer to
The term "substantially amorphous" as used herein refers to a solid material having little or no long-range order in its molecular position. For example, a substantially amorphous material has a crystallinity of less than about 15% (e.g., less than about 10% crystallinity or less than about 5% crystallinity). It should also be noted that the term "substantially amorphous" includes the descriptor "amorphous," which refers to a material that does not have (0%) crystallinity.
The term "substantially crystalline" (as in the phrase substantially
As used herein, the term "crystalline" and related terms, when used in reference to a substance, component, product or form, mean 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) ("Remington: science and practice of pharmacy, 21st edition, Leipikott Williams Wilkins publishing Co., Baltimore, Md., 2003); the United States Pharmacopeia, 23rded., 1843-1844(1995) ("United states Pharmacopeia, 23 rd edition, pages 1843-1844, 1995)).
As used herein, "excipient" includes both functional and non-functional ingredients in a pharmaceutical composition.
As used herein, a "disintegrant" is an excipient that hydrates a pharmaceutical composition and aids in the dispersion of a tablet. As used herein, a "diluent" or "filler" is an excipient that adds bulk to a pharmaceutical composition.
As used herein, a "surfactant" is an excipient that imparts enhanced solubility and/or wettability to a pharmaceutical composition.
As used herein, a "binder" is an excipient that imparts enhanced cohesive or tensile strength (e.g., hardness) to a pharmaceutical composition.
As used herein, a "glidant" is an excipient that imparts enhanced flow properties to a pharmaceutical composition.
As used herein, a "colorant" is an excipient that imparts a desired color to a pharmaceutical composition, such as a tablet. Examples of colorants include commercially available pigments such as FD &
As used herein, a "lubricant" is an excipient added to a pharmaceutical composition that is compressed into a tablet. The lubricant aids in compressing the granules into tablets and allows tablets of the pharmaceutical composition to be ejected from the die press.
As used herein, "cubic centimeters" and "cc" are used interchangeably to refer to units of volume. Note that 1cc equals 1 mL.
As used herein, "kips" and "kP" are used interchangeably and represent a measure of force, where 1kP is about 9.8 newtons.
As used herein, "friability" refers to the property of a tablet to remain intact and maintain its form despite external pressure. Brittleness can be quantified using the mathematical expression shown in equation 1:
wherein W0Is the initial weight of the tablet, and WfIs the final weight of the tablet after it is placed through the friability apparatus. Friability was measured using a standard USP testing apparatus which tumbles the test 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.
As used herein, "average particle size" is the average particle size as measured using techniques such as laser light scattering, image analysis, or sieve analysis. In one embodiment, the particles used to prepare the pharmaceutical compositions provided herein have an average particle size of less than 1.0 mm.
As used herein, "bulk density" is the mass of particles of a material divided by the total volume occupied by the particles. The total volume includes the particle volume, the volume of inter-particle voids, and the internal pore volume. Bulk density is not an inherent property of a material; it may vary depending on the way the material is processed. In one embodiment, the particles used to prepare the pharmaceutical compositions provided herein have a bulk density of about 0.5 to 0.7 g/cc.
The "effective" amount or "therapeutically effective amount" of a pharmaceutical compound of the invention may vary depending on, for example, the following factors: disease state, age and weight of the subject, and the ability of the compounds of the invention to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. An effective amount is also an amount that outweighs any toxic or deleterious (e.g., side effects) effects of the compounds of the present invention.
As used herein, and unless otherwise indicated, the terms "therapeutically effective amount" and "effective amount" of a compound refer to an amount sufficient to provide a therapeutic benefit or to delay or minimize one or more symptoms associated with a disease or condition in the treatment or management of the disease or condition. "therapeutically effective amount" and "effective amount" of a compound refers to the amount of a therapeutic agent, alone or in combination with one or more other agents, that provides a therapeutic benefit in the treatment or management of a disease or disorder. The terms "therapeutically effective amount" and "effective amount" can encompass an amount that improves the overall treatment, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.
As used in the phrase "substantially
For
Pharmaceutical composition
The present invention provides a pharmaceutical
Accordingly, in one aspect, the present invention provides a pharmaceutical composition comprising:
a.
b. a substantially amorphous solid dispersion of
c. a filler;
d. a disintegrant;
e. a surfactant; and
f. and (3) an adhesive.
In one embodiment of this aspect, the pharmaceutical composition comprises 25mg of
In one embodiment of this aspect, the pharmaceutical composition comprises 25mg of substantially
In some embodiments, the pharmaceutical composition comprises
In some embodiments, the pharmaceutical composition comprises substantially
In some embodiments, the pharmaceutical composition comprises
The concentration of
In another embodiment, the pharmaceutical composition comprises
As noted, in addition to the solid dispersion of
Fillers suitable for use in the present invention are compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, hardness, chemical stability, physical stability, or biological activity of the pharmaceutical composition. Exemplary fillers include: cellulose, modified cellulose (e.g., sodium carboxymethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose), cellulose acetate, microcrystalline cellulose, calcium phosphate, dibasic calcium phosphate, starch (e.g., corn starch, potato starch), sugar (e.g., sorbitol, lactose, sucrose, etc.), or any combination thereof.
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 the 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%) microcrystalline cellulose, such as MCC avicel ph102, by weight of the composition. In 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 microcrystalline cellulose by weight of the composition.
Disintegrants suitable for use in the present invention improve the dispersibility of the pharmaceutical composition and match the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the chemical stability, physical stability, hardness, or biological activity of the pharmaceutical composition. Exemplary disintegrants include croscarmellose sodium, sodium starch glycolate, or combinations thereof.
Thus, in one embodiment, the pharmaceutical composition comprises a disintegrant in an amount of about 10 wt% or less by weight of the composition (e.g., about 7 wt% or less, about 6 wt% or less, or about 5 wt% or less). For example, the pharmaceutical composition comprises about 1 wt% to about 10 wt% (e.g., about 1.5 wt% to about 7.5 wt% or 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 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 about 0.1% to about 10% (e.g., about 0.5% to about 7.5% or about 1.5% to about 6%) by weight of the composition of a disintegrant. In other examples, the pharmaceutical composition comprises about 0.5% to about 10% (e.g., about 1.5% to about 7.5% or about 2.5% to about 6%) by weight of the composition of a disintegrant.
Surfactants suitable for use in the present invention improve the wettability of the pharmaceutical composition and match the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the chemical stability, physical stability, hardness, or biological activity of the pharmaceutical composition. Exemplary surfactants include Sodium Lauryl Sulfate (SLS), Sodium Stearyl Fumarate (SSF),
Thus, in one embodiment, the pharmaceutical composition comprises a surfactant in an amount of about 10 wt% or less by weight of the composition (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). 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 a 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 another example, the pharmaceutical composition comprises about 10 wt% to about 0.1 wt% (e.g., about 5 wt% to about 0.2 wt% or about 2 wt% to about 0.3 wt%) of sodium lauryl sulfate by weight of the composition.
The binders suitable for use in the present invention enhance the tablet strength of the pharmaceutical composition and match the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the chemical stability, physical stability or 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.
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 the 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 another example, the pharmaceutical composition comprises the glidant polyvinylpyrrolidone in an amount 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%) by weight of the composition.
Diluents suitable for use in the present invention may add the necessary volume to the formulation to prepare a tablet of the desired size and generally match the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, hardness, chemical stability, physical stability or biological activity of the pharmaceutical composition. Exemplary diluents include: sugars, such as sugar powder (confectioner's), compressible sugar (compressible sugar), dextran, dextrin, dextrose, lactose, mannitol, sorbitol, cellulose, and modified celluloses, such as powdered cellulose, talc, calcium phosphate, starch, or any combination thereof.
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 about 40 wt% to about 1 wt% (e.g., about 35 wt% to about 5 wt% or about 30 wt% to about 7 wt%, about 25 wt% to about 10 wt%, about 20 wt% to about 15 wt%) of a 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) mannitol by weight of the composition. In another example, the pharmaceutical composition comprises about 35 wt% to about 1 wt% (e.g., about 30 wt% to about 5 wt% or about 25 wt% to about 10 wt%) mannitol by weight of the composition.
Glidants suitable for use in the present invention improve the flow properties of a pharmaceutical composition and match the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, hardness, chemical stability, physical stability or biological activity of the pharmaceutical composition. Exemplary glidants include colloidal silicon dioxide, talc, or combinations thereof.
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 about 2 wt% to about 0.05 wt% (e.g., about 1.5 wt% to about 0.07 wt% or about 1.0 wt% to about 0.09 wt%) of a 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 silica by weight of the composition. In 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 silica, by weight of the composition.
In some embodiments, the pharmaceutical composition may comprise an oral solid pharmaceutical dosage form that may contain a lubricant that may prevent the granulation microbead admixture from adhering to surfaces (e.g., surfaces of a compounding bowl, die and/or punch press). Lubricants may also reduce inter-granular friction within the granules and improve compression of the pharmaceutical composition and release of the compressed pharmaceutical composition from the molding press. Lubricants are also compatible with the ingredients of the pharmaceutical composition, i.e., they do not substantially reduce the solubility, hardness, or 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% to about 0.10% (e.g., from about 4.5% to about 0.5% or from about 3% to about 1%) by weight of the composition of the lubricant. In another example, the pharmaceutical composition comprises 5% or less (e.g., 4.0% or less, 3.0% or less, or 2.0% or less, or 1.0% or less) by weight of the composition of magnesium stearate. In another example, the pharmaceutical composition comprises from about 5% to about 0.10% (e.g., from about 4.5% to about 0.15% or from about 3.0% to about 0.50%) by weight of the composition of magnesium stearate.
The pharmaceutical compositions of the present invention may optionally comprise one or more coloring, flavoring and/or perfuming agents to enhance the visual appeal, taste and/or aroma of the composition. Suitable coloring, flavoring or perfuming agents are compatible with the ingredients of the pharmaceutical composition, i.e. they do not substantially detract from the solubility, chemical stability, physical stability, hardness or biological activity of the pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a colorant, a flavoring agent, and/or an aroma. In one embodiment, the pharmaceutical composition provided by the present invention is purple in color.
In some embodiments, the pharmaceutical composition comprises or can be formulated into a tablet, and the tablet can be coated with a colorant and optionally marked with a logo, other images, and/or text using a suitable ink. In still other embodiments, the pharmaceutical composition comprises or can be formulated into a tablet, and the tablet can be coated with a colorant, waxed, and optionally marked with a logo, other images, 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, chemical stability, physical stability, hardness, or biological activity of the pharmaceutical composition. Suitable colorants and inks may be of any color and be water-based or solvent-based. In one embodiment, tablets prepared from the pharmaceutical composition are coated with a colorant and then labeled with a logo, other images, and/or text using a suitable ink. For example, a tablet comprising a pharmaceutical composition as described herein may be coated with about 3% by weight (e.g., less than about 6% or less than about 4% by weight) of a film coating comprising a colorant. The colored tablets may be marked with logos and words using suitable inks to indicate the strength of the active ingredient in the tablet. In another example, a tablet comprising a pharmaceutical composition as described herein can be coated with about 3% by weight (e.g., less than about 6% or less than about 4% by weight) of a film coating comprising a colorant.
In another embodiment, tablets prepared from the pharmaceutical composition are coated with a colorant, waxed, and then labeled with a logo, other images, and/or text using a suitable ink. For example, a tablet comprising a pharmaceutical composition as described herein may be used at about 3 wt% (e.g., less than about 6 wt%)Amount% or less than about 4 wt.%) of a film coating comprising a colorant. The coloured tablets may be waxed with carnauba wax powder weighed in an amount of about 0.01% w/w of the weight of the starting core. The waxed tablets may be marked with logos and words with a suitable ink to indicate the strength of the active ingredient in the tablet. In another example, a tablet comprising a pharmaceutical composition as described herein can be coated with about 3% by weight (e.g., less than about 6% or less than about 4% by weight) of a film coating comprising a colorant. The coloured tablets may be waxed with carnauba wax powder weighed in an amount of about 0.01% w/w of the weight of the starting core. The waxed tablets may be coated with a pharmaceutical grade ink such as black ink (e.g. black ink)S-1-17823, a solvent-based ink, commercially available from Colorcon, inc., WestPoint, PA.) is labeled with logos and words to indicate the strength of the active ingredient in the tablet.
An exemplary pharmaceutical composition comprises about 15 wt% to about 70 wt% (e.g., about 15 wt% to about 60 wt%, about 15 wt% to about 50 wt%, or about 20 wt% to about 70 wt%, or about 30 wt% to about 70 wt%) of
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
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., from about 20 to 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; 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; about 2 wt% to about 0.1 wt% of a lubricant; about 2 wt% to about 4 wt% colorant; and from about 0.005 wt% to about 0.015 wt% wax.
In one embodiment, the invention is a particulate pharmaceutical composition comprising:
a. about 43 wt%, by weight of the composition, of
b. about 34 wt% by weight of the composition of a solid dispersion comprising substantially
c. about 17 wt% microcrystalline cellulose by weight of the composition;
d. about 2 wt% croscarmellose sodium, by weight of the composition;
e. about 1 wt% sodium lauryl sulfate by weight of the composition; and
f. about 3 wt% polyvinylpyrrolidone by weight of the composition.
In one embodiment, the invention is a tablet comprising:
a. about 35 wt
b. about 28 wt% by weight of the composition of a solid dispersion comprising substantially
c. about 26 wt% microcrystalline cellulose by weight of the composition;
d. about 6 wt% croscarmellose sodium, by weight of the composition;
e. about 3 wt% polyvinylpyrrolidone, by weight of the composition;
f. about 1 wt% sodium lauryl sulfate by weight of the composition; and
g. about 1 wt% magnesium stearate by weight of the composition.
In one embodiment, the invention is a tablet comprising:
a. about 34 wt
b. about 27 wt% by weight of the composition of a solid dispersion comprising substantially
c. about 26 wt% microcrystalline cellulose by weight of the composition;
d. about 6 wt% croscarmellose sodium, by weight of the composition;
e. about 2 wt% polyvinylpyrrolidone, by weight of the composition;
f. about 1 wt% sodium lauryl sulfate by weight of the composition;
g. about 1 wt% magnesium stearate by weight of the composition;
h. about 3 wt% of a colorant, by weight of the composition; and
i. about 0.010 wt% wax, by weight of the composition.
Another tablet of the invention comprises:
a. about 150-250 mg of
b. about 100-150 mg of substantially
c. about 125-175 mg microcrystalline cellulose;
d. about 20-40 mg of croscarmellose sodium;
e. about 10-20 mg of polyvinylpyrrolidone;
f. about 2-6 mg of sodium lauryl sulfate; and
g. about 3-7 mg of magnesium stearate.
Another tablet of the invention comprises:
a. about 200mg of
b. about 125mg of substantially
c. about 150mg of microcrystalline cellulose;
d. about 34mg of croscarmellose sodium;
e. about 15mg of polyvinylpyrrolidone;
f. about 4mg of sodium lauryl sulfate; and
g. about 6mg of magnesium stearate.
Another tablet of the invention comprises:
a. about 200mg of
b. about 125mg of substantially
c. about 150mg of microcrystalline cellulose;
d. about 34mg of croscarmellose sodium;
e. about 15mg of polyvinylpyrrolidone;
f. about 4mg of sodium lauryl sulfate;
g. about 6mg of magnesium stearate;
h. about 17mg of a colorant; and
i. about 0.06mg of wax.
In one embodiment, the invention is a particulate pharmaceutical composition comprising:
a. about 38 wt
b. about 40 wt% by weight of the composition of a solid dispersion comprising substantially
c. about 16 wt% microcrystalline cellulose by weight of the composition;
d. about 2 wt% croscarmellose sodium, by weight of the composition;
e. about 1 wt% sodium lauryl sulfate by weight of the composition; and
f. about 3 wt% polyvinylpyrrolidone by weight of the composition.
In one embodiment, the invention is a tablet comprising:
a. about 31 wt
b. about 32 wt% by weight of the composition of a solid dispersion comprising substantially
c. about 26 wt% microcrystalline cellulose by weight of the composition;
d. about 6 wt% croscarmellose sodium, by weight of the composition;
e. about 3 wt% polyvinylpyrrolidone, by weight of the composition;
f. about 1 wt% sodium lauryl sulfate by weight of the composition;
g. about 1 wt% magnesium stearate by weight of the composition; and
h. about 3 wt% colorant, by weight of the composition.
Another tablet of the invention comprises:
a. about 100-200 mg of
b. about 100-150 mg of substantially
c. about 100-150 mg microcrystalline cellulose;
d. about 20-40 mg of croscarmellose sodium;
e. about 10-20 mg of polyvinylpyrrolidone;
f. about 2-6 mg of sodium lauryl sulfate; and
g. about 3-7 mg of magnesium stearate.
Another tablet of the invention comprises:
a. about 150mg of
b. about 125mg of substantially
c. about 129mg of microcrystalline cellulose;
d. about 29mg of croscarmellose sodium;
e. about 13mg of polyvinylpyrrolidone;
f. about 4mg of sodium lauryl sulfate;
g. about 5mg of magnesium stearate; and
h. about 15mg of colorant.
The pharmaceutical compositions of the present invention may be processed into tablet forms, capsule forms, sachet forms, lozenge forms or other solid forms suitable for oral administration. Thus, in some embodiments, the pharmaceutical composition is in the form of a tablet.
Another aspect of the invention provides a pharmaceutical formulation consisting of a
In one example, the pharmaceutical composition consists of a tablet comprising:
Dissolution can be measured by a standard usp type II instrument using the following conditions: 0.1% CTAB dissolved in 900mL of deionized water (buffered to pH 6.8 with 50mM potassium dihydrogen phosphate) as dissolution medium, stirred at about 50-75rpm at a temperature of about 37 ℃. A single test tablet was tested in each test vessel of the instrument. Dissolution may also be measured by a standard usp type II instrument using the following conditions: 0.7% sodium lauryl sulfate dissolved in 900mL of 50mM sodium phosphate buffer (pH 6.8) was used as the dissolution medium and stirred at about 65rpm at a temperature of about 37 ℃. A single test tablet was tested in each test vessel of the instrument. Dissolution may also be measured by a standard usp type II instrument using the following conditions: 0.5% sodium lauryl sulfate dissolved in 900mL of 50mM sodium phosphate buffer (pH 6.8) was used as the dissolution medium and stirred at about 65rpm at a temperature of about 37 ℃. A single test tablet was tested in each test vessel of the instrument.
Process for preparing
Starting
Scheme 4.formation of the acid salt of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid.
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 prepare 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 a suitable solvent for an effective period of time. Other salts of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid, for example salts derived from other mineral or organic acids, may be used. Other salts are obtained by acid mediated hydrolysis of the tert-butyl ester moiety. Salts derived from other acids may include, for example, nitric acid, sulfuric acid, phosphoric acid, boric acid, acetic acid, benzoic acid, and malonic acid. These salt forms of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid may be soluble or insoluble depending on the solvent used, but insufficient solubility does not hinder the formation of
The effective length of time for forming
In one embodiment,
In some embodiments, the D90 particle size distribution is about 82 μm or less for
Scheme 5: synthesis of 4-oxo-dihydroquinolinecarboxylic acid moieties.
Phenyl ethers
HCl (aqueous solution) or H2SO4(aqueous solution)
Scheme 6: synthesis of amine moieties.
Scheme 7: coupling of 4-oxo-dihydroquinolinecarboxylic acids with amine moieties.
Solid dispersion comprising substantially
The amorphous form of
Spray drying typically uses a solid material (i.e., drug and excipient) loading of about 3% to about 30% by weight, for example about 4% to about 20% by weight, preferably at least about 10%. Generally, the upper limit of the solids loading is determined by the viscosity (e.g., pumpability) of the resulting solution and the solubility of the ingredients in the solution. In general, the concentration of the solution may determine the size of the particles in the resulting powder product.
Techniques and methods for spray drying may be found in Perry's Chemical Engineering Handbook, 6 th edition, r.h.perry, d.w.green & j.o.maloney, eds.), McGraw-Hill book co (1984); and Marshall "Atomization and spread-Drying" 50, chem.Eng.prog.monogr.series 2 (1954). Generally, spray drying is carried out using an inlet temperature of from about 60 ℃ to about 200 ℃, e.g., from about 95 ℃ to about 185 ℃, from about 110 ℃ to about 182 ℃, from about 96 ℃ to about 180 ℃, e.g., about 145 ℃. Spray drying is generally carried out using an outlet temperature of from about 30 ℃ to about 90 ℃, e.g., from about 40 ℃ to about 80 ℃, from about 45 ℃ to about 80 ℃, e.g., about 75 ℃. The atomization flow rate is generally from about 4kg/h to about 12kg/h, for example from about 4.3kg/h to about 10.5kg/h, for example about 6kg/h or about 10.5 kg/h. The feed rate is generally from about 3kg/h to about 10kg/h, for example from about 3.5kg/h to about 9.0kg/h, for example about 8kg/h or about 7.1 kg/h. The atomization rate is generally from about 0.3 to about 1.7, such as from about 0.5 to about 1.5, for example about 0.8 or about 1.5.
Removal of the solvent may require a subsequent drying step, such as tray drying, fluidized bed drying (e.g., from about room temperature to about 100 ℃), vacuum drying, microwave drying, drum drying, or double cone vacuum drying (e.g., from about room temperature to about 200 ℃).
In one embodiment, the spray dried dispersion is fluid bed dried.
In one process, the solvent comprises a volatile solvent, such as a solvent having a boiling point less than about 100 ℃. In some embodiments, the solvent comprises a mixture of solvents, such as a mixture of volatile solvents or a mixture of volatile and non-volatile solvents. If a solvent mixture is used, the mixture may include one or more volatile solvents, for example, where the non-volatile solvent is present in the mixture at less than about 15%, such as 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%.
Preferred solvents are those in which
Exemplary solvents that can be tested include acetone, cyclohexane, dichloromethane, N-Dimethylacetamide (DMA), N-Dimethylformamide (DMF), 1, 3-dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide (DMSO), dioxane, ethyl acetate, diethyl ether, glacial acetic acid (HAc), Methyl Ethyl Ketone (MEK), N-methyl-2-pyrrolidone (NMP), methyl tert-butyl ether (MTBE), Tetrahydrofuran (THF), pentane, acetonitrile, methanol, ethanol, isopropanol, 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 solvent may be present at about 0.1% to about 99.9%. In some preferred embodiments, water is a co-solvent for acetone, wherein water is present at about 0.1% to about 15%, such as about 9% to about 11%, such as about 10%. In some preferred embodiments, water is a co-solvent for MEK, wherein water is present at about 0.1% to about 15%, such as about 9% to about 11%, for example about 10%. In some embodiments, the solvent solution includes three solvents. For example, acetone and water may be mixed with a third solvent such as DMA, DMF, DMI, DMSO, or HAc. In the case where substantially
The particle size and temperature drying range can be varied to produce the optimum spray dried dispersion. As can be appreciated by those skilled in the art, small particle size results in improved solvent removal. However, applicants have found that small particles can produce loose particles, and in some cases, do not provide an optimal spray-dried dispersion for downstream processing, such as tableting. At higher temperatures, substantially
Generally, the particle size is such that D10(μm) is less than about 5, such as less than about 4.5, less than about 4.0, or less than about 3.5, D50(μm) is generally less than about 17, such as less than about 16, less than about 15, less than about 14, less than about 13; and D90(μm) is generally less than about 175, such as 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 50. Generally, the bulk density of the spray-dried particles is from about 0.08g/cc to about 0.20g/cc, such as from about 0.10 to about 0.15g/cc, such as about 0.11g/cc or about 0.14 g/cc. The tap density of the spray dried particles is generally from about 0.08g/cc to about 0.20g/cc, for example from about 0.10 to about 0.15g/cc, for example about 0.11g/cc or about 0.14g/cc, for 10 light shakes; for 500 light shakes, typically from about 0.10g/cc to about 0.25g/cc, such as from about 0.11 to about 0.21g/cc, such as about 0.15g/cc, about 0.19g/cc, or about 0.21 g/cc; for 1250 light oscillations, generally about 0.15g/cc to about 0.27g/cc, such as about 0.18 to about 0.24g/cc, such as about 0.18g/cc, about 0.19g/cc, about 0.20g/cc, or about 0.24 g/cc; and generally from about 0.15g/cc to about 0.27g/cc, such as from about 0.18 to about 0.24g/cc, such as about 0.18g/cc, about 0.21g/cc, about 0.23g/cc, or about 0.24g/cc for 2500 light vibrations.
Polymer and method of making same
Also included herein are spray-dried dispersions comprising
In one embodiment, the polymer is capable of being dissolved in an aqueous medium. The solubility of the polymer may be pH independent or pH-dependent. The latter includes one or more enteric polymers. The term "enteric" refers to polymers that dissolve preferentially in the less acidic intestinal environment relative to the more acidic environment of the stomach, e.g., polymers that are insoluble in acidic aqueous media and soluble at pH above 5-6. Suitable polymers should be chemically and biologically inert. To improve the physical stability of the spray-dried dispersion, the glass transition temperature (T) of the polymerg) Should be as high as possible. For example, preferred polymers have a glass transition temperature that is 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 in the range of about 10 to about 15 ℃ of the drug (i.e., compound 2). Examples of suitable glass transition temperatures for the polymer include at least about 90 ℃, at least about 95 ℃, at least about 100 ℃, at least about 105 ℃, at least about 110 ℃, at least about 115 ℃, at least about 120 ℃, at least about 125 ℃, at least about 130 ℃, at least about 135 ℃, at least about 140 ℃, at least about 145 ℃, at least about 150 ℃, at least about 155 ℃, at least about 160 ℃, at least about 165 ℃, at least about 170 ℃ or at least about 175 ℃ (as determined under dry conditions). Without wishing to be bound by theory, it is believed that the underlying mechanism is to have a higher TgGenerally have a low molecular flow at room temperatureThis may be a decisive factor in stabilizing the physical stability of the amorphous spray-dried dispersion.
In addition, the hygroscopicity of the polymer is as low as, for example, less than about 10%. For purposes of comparison in this application, the hygroscopicity of a polymer or composition is characterized by a relative humidity of about 60%. In some preferred embodiments, the polymer has a water absorption of less than about 10%, such as 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%. Hygroscopicity can also affect the physical stability of spray dried dispersions. In general, moisture adsorbed in the polymer can significantly reduce the T of the polymer and the resulting spray-dried dispersiongThis may further reduce the physical stability of the spray dried dispersion as described above.
In one embodiment, the polymer is one or more water soluble polymers or partially water soluble polymers. Water soluble or partially water soluble polymers include, but are not limited to, cellulose derivatives (e.g., hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC)) or ethylcellulose; polyvinylpyrrolidone (PVP); polyethylene glycol (PEG); polyvinyl alcohol (PVA); acrylates, such as polymethacrylates (e.g.,
E) cyclodextrins (e.g., β -cyclodextrin) and copolymers and derivatives thereof, including, for example, PVP-VA (polyvinylpyrrolidone-vinyl acetate).In some embodiments, the polymer is hydroxypropyl methylcellulose (HPMC), e.g., HPMCAS, HPMCE50, HPMCE15, or HPMC60SH 50).
As discussed herein, the polymer may 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)), hydroxypropylmethylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose acetate succinate (HPMCAS), carboxymethylcellulose (CMC), or salts thereof (e.g.Such as sodium salt, e.g. (CMC-Na)); cellulose Acetate Trimellitate (CAT), hydroxypropyl cellulose acetate phthalate (HPCAP), hydroxypropyl methyl cellulose acetate phthalate (HPMCAP), and Methyl Cellulose Acetate Phthalate (MCAP) or polymethacrylates (e.g.,s). In some embodiments, the polymer is hydroxypropyl methyl cellulose acetate succinate (HPMCAS). In some embodiments, the polymer is HG grade hydroxypropyl methylcellulose acetate succinate (HPMCAS-HG).
In another embodiment, the polymer is a polyvinylpyrrolidone copolymer, such as vinylpyrrolidone/vinyl acetate copolymer (PVP/VA).
In embodiments wherein
In some embodiments, substantially
In some embodiments, the combined substantially
In some embodiments, the dispersion further includes an additional minor ingredient, such as a surfactant (e.g., SLS). In some embodiments, the surfactant is present in the dispersion in an amount less than about 10%, such as 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%.
In embodiments that include a polymer, the polymer should be present in an amount effective to stabilize the spray-dried dispersion. Stabilizing includes inhibiting or preventing crystallization of substantially
Suitable polymers for combination with
the glass transition temperature of the polymer has a temperature of about 10-15 ℃ not less than the glass transition temperature of substantially
The polymer should be relatively non-hygroscopic. For example, the polymer should absorb less than about 10% water, such as 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, when stored under standard conditions. Preferably, the polymer should be substantially non-hygroscopic when stored under standard conditions.
The polymer should have similar or better solubility than
When combined with substantially
The polymer should reduce the bulk fraction of amorphous material.
The polymer should increase the physical and/or chemical stability of substantially
The polymer should improve the manufacturability of substantially
The polymer should improve one or more of the handling, application, or storage characteristics of substantially
The polymer should not interact adversely with other pharmaceutical ingredients such as excipients.
The suitability of the candidate polymer (or other ingredient) in forming an amorphous composition can be tested using the spray drying method (or other methods) described herein. The stability, resistance to crystal formation, or other characteristics of the candidate compositions can be compared and compared to a reference preparation, e.g., the preparation of pure
Surface active agent
The spray dried dispersion may include a surfactant. The surfactant or surfactant mixture can generally reduce the interfacial tension between the spray dried dispersion and the aqueous medium. Suitable surfactants or surfactant mixtures may also enhance the water solubility and bioavailability of
The surfactant (e.g., SLS) may be present in an amount of 0.1 to 15% relative to the total weight of the spray-dried dispersion. Preferably, it is from about 0.5% to about 10%, more preferably from about 0.5 to about 5%, for example from about 0.5 to 4%, from about 0.5 to 3%, from about 0.5 to 2%, from about 0.5 to 1% or about 0.5%.
In some embodiments, the surfactant is present in an amount of at least about 0.1%, preferably about 0.5%, relative to the total weight of the spray-dried dispersion. In these embodiments, the surfactant may 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 is preferred wherein the amount of surfactant is about 0.5% by weight.
The suitability of candidate surfactants (or other ingredients) for use in the present invention can be tested in a manner similar to that described for testing polymers.
Process for preparing pharmaceutical composition
Dosage unit forms of the invention can be prepared by compressing or compacting a compound or composition (e.g., 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.
The term "tablet" as used herein refers to a physically discrete unit of active agent suitable for the patient being treated. Generally, the density of the compressed mixture is greater than the density of the mixture prior to compression. Dosage tablets of the invention may have almost any shape, including concave and/or convex surfaces, rounded or wedge-shaped angles, and rounded to rectilinear shapes. In some embodiments, the compressed tablets of the invention comprise circular tablets having a flat surface. The tablets of the present invention may be prepared by any compression and compression method known to those of ordinary skill in the art to form compressed solid pharmaceutical dosage forms. In particular embodiments, the formulations provided herein can be prepared using conventional methods known to those skilled in the art of pharmaceutical formulation, as described, for example, in related texts. See, e.g., Remington, The Science and Practice of Pharmacy, 21stEd., Lippincott Williams&Wilkins, Baltimore, Md. (2003) ("Remington: science and practice of pharmacy, 21st edition, Leipikott Williams Wilkins publishing Co., Baltimore, Md., 2003); ansel et al, Pharmaceutical document Forms And Drug Delivery Systems, 7th Edition, Lippincott Williams&Wilkins, (1999) (Ansel et al, drug dosage forms and drug delivery systems, 7th edition, Lipocott Williams Wilkins publishing, 1999); the Handbook of pharmaceutical excipients, 4thedition, Rowe et al, eds., American Pharmaceuticals Association (2003) ("handbook of pharmaceutical excipients", 4 th edition, edited by Rowe et al, American society of medicine, 2003); gibson, Pharmaceutical Preformulation And Formulation, CRC Press (2001) (Gibson, Pharmaceutical Preformulation And Formulation, CRC Press, 2001) the references are hereby incorporated by reference in their entiretyAre incorporated herein.
Granulating and compressing
In some embodiments, the ingredients are weighed out according to the formulations set forth herein. Next, all intragranular ingredients were sieved and mixed thoroughly. These ingredients may be lubricated with a suitable lubricant, such as magnesium stearate. The next step may include compressing/slugging the powder compound and sieved ingredients. Next, the compressed or slugged blend is milled into granules and sieved to obtain the desired size. Next, the granules are further lubricated with, for example, magnesium stearate. Next, the granular composition of the present invention may be compressed on a suitable punch to form various pharmaceutical formulations according to the present invention. Optionally, the tablets may be coated with a film coat, colorant, or other coating.
Another aspect of the invention provides a method of preparing a pharmaceutical composition, the method comprising: providing a compound of a composition, wherein the composition comprises
In another embodiment, a wet granulation process is performed to obtain the pharmaceutical formulation of the present invention from a blend of powder and liquid ingredients. For example, a pharmaceutical composition comprising a compound of a
In a particularly advantageous embodiment, the pharmaceutical composition of the invention is prepared by a continuous Twin Screw Wet Granulation (TSWG) process. Continuous manufacturing provides high quality and highly consistent products through on-line monitoring and control. Continuous manufacturing also benefits quality by design development with a "data rich" design space, and facilitates understanding of the impact of upstream variables on downstream processes and final product quality. In addition, the pharmaceutical compositions of the present invention can be shaped early on a commercial scale device, which avoids the risk of process upscaling and development of late-stage formulation changes. Finally, continuous manufacturing has commercial manufacturing advantages such as improved process control, reduced product handling, and real-time release efficiency. The overall result is a more robust, controllable and scalable process with fewer process checks, thereby enabling an increase in product quality and thus greater patient safety. These advantages address the concern of Janet Woodcock (a director of the Center for Drug Evaluation and Research (CDER)), namely that chemistry, manufacture and control (CMC) is not able to maintain rapid clinical development of highly effective therapies ("at world search of the high temperature treatment of the patient and the like," 24/7/2013, Friends of Cancer family consistency designing "analysis of the patient and the like: Expediting Life-science and Research procedures to medicine" to discrete the Food and Drug addition's Break high temperature treatment design).
For example, High Shear Granulation (HSG), a common granulation technique, is well known to risk over-granulation and poor process control. Scaling up of the process is extremely challenging and involves significant risks. Changing from the HSG process to the continuous TSWG process allows for scaling up using the same equipment to produce different batches by running for longer periods of time. This eliminates the scaling risks normally encountered with wet granulation processes. In addition, the TSWG process was found to be more robust and less prone to over-granulation. As can be seen in figure 3 of the
In one embodiment, the continuous process begins with separate excipients and
For example, in one embodiment, a
Each ingredient of this exemplary compound is described above and in the examples below. In addition, the compound may contain optional additives such as one or more colorants, one or more flavorants, and/or one or more fragrances as described above and in the examples below. In some embodiments, the examples above and below also provide the relative concentrations (e.g., wt%) of each of these ingredients (and any optional additives) in the compound. The ingredients that make up the compound may be provided sequentially or in any additive combination; and these ingredients or combinations of ingredients may be provided in any order. In one embodiment, the lubricant is the final ingredient of the last added compound.
In another embodiment, the compound comprises a composition of
In another embodiment, compressing the compound into a tablet is accomplished by: a form (e.g., a mold) is filled with the compound and pressure is applied to the compound. This may be accomplished using a die press or other similar device. In some embodiments, a compound of
In some embodiments, a tablet comprising a pharmaceutical composition as described herein may be coated with a film coat of about 3.0% by weight, based on the weight of the tablet, wherein the film coat comprises a colorant. In some cases, the colorant suspension or solution used to coat the tablets contains about 20% w/w solids by weight of the colorant suspension or solution. In other cases, the coated pieces may be marked with logos, other images, or text.
In another embodiment, a method for producing a pharmaceutical composition comprises: providing a compound in solid form, e.g., a compound of powder and/or liquid ingredients, the compound comprising a solid
In some embodiments, the compounds are mixed by stirring, blending, shaking, or the like, using hand mixing, mixers, blenders, any combination thereof, or the like. When ingredients or combinations of ingredients are added sequentially, mixing can occur between sequential additions, continuously throughout the addition of ingredients, after addition of all ingredients or combinations of ingredients, or in any combination thereof. The compound is mixed until it has a substantially uniform composition.
In another embodiment, the invention comprises jet milling a pharmaceutical
The formulations of the present invention provide fixed doses of two APIs in order to effectively treat cystic fibrosis, a combination that has received only one of two FDA-approved breakthrough therapies and that does have surprising stability for both as determined by a small loss of amorphous solid form of
Dosage forms prepared as described above may be subjected to in vitro Dissolution evaluation according to Test 711 "Dissolution" in United States Pharmacopeia 29, United States Pharmacopeial Convention, Inc., Rockville, Md., 2005 ("USP") (United States Pharmacopeia 29 th edition 711 Dissolution Test, Rockville, Md., 2005) to determine the rate of release of the active agent from the dosage form. The active substance content and impurity levels are conveniently determined by techniques such as High Performance Liquid Chromatography (HPLC).
In some embodiments, the invention includes containers and closures using packaging materials such as High Density Polyethylene (HDPE), Low Density Polyethylene (LDPE) and or polypropylene and/or glass, cellophane sheets, aluminum foil pouches and blisters or strips composed of aluminum or high density polyvinyl chloride (PVC), optionally including desiccants, Polyethylene (PE), polyvinylidene chloride (PVDC), PVC/PE/PVDC and the like. These packaging materials can be used to store various pharmaceutical compositions and formulations in an aseptic manner after appropriate sterilization of the packaging and its contents using chemical or physical sterilization techniques commonly used in the pharmaceutical arts.
Methods of administering pharmaceutical compositions
In one aspect, a pharmaceutical composition of the invention can be administered to a patient once daily or approximately every 24 hours. Alternatively, the pharmaceutical composition of the present invention may be administered to a patient twice daily. Alternatively, the pharmaceutical composition of the present invention is administered to the patient about once every 12 hours. Administering these pharmaceutical compositions in an oral formulation comprising about 25mg, 50mg, 100mg, 125mg, 150mg, 200mg, 250mg, 300mg, or 400mg of
It will be appreciated that the compounds of the invention, as well as pharmaceutically acceptable compositions and formulations, may be used in combination therapy; that is, substantially
In one embodiment, the additional therapeutic agent is selected from the group consisting of a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent,
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) cyclopropanecarboxamide. 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 1.
In another embodiment, the additional agent is any combination of the agents described above. For example, the combination may comprise a pharmaceutical composition or tablet of the
In another embodiment, the additional agent is selected from table 1:
in another embodiment, the additional agent is selected from table 2:
in one embodiment, the additional therapeutic agent is an antibiotic. Exemplary antibiotics for use herein include tobramycin (including Tobramycin Inhalation Powder (TIP)), azithromycin, aztreonam (including an aerosolized form of aztreonam), amikacin (including liposomal formulations thereof), ciprofloxacin (including formulations thereof suitable for administration by inhalation), levofloxacin (including aerosolized formulations thereof), and combinations of two antibiotics (e.g., fosfomycin and tobramycin).
In another embodiment, the additional agent is a mucolytic agent (mucolytice). Exemplary mucolytic agents useful herein include
In another embodiment, the additional agent is a bronchodilator. Exemplary bronchodilators include salbutamol, metaproterol sulfate, pirbuterol acetate, salmeterol, or terbutaline sulfate.
In another embodiment, the additional agent is effective to restore fluid to the surface of the pulmonary airways. Such agents improve salt access to the cells, thereby making mucus in the lung airways more hydrated and therefore more easily cleared. Exemplary such agents include hypertonic saline, denufosol sodium ([ [ (3S, 5R) -5- (4-amino-2-oxopyrimidin-1-yl) -3-hydroxyoxolan-2-yl ] methoxy-hydroxyphosphoryl ] [ [ [ (2R, 3S, 4R, 5R) -5- (2, 4-dioxopyrimidin-1-yl) -3, 4-dihydroxyoxolan-2-yl ] methoxy-hydroxyphosphoryl ] oxy-hydroxyphosphoryl ] hydrogen phosphate) or bronchitol (inhalation formulation of mannitol).
In another embodiment, the additional agent is an anti-inflammatory agent, i.e., an agent that reduces lung inflammation. Exemplary such agents useful herein include: ibuprofen, docosahexaenoic acid (DHA), sildenafil, inhaled glutathione, pioglitazone, hydroxychloroquine or simvastatin.
In another embodiment, the additional agent is an agent that increases or induces CFTR activity other than
In another embodiment, the additional agent is a nutritional agent. Exemplary nutritional agents include pancreatic lipase (pancreatic enzyme substitute),comprises that
Or(formerly)、Or a glutathione inhalant. In one embodiment, the additional nutrient is pancrelipase.In another embodiment, the additional agent is a compound selected from the group consisting of: gentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, meglumine, felodipine, nimodipine, philixin B, genistein, apigenin, cAMP/cGMP enhancers or inducers such as rolipram, sildenafil, milrinone, tadalafil, amrinone, isoproterenol, salbutamol and salmeterol, deoxyspergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxygenase (epoxomicin), lactacystin, and the like.
In another embodiment, the additional agent is a compound selected from the group consisting of: 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] bipyridine-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- (trifluoromethyl) picolinamide; 3-amino-6-methoxy-N- (3, 3, 3-trifluoro-2-hydroxy-2- (trifluoromethyl) propyl) -5- (trifluoromethyl) 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-, (S) 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- (trifluoromethyl) 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 a pharmaceutically acceptable salt thereof. In another embodiment, the additional agent is a compound disclosed in U.S. patent No. US8, 247, 436 and international PCT publication No. WO 2011113894, each of which is incorporated herein by reference in its entirety.
In another embodiment, the additional agent may be an epithelial sodium channel (ENac) modulator disclosed in PCT publication nos. WO2012035158, WO2009074575, WO2011028740, WO2009150137, WO2011079087, or WO2008135557, each of which is incorporated by reference herein in its entirety.
In other embodiments, the additional agent is a compound disclosed in WO 2004028480, WO2004110352, WO2005094374, WO 2005120497, or WO 2006101740, each of which is incorporated herein by reference in its entirety. In another embodiment, the additional agent is a benzo [ c ] quinolizinium derivative exhibiting CFTR inducing or enhancing activity or a benzopyran derivative exhibiting CFTR inducing or enhancing activity. In another embodiment, the additional agent is a compound disclosed in U.S. patent nos. US7, 202, 262, US6, 992, 096, US20060148864, US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456, WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502, each of which is incorporated herein by reference in its entirety. In another embodiment, the additional agent is a compound disclosed in WO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256, WO2006127588, or WO2007044560, each of which is incorporated herein by reference in its entirety.
In one embodiment, 400mg of
In one embodiment, 400mg of
In one embodiment, 300mg of
In one embodiment, 600mg of
In one embodiment, 800mg of
In one embodiment, 600mg of
In one embodiment, 600mg of
These combinations are useful in the treatment of diseases described herein, including cystic fibrosis. These compositions are also useful in the kits described herein. In another aspect, the invention features a kit that includes a pharmaceutical composition or tablet of the invention (including
The amount of additional therapeutic agent present in the compositions of the present invention will not exceed that normally administered in compositions containing the therapeutic agent as the only active agent. Preferably, the amount of additional therapeutic agent in the disclosed compositions will be in the range of about 50% to 100% of the amount typically present in compositions comprising the agent as the sole therapeutically active agent.
Therapeutic uses of compositions
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 to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the disease is selected from: cystic fibrosis, asthma, smoking-induced COPD, chronic bronchitis, sinusitis, constipation, pancreatitis, pancreatic insufficiency, male infertility due to congenital bilateral vasectomy (CBAVD), mild lung disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis defects (e.g., protein C deficiency), hereditary angioedema type 1, lipid processing defects (e.g., familial hypercholesterolemia), chylomicronemia type 1, betalipoproteinemia, lysosomal storage disorders (e.g., I-cell disease/pseudoherler syndrome), mucopolysaccharidosis, Sandhff/Tay-saxophone disease, Creutzfeldt-Jakob syndrome type II, polyendocrinopathy/hyperinsulinemia, diabetes, Ladwarfism, Myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, polyoses disease type CDG1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, Charpy-Marek syndrome, Palmer's disease, neurodegenerative diseases (e.g., Alzheimer's disease), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders (e.g., Huntington's disease), spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and myotonic dystrophy, and spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease (caused by prion protein processing defects), Fabry's disease, Guillain-Straus-Scheinker syndrome, Gongzi-Scheinker syndrome, Gottzeuginemia, and Creutzfeldt-Jakob disease, COPD, dry eye or sjogren's syndrome, osteoporosis, osteopenia, bone healing and bone growth (including bone repair, bone regeneration, decreased bone resorption and increased bone resorption), gorem syndrome, chloride channel pathologies such as congenital myotonia (thomson and becker), barter syndrome type III, hunter disease, excessive convulsions, epilepsy, lysosomal storage diseases, angmann-syndrome and Primary Ciliary Dyskinesia (PCD) (a term for ciliary structural and/or functional genetic disorders), including PCD with left and right transposition (also known as carter's syndrome), absence of left and right transposition and ciliary dysplasia.
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 to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the disease is selected from: global epilepsy with febrile convulsion adjunctive disorder (GEFS +), global epilepsy with febrile and non-febrile convulsions, myotonia, congenital paramyositis, potassium aggravated myotonia values, high potassium type periodic paralysis, LQTS/brucah syndrome, autosomal dominant LQTS with deafness, autosomal recessive LQTS, LQTS associated physical deficiency, congenital and acquired LQTS, temoci syndrome, persistent pediatric type insulin hypersecretory hypoglycemia, dilated cardiomyopathy, autosomal dominant LQTS, Dent's disease, osteopetrosis, barter syndrome type III, central axial empty disease, malignant hyperthermia and catecholamine sensitive polymorphic tachycardia.
In one aspect, the present invention relates to a method of treating, or symptomatically treating, cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a genetic mutation in CFTR, N1303K, Δ I507, or R560T thereof.
In one aspect, the present invention relates to a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has the CFTR genetic mutation, G551D thereof. In another embodiment, the patient is homozygous for G551D. In another embodiment, the patient is heterozygous G551D wherein the further CFTR genetic mutation is any one of Δ F508, G542X, N1303K, W1282X, R117H, R553X, 1717-1G- > a, 621+1G- > T, 2789+5G- > a, 3849+10kbC- > T, R1162X, G85E, 3120+1G- > a, Δ I507, 1898+1G- > a, 3659delC, R347P, R560T, R334W, a455E, 2184delA or 711+1G- > T.
In one aspect, the present invention relates to a method of treating, or symptomatically treating, cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has the genetic mutation CFTR, Δ F508 thereof. In another embodiment, the patient is homozygous for Δ F508. In another embodiment, the patient is heterozygous for Δ F508, wherein the further CFTR genetic mutation is any one of G551D, G542X, N1303K, W1282X, R117H, R553X, 1717-1G- > a, 621+1G- > T, 2789+5G- > a, 3849+10kbC- > T, R1162X, G85E, 3120+1G- > a, Δ I507, 1898+1G- > a, 3659delC, R347P, R560T, R334W, a455E, 2184delA or 711+1G- > T.
In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation of CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549 28, S549 9, S1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56, P67Q, L Q, a 455Q, D579Q, S1235Q, S945Q, R Q, F1073672, D1523672, D1270, D2Q, 1717-1G- > a, 3121G- > 1G-1G + 5G-b 1G-b 1G + G1-b 1G + G1-b 1G + G1-b 1G + b 1G 1-b 1G + b 1G 1, G + b 1G + b 1, G1, 712-1G- > T, 1248+1G- > A, 1341+1G- > A, 3121-1G- > A, 4374+1G- > T, 3850-1G- > A, 2789+5G- > A, 3849+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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 relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 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
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of 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 ion transport of greater than or equal to 10% of the baseline chloride ion transport.
In one aspect, the invention relates to a method of treating or opportunistically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 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+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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 invention relates to a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 1717-1G- > a, 1811+1.6kbA- > G, 2789+5G- > a, 3272-26A- > G, and 3849+10kbC- > T. In one aspect, the present invention relates to a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 2789+5G- > a and 3272-26A- > G.
In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation of CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549 28, S549 9, S1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56, P67Q, L Q, a 455Q, D579Q, S1235Q, S945Q, R Q, F1073672, D1523672, D1270, D2Q, 1717-1G- > a, 3121G- > 1G +5, G-b 1G-b 1G + b > 1G 1-b > G1G + b > 1G + b > G1-b > G1G + b > G1G + b > G1G 3, b > b, 712-1G- > T, 1248+1G- > A, 1341+1G- > A, 3121-1G- > A, 4374+1G- > T, 3850-1G- > A, 2789+5G- > A, 3849+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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, 18 +1G- > T, 4005+2T- > C and 621G- > A- > G-, and the patient has a human CFTR mutation selected from Δ F508, R117H, and G551D.
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V, and G1069R, and the patient has a human CFTR mutation selected from the group consisting of Δ F508, R117H, and G551D. In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, and S1251N, and the patient has a mutation in human CFTR selected from the group consisting of Δ F508, R117H, and G125 551D. In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of E193K, F1052V and G1069R, and the patient has a human CFTR mutation selected from the group consisting of Δ F508, R117H and G551D. In some embodiments of this aspect, the method produces a 10-fold increase in chloride ion transport greater than baseline chloride ion transport.
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of R117C, D110H, R347H, R352Q, E56K, P67L, L206W, a455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, and D1152H, and the patient has a human CFTR mutation selected from the group consisting of Δ F508, R117H, and G551D. In one embodiment of this aspect, the method produces an increase in chloride ion transport greater than or equal to 10% or greater of the baseline chloride ion transport.
In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 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+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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 the patient has a human CFTR mutation selected from Δ F508, R117H, and G551D. In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition of the invention or wherein the patient has a CFTR genetic mutation selected from the group consisting of 1717-1G- > a, 1811+1.6kbA- > G, 2789+5G- > a, 3272-26A- > G, and 3849+10kbC- > T and the patient has a human CFTR mutation selected from Δ F508, R117H, and G551D. In one aspect, the present invention relates to a method of treating, reducing the severity of, cystic fibrosis in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 2789+5G- > a and 3272-26A- > G, and the patient has a human CFTR mutation selected from the group consisting of Δ F508, R117H.
In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation of CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549 28, S549 9, S1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56, P67Q, L Q, a 455Q, D579Q, S1235Q, S945Q, R Q, F4Q, D1523672, D1270, D2Q, 1717-1G- > a, 3121G- > 1G-b > 1G + b > G1-b > G1G + b > G1G + b > b, 712-1G- > T, 1248+1G- > A, 1341+1G- > A, 3121-1G- > A, 4374+1G- > T, 3850-1G- > A, 2789+5G- > A, 3849+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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, 18 +1G- > T, 4005+2T- > C and 4003G- > A, and the patient has a human CFTR mutation selected from Δ F508, R117H, and G551D.
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V, and G1069R. In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, and S1251N. In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity, in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of E193K, F1052V, and G1069R. In some embodiments of this aspect, the method produces a 10-fold increase in chloride ion transport greater than baseline chloride ion transport.
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of 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 ion transport greater than or equal to 10% or greater of the baseline chloride ion transport.
In one aspect, the invention relates to a method of treating or opportunistically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 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+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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 invention relates to a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 1717-1G- > a, 1811+1.6kbA- > G, 2789+5G- > a, 3272-26A- > G, and 3849+10kbC- > T. In one aspect, the present invention relates to a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 2789+5G- > a and 3272-26A- > G.
In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation of CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549 28, S549 9, S1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56, P67Q, L Q, a 455Q, D579Q, S1235Q, S945Q, R Q, F1073672, D1523672, D1270, D2Q, 1717-1G- > a, 3121G- > 1G +5, G-b 1G-b 1G + b > 1G 1-b > G1G + b > 1G + b > G1-b > G1G + b > G1G + b > G1G 3, b > b, 712-1G- > T, 1248+1G- > A, 1341+1G- > A, 3121-1G- > A, 4374+1G- > T, 3850-1G- > A, 2789+5G- > A, 3849+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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, 18 +1G- > T, 4005+2T- > C and 4003G- > A, and the patient has a human CFTR mutation selected from Δ F508, R117H, and G551D, and the patient has one or more human CFTR mutations selected from Δ F508, R117H, and G551D.
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V, and G1069R, and the patient has one or more mutations in human CFTR selected from the group consisting of Δ F508, R117H, and G551D. In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, and S1251N, and the patient has one or more mutations in human CFTR selected from the group consisting of Δ F508, R117H, and G551D. In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of E193K, F1052V and G1069R, and the patient has one or more human CFTR mutations selected from the group consisting of Δ F508, R117H and G551D. In some embodiments of this aspect, the method produces a 10-fold increase in chloride ion transport greater than baseline chloride ion transport.
In one aspect, the present invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of R117C, D110H, R347H, R352Q, E56K, P67L, L206W, a455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, and D1152H, and the patient has one or more human CFTR mutations selected from the group consisting of Δ F508, R117H, and G551D. The method produces an increase in chloride ion transport greater than or equal to 10% or greater of the baseline chloride ion transport.
In one aspect, the invention relates to a method of treating or opportunistically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 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+10kbC- > T, 3272-26A- > G, 711+5G- > A, 3120G- > A, 1811+1.6kbA- > 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 the patient has one or more human CFTR mutations selected from Δ F508, R117H, and G551D. In one aspect, the invention relates to a method of treating or symptomatically treating cystic fibrosis, reducing the severity thereof, in a patient, preferably a mammal, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the invention, wherein the patient has a CFTR genetic mutation selected from the group consisting of 1717-1G- > a, 1811+1.6kbA- > G, 2789+5G- > a, 3272-26A- > G, and 3849+10kbC- > T, and the patient has one or more human CFTR mutations selected from the group consisting of Δ F508, R117H, and G551D. In one aspect, the present invention relates to a method of treating, or symptomatically treating, cystic fibrosis, lessening the severity in a patient, comprising administering to the patient, preferably a mammal, an effective amount of a pharmaceutical composition or tablet of the present invention, wherein the patient has a genetic mutation in CFTR selected from the group consisting of 2789+5G- > a and 3272-26A- > G, and the patient has one or more mutations in human CFTR selected from the group consisting of Δ F508, R117H, and G551D.
In some embodiments, a pharmaceutically acceptable composition or tablet of the
In another embodiment, the compounds and compositions of the invention are useful for treating or lessening the severity of cystic fibrosis in a patient having induced or enhanced residual CFTR activity, such residual CFTR inducers or enhancers being achievable using pharmacological means. In another embodiment, the compounds and compositions of the present invention are useful for treating or lessening the severity of cystic fibrosis in a patient having residual CFTR activity induced or enhanced using gene therapy. Such methods increase the amount of CFTR present on the cell surface, thereby inducing CFTR activity in patients that is not present to date or enhancing existing levels of residual CFTR activity in patients.
In one embodiment, the pharmaceutical compositions and tablets of the
In one embodiment, the pharmaceutical compositions and tablets of the
In one embodiment, the pharmaceutical compositions and tablets of the
In one embodiment, the pharmaceutical compositions and tablets of the
In addition to cystic fibrosis, modulation of CFTR activity may also be beneficial in other diseases not directly caused by mutations in CFTR, such as secretory diseases and other protein folding diseases mediated by CFTR. These diseases 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. 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 common in COPD. In particular, increasing anion secretion across CFTR may facilitate fluid transport to the inlet surface fluid to hydrate the mucus and optimize fluid viscosity around cilia. This will result in enhanced mucociliary clearance and a reduction in symptoms associated with COPD. Dry eye disease is characterized by reduced tear production and abnormal tear film lipid, protein and mucin behavior. There are many causes of dry eye, some of which include age, Lasik eye surgery, arthritis, medications, chemical/thermal burns, allergies, and diseases such as cystic fibrosis and sjogren's syndrome. Increasing anion secretion via CFTR will enhance transport of body fluids from corneal endothelial cells and secretory glands surrounding the eye, thereby increasing hydration of the cornea. This will help to alleviate symptoms associated with dry eye. Sjogren's syndrome is an autoimmune disease in which the immune system attacks glands that produce water everywhere in the body, including the eye, mouth, skin, respiratory tissues, liver, vagina, and intestine. Symptoms include dry eyes, mouth and vagina, and lung disease. The disease is also associated with rheumatoid arthritis, systemic lupus, systemic sclerosis and polymyositis/dermatomyositis. Defective protein trafficking is believed to lead to the disease, and treatment options are limited. Enhancers or inducers of CFTR activity may hydrate the various organs affected by the disease and help to ameliorate the associated symptoms.
In one embodiment, the invention relates to a method of enhancing or inducing the activity of an anion channel in vitro or in vivo, comprising contacting the channel with any one of the pharmaceutical compositions PC-I-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.
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 present invention are preferably formulated in unit dosage form for ease of administration and to maintain uniform dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of medicament suitable for the patient to be treated. It will be understood, however, that the total daily amount of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend upon a variety of factors including: the condition treated and the severity of the condition; the activity of the particular compound used; the specific composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; the duration of treatment; drugs used in combination or concomitantly with the specific compound employed, and similar factors well known in the medical arts. The term "patient" as used herein means an animal, preferably a mammal, and most preferably a human.
In the present application, if the name of a compound fails to correctly describe the structure of the compound, the structure replaces the name, subject to the structure.
Detailed description of the preferred embodiments
XRPD (X-ray powder diffraction)
X-ray diffraction (XRD) data for
Differential Scanning Calorimetry (DSC)
Differential Scanning Calorimetry (DSC) data for
Diffraction data were obtained on a Bruker Apex II diffractometer equipped with a sealed tube Cu K α source and an Apex II CCD detector the structure was analyzed and refined using the SHELX program (Sheldrick, G.M., Acta Crystal., (2008) A64, 112-122(Sheldrick, G.M., Crystal journal, 2008, phase A64, page 112-122).)1Structure in/n space group.
Sodium (dihydrobis (2-methoxyethoxy) aluminate [ or NaAlH)2(OCH2CH2OCH3)2]65 wt% toluene solution) was purchased from Drick chemical company (Aldrich Chemicals).
2, 2-difluoro-1, 3-benzodioxole-5-carboxylic acid was purchased from Saltigo (Lanxess Corp.).
Preparation of
Preparation of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -methanol.
Commercially available 2, 2-difluoro-1, 3-benzodioxole-5-carboxylic acid (1.0 equivalent) was slurried in toluene (10 volumes). Will be provided with
(2 equivalents) was added via addition funnel at a rate to maintain the temperature at 15-25 deg.C at the end of the addition, the temperature was increased to 40 deg.C for 2 hours (h), then 10% (w/w) aqueous NaOH (aq, 4.0 equivalents) was carefully added via addition funnel, the temperature was maintained at 40-50 deg.C, after stirring for an additional 30 minutes (min), the layers were allowed to separate at 40 deg.C, the organic phase was cooled to 20 deg.C, then washed with water (2 × 1.5.5 volumes), dried (Na)2SO4) Filtered and concentrated to give crude (2, 2-difluoro-1, 3-benzodioxol-5-yl) -methanol which was used directly in the next step.Preparation of 5-chloromethyl-2, 2-difluoro-1, 3-benzodioxole.
(2, 2-difluoro-1, 3-benzodioxol-5-yl) -methanol (1.0 eq) was dissolved in MTBE (5 volumes). A catalytic amount of 4- (N, N-dimethyl) aminopyridine (DMAP) (1 mol%) was added and SOCl was added via the addition funnel2(1.2 equiv.) was added. Adding SOCl2At a rate to maintain the temperature in the reactor at 15-25 ℃. Increasing the temperature to 30 ℃ for 1h, and then coolingTo 20 ℃. Water (4 volumes) was added via an addition funnel while maintaining the temperature below 30 ℃. After stirring for another 30min, the layers were separated. The organic layer was stirred and 10% (w/v) aqueous NaOH (4.4 volumes) was added. After stirring for 15 to 20min, the layers were separated. The organic phase is then dried (Na)2SO4) Filtered and concentrated to give crude 5-chloromethyl-2, 2-difluoro-1, 3-benzodioxole which was used directly in the next step.
Preparation of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile.
A solution of 5-chloromethyl-2, 2-difluoro-1, 3-benzodioxole (1 eq) in DMSO (1.25 volumes) was added to a slurry of NaCN (1.4 eq) in DMSO (3 volumes) while maintaining the temperature between 30-40 ℃. The mixture was stirred for 1h, then water (6 volumes) was added and methyl tert-butyl ether (MTBE) (4 volumes) was added. After stirring for 30min, the layers were separated. The aqueous layer was extracted with MTBE (1.8 volumes). The combined organic layers were washed with water (1.8 volumes) and dried (Na)2SO4) Filtered and concentrated to give crude (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile (95%) which was used directly in the next step.
Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -1-acetic acid ethyl ester-acetonitrile
The reactor was purged with nitrogen and then 900mL of toluene was added. The solvent was degassed by bubbling nitrogen for not shorter than 16 h. Then adding Na into the reactor3PO4(155.7g, 949.5mmol) and additional bis (dibenzylideneacetone) palladium (0) (7.28g, 12.66mmol) was added. A10% w/w solution of tert-butylphosphine in hexane (51.23g, 25.32mmol) was added at 23 ℃ over 10min via an addition funnel purged with nitrogen. The mixture was stirred for 50min, at which time 5-bromo-2, 2-difluoro-1, 3-benzodioxole (75g, 316.5mmol) was added over 1min, after stirring for an additional 50min, ethyl cyanoacetate (71.6g, 633.0mmol) was added to the mixture over 5min, then water (4.5mL) was added in one portion, the mixture was heated to 70 ℃ over 40min, the percent conversion of the reactants to the product was analyzed by HPLC every 1-2h after the conversion was observed to be complete (typically 100% conversion after 5-8 h), the mixture was cooled to 20-25 ℃, then filtered through a pad of celite, the pad was rinsed with toluene (2 × 450mL), the combined organics were concentrated to 300mL under vacuum at 60-65 ℃, 225mL of DMSO was added to the concentrate, concentrated under vacuum at 70-80 ℃ until active solvent distillation ceased, the solution was cooled to 20-25 ℃, diluted to 900mL with DMSO to prepare for step 2.1H NMR(500MHz,CDCl3)7.16–7.10(m,2H),7.03(d,J=8.2Hz,1H),4.63(s,1H),4.19(m,2H),1.23(t,J=7.1Hz,3H).
Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile.
To the above-obtained DMSO solution of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -1-acetic acid ethyl ester-acetonitrile was added 3N HCl (617.3mL, 1.85mol) over 20min while maintaining the internal temperature<At 40 ℃. The mixture was then heated to 75 ℃ over 1h and analyzed by HPLC for percent conversion every 1-2 h. When conversion is observed>At 99% (typically after 5-6 h), the reaction was cooled to 20-25 deg.C and extracted with MTBE (2 × 525mL) for a sufficient time to allow complete phase separation during extraction, the combined organic extracts were washed with 5% NaCl (2 × 375mL), the solution was then transferred to an apparatus suitable for 1.5-2.5 torr vacuum distillation equipped with a cooled receiving flask, the solution was subjected to vacuum and extracted with MTBE (2 × mL) for a sufficient time to allow complete phase separation during extraction<Concentrate at 60 ℃ to remove the solvent. (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile was then distilled from the resulting oil at 125 ℃ and 130 ℃ (oven temperature) at 1.5-2.0 torr. (2, 2-difluoro-1, 3-benzoDioxol-5-yl) -acetonitrile was isolated from 5-bromo-2, 2-difluoro-1, 3-benzodioxole (step 2) as a clear oil in 66% yield with an HPLC purity of 91.5% AUC (determined corresponding to 95% by weight).1H NMR(500MHz,DMSO)7.44(brs,1H),7.43(d,J=8.4Hz,1H),7.22(dd,J=8.2,1.8Hz,1H),4.07(s,2H).
Preparation of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarbonitrile.
(2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile (1.0 equiv.), 50% by weight aqueous KOH (5.0 equiv.), 1-bromo-2-chloroethane (1.5 equiv.), and Oct4A mixture of NBr (0.02 eq.) was heated at 70 ℃ for 1 h. The reaction mixture was cooled and then worked up with MTBE and water. The organic phase was washed with water and brine. The solvent is removed to give (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarbonitrile.
Preparation of 1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid.
(2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarbonitrile was hydrolyzed with 6M NaOH (8 equivalents) in ethanol (5 volumes) overnight at 80 ℃. The mixture was cooled to room temperature and the ethanol was evaporated under vacuum. The residue was taken up in water and MTBE, 1M HCl was added and the layers were separated. The MTBE layer was then treated with Dicyclohexylamine (DCHA) (0.97 eq). The slurry was cooled to 0 ℃, filtered and washed with heptane to give the corresponding DCHA salt. The salt was absorbed into MTBE and 10% citric acid and stirred until all solids dissolved. The layers were separated and the MTBE layer was washed with water and brine. The solvent was exchanged for heptane and filtered and dried in a vacuum oven at 50 ℃ overnight to yield 1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid.
1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -preparation of carbonyl chloride herein.
1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid (1.2 equivalents) was slurried in toluene (2.5 volumes) and the mixture heated to 60 ℃. Adding SOCl2(1.4 eq) was added via addition funnel. After 30 minutes toluene and SOCl were added2Distilled out of the reaction mixture. Additional toluene (2.5 volumes) was added and the resulting mixture was again distilled, leaving the acid chloride as an oil, which was used without further purification.
Preparation of tert-butyl 3- (3-methylpyridin-2-yl) benzoate.
2-bromo-3-methylpyridine (1.0 eq) was dissolved in toluene (12 volumes). Will K2CO3(4.8 equiv.) and then water (3.5 volumes) was added. The resulting mixture is stirred under N2The mixture was heated to 65 ℃ under reduced pressure for 1 hour. 3- (tert-Butoxycarbonyl) phenylboronic acid (1.05 eq.) and Pd (dppf) Cl2·CH2Cl2(0.015 eq.) was added and the mixture was heated to 80 ℃. After 2 hours, the heating was stopped, water was added (3.5 volumes) and the layers were separated. The organic phase was then washed with water (3.5 volumes) and extracted with 10% aqueous methanesulfonic acid (2 equivalents MsOH, 7.7 volumes). The aqueous phase was made basic by 50% aqueous NaOH (2 eq) and extracted with EtOAc (8 vol). The organic layer was concentrated to give crude tert-butyl 3- (3-methylpyridin-2-yl) benzoate (82%) which was used directly in the next step.
Preparation of 2- (3- (tert-butoxycarbonyl) phenyl) -3-methylpyridine-1-oxide
Tert-butyl 3- (3-methylpyridin-2-yl) benzoate (1.0 eq) was dissolved in EtOAc (6 vol). Water (0.3 volumes) was added followed by urea-hydrogen peroxide (3 equivalents). Phthalic anhydride (3 equivalents) was then added in portions to the mixture as a solid at a rate that maintained the temperature in the reactor below 45 ℃. After the addition of phthalic anhydride was complete, the mixture was heated to 45 ℃. After stirring for a further 4 hours, the heating was stopped. Mixing 10% w/w of Na2SO3The aqueous solution (1.5 eq) was added via addition funnel. After completing Na2SO3After addition of (b), the mixture was stirred for a further 30min and the layers were then separated. The organic layer was stirred and 10% w/w Na2CO3Aqueous solution (2 eq) was added. After stirring for 30 minutes, the layers were separated. The organic phase was washed with 13% w/v aqueous NaCl solution. The organic phase was then filtered and concentrated to give crude 2- (3- (tert-butoxycarbonyl) phenyl) -3-methylpyridine-1-oxide (95%) which was used directly in the next step.
Preparation of tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate.
Heating a solution of 2- (3- (tert-butoxycarbonyl) phenyl) -3-methylpyridine-1-oxide (1 eq) and pyridine (4 eq) in acetonitrile (8 vol.) to 70 ℃ adding a solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol.) via an addition funnel over 50min while maintaining the temperature below 75 ℃ after addition is complete the mixture is stirred for a further 0.5 h then the mixture is cooled to ambient temperature ethanolamine (10 eq.) is added via an addition funnel after stirring for 2h water (6 vol.) is added and the mixture is cooled to 10 ℃ after stirring for 3 h the solid is collected by filtration, washed with water (3 vol.), 2:1 acetonitrile/water (3 vol.) and acetonitrile (2 × 1.5.5 vol.) the solid is passed through a gentle N at 50 ℃ in a vacuum oven2Flow-drying to constant weight (<1% difference) to give 3- (6) as a red-yellow solid-amino-3-methylpyridin-2-yl) benzoic acid tert-butyl ester (53% yield).
Preparation of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) -cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester.
The crude acid chloride described above was dissolved in toluene (2.5 volumes based on acid chloride) and added via an addition funnel to a mixture of tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate (1 equivalent), DMAP (0.02 equivalent) and triethylamine (3.0 equivalent) in toluene (4 volumes based on tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate). After 2 hours, water (4 volumes based on 3- (6-amino-3-methylpyridin-2-yl) benzoic acid tert-butyl ester) was added to the reaction mixture. After stirring for 30 minutes, the layers were separated. The organic phase is then filtered and concentrated to give 3- (6- (1- (2, 2-difluorobenzo [ d ])][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester (quantitative crude yield) thick oil acetonitrile (3 volumes based on crude product) was added and distilled until crystallization occurred water (2 volumes based on crude product) was added and the mixture was stirred for 2h the solid was collected by filtration, washed with 1:1 (volume ratio) acetonitrile/water (2 × 1 volumes based on crude product) and then partially dried under vacuum on the filter the solid was passed through a slight N in a vacuum oven at 60 ℃ under vacuum2Flow-drying to constant weight (<1% difference) to give 3- (6- (1- (2, 2-difluorobenzo [ d ] as a brown solid][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester.
Preparation of 3- (6- (1- (2, 2-difluorobenzo [ d ] [1, 3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid HCl salt.
To 3- (6)- (1- (2, 2-difluorobenzo [ d ]][1,3]Dioxolen-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester (1.0 equivalent) slurry in MeCN (3.0 volumes) Water (0.83 volumes) was added followed by concentrated aqueous hydrochloric acid (0.83 volumes). the mixture was heated to 45 + -5 deg.C and stirred for 24 to 48h, after which the reaction was complete and the mixture was allowed to cool to ambient temperature Water (1.33 volumes) was added, the mixture was stirred the solid was collected by filtration, washed with water (2 × 0.3 volumes) and then partially dried under vacuum on a filter the solid was passed through a slight N at 60 deg.C in a vacuum oven2Flow-drying to constant weight (<1% difference) to give 3- (6- (1- (2, 2-difluorobenzo [ d ] as an off-white solid][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid HCl.
Process for preparation of
The following Table 2 describes the compounds I1HNMR data.
Table 2.
Preparation of
Preparation of
Reacting 3- (6- (1- (2, 2-difluorobenzo [ d ]][1,3]Dioxolen-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid HCl (1 equivalent) slurry in water (10 volumes) was stirred at ambient temperature after stirring for 24h samples were taken samples were filtered, the solids were washed with water (2 times) the solid samples were subjected to DSC analysis when DSC analysis indicated complete conversion to form I, the solids were collected by filtration, washed with water (2 × 1.0 volumes) and partially dried under vacuum on the filterPassing a slight N gas at 60 ℃ in a vacuum oven2Flow-drying to constant weight (<1% difference) to give
Preparation of
Reacting 3- (6- (1- (2, 2-difluorobenzo [ d ]][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester (1.0 equiv) in formic acid (3.0 volumes) was heated to 70. + -. 10 ℃ for 8h with stirring. 3- (6- (1- (2, 2-difluorobenzo [ d ]) when chromatography indicates an AUC of not more than 1.0%][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester) remains, the reaction is deemed complete. The mixture was allowed to cool to ambient temperature. The solution was added to water (6 volumes), heated at 50 ℃ and the mixture was stirred. The mixture was then heated to 70 + -10 deg.C until 3- (6- (1- (2, 2-difluorobenzo [ d ]) was reached][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) -benzoic acid tert-butyl ester content not exceeding 0.8% (AUC.) the solid was collected by filtration, washed with water (2 × 3 times by volume) and then partially dried under vacuum on the filter the solid was passed through a slight N in a vacuum oven at 60 deg.C2Flow-drying to constant weight (<1% difference) to yield
The DSC curve of
The X-ray diffraction pattern was calculated from the single crystal structure of
Table 3.
Peak ordering
2 theta angle]
Relative 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
The actual X-ray powder diffraction pattern of
Table 4.
Peak ordering
2 theta angle]
Relative 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
Colorless crystals of
Obtaining a reciprocal space diffraction data set using 0.5 ° stepping at 30s exposure per frame, up toThe resolution of (2). Data is collected at 100(2) K. Integration of intensity and refinement of unit cell parameters was done using APEXII software. Observation of the crystals after data acquisition showed no evidence of decomposition.
A conformational diagram of
Preparation of
Synthesis of 4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (26)
Process for preparing 4-oxo-1, 4-dihydroquinoline-3-carboxylic acid ethyl ester (25)
Compound 23(4.77g, 47.7mmol) was added dropwise to compound 22(10g, 46.3mmol), then N was introduced2The ethanol was driven off with a stream of air at below 30 ℃ for 0.5 h. The solution was then heated to 100 ℃ and 110 ℃ and stirred for 2.5 hours. After cooling the mixture to below 60 ℃, diphenyl ether was added. The resulting solution was added dropwise to diphenyl ether which had been heated to 228-2The gas flow is used to drive off the ethanol. The mixture was stirred at 228-232 ℃ for a further 2 hours, cooled to below 100 ℃ and heptane was then added to precipitate the product. The resulting slurry was stirred at 30 ℃ for 0.5 hour. The solid was then filtered, the cake washed with heptane and dried in vacuo to give compound 25 as a brown solid.1H NMR(DMSO-d6;400MHz)12.25(s),8.49(d),8.10(m),7.64(m),7.55(m),7.34(m),4.16(q),1.23(t).
Process for preparing 4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (26)
Compound 25(1.0 equiv.) is suspended in HCl (10.0 equiv.) and H2O (11.6 volumes). The slurry is heated to 85-90 ℃, although alternative temperatures are suitable for the hydrolysis step. For example, the hydrolysis may be selected to be carried out at a temperature of from about 75 to about 100 ℃. In some cases, the hydrolysis is carried out at a temperature of about 80 to about 95 ℃. In other instances, the hydrolysis step is carried out at a temperature of about 82 to about 93 ℃ (e.g., about 82.5 to about 92.5 ℃ or about 86 to about 89 ℃). After stirring at 85-90 ℃ for about 6.5 hours, the reaction system was sampled as the reaction was complete. Stirring may be carried out at any temperature suitable for hydrolysis. The solution was then cooled to 20-25 ℃ and filtered. By H2O (2 volumes x 2) flushes the reactor/cake. Then 2 volumes H2O washing the cake to a pH of 3.0 or more. The cake was then dried under vacuum at 60 ℃ to give compound 26.
Compound 25(11.3g, 52mmol) was added to a mixture of 10% NaOH (aq) (10mL) and ethanol (100 mL). Heating the solution to reflux for 16 hours, cooling to 20-25 deg.C, then 8%HCl adjusts the pH to 2-3. The mixture was then stirred for 0.5 hour and filtered. The cake was washed with water (50mL) and then dried in vacuo to give compound 26 as a brown solid.1H NMR(DMSO-d6;400MHz)15.33(s),13.39(s),8.87(s),8.26(m),7.87(m),7.80(m),7.56(m).
Overall Synthesis of N- (2, 4-di-tert-butyl-5-hydroxyphenyl) -4-oxo-1, 4-dihydroquinoline-3-carboxamide (Compound 2)
Method for preparing 2, 4-di-tert-butyl phenyl methyl carbonate (30)
To a solution of 2, 4-di-tert-butylphenol 29(10g, 48.5mmol) in diethyl ether (100mL) and triethylamine (10.1mL, 72.8mmol) was added methyl chloroformate (7.46mL, 97mmol) dropwise at 0 ℃. The mixture was then warmed to room temperature and stirred for an additional 2 hours. Then 5mL of triethylamine and 3.7mL of methyl chloroformate were added and the reaction was stirred overnight. The reaction system was then filtered, the filtrate was cooled to 0 ℃, then 5mL of triethylamine and 3.7mL of methyl chloroformate were added, the reaction system was warmed to room temperature, and then stirred for another 1 hour. At this point, the reaction was nearly complete, worked up by filtration, then washed with water (2 ×), then brine. The solution was then concentrated to give a yellow oil, which was purified using column chromatography to give compound 30.1H NMR(400MHz,DMSO-d6)7.35(d,J=2.4Hz,1H),7.29(dd,J=8.4,2.4Hz,1H),7.06(d,J=8.4Hz,1H),3.85(s,3H),1.30(s,9H),1.29(s,9H).
To a reactor vessel were added 4-dimethylaminopyridine (DMAP, 3.16g, 25.7mmol) and 2, 4-di-tert-butylphenol (compound 29, 103.5g, 501.6mmol), dichloromethane (415g, 313mL) was added and the solution was stirred until all the solid had dissolved. Triethylamine (76g, 751mmol) was then added and the solution cooled to 0-5 ℃. Methyl chloroformate (52g, 550.3mmol) was then added dropwise over 2.5-4 hours while maintaining the solution temperature at 0-5 ℃. The reaction mixture was then slowly heated to 23-28 ℃ and stirred for 20 hours. The reaction was then cooled to 10-15 deg.C and 150mL of water was added. The mixture was stirred at 15-20 ℃ for 35-45 minutes, then the aqueous layer was separated and extracted with 150mL of dichloromethane. The combined organic layers were neutralized with 2.5% HCl (aq) at 5-20 deg.C to give a final pH of 5-6. The organic layer was then washed with water and concentrated to 150mL under vacuum at a temperature below 20 ℃ to give
Method for preparing 5-nitro-2, 4-di-tert-butyl phenyl methyl carbonate (31)
To stirred compound 30(6.77g, 25.6mmol) was added dropwise 6mL of 1:1 sulfuric acid and nitric acid. The mixture was warmed to room temperature and stirred for 1 hour. The product was purified using liquid chromatography (ISCO, 120g, 0-7% EtOAc/hexanes, 38min) to yield about 8: 1-10: 1 as a white solid.1H NMR(400MHz,DMSO-d6)7.63(s,1H),7.56(s,1H),3.87(s,3H),1.36(s,9H),1.32(s,9H).HPLCret.time 3.92min 10-99%CH3CN,5min run;ESI-MS 310m/z(MH)+.
To compound 30(100g, 378mmol) was added DCM (540g, 408 mL). The mixture was stirred until all the solids dissolved and then cooled to-5-0 ℃. Then, concentrated sulfuric acid (163g) was added dropwise while maintaining the internal temperature of the reaction, and the mixture was stirred for 4.5 hours. Nitric acid (62g) was then added dropwise over 2-4 hours while maintaining the internal temperature of the reaction, and then stirred at that temperature for an additional 4.5 hours. The reaction mixture was then slowly added to cold water, maintaining the temperature below 5 ℃. Then heating the quenched reaction system to 25 ℃, and takingThe aqueous layer was extracted with dichloromethane. The combined organic layers were washed with water and Na2SO4Dried and concentrated to 124-155 mL. Hexane (48g) was added and the resulting mixture was further concentrated to 124-155 mL. Hexane (160g) was then added to the mixture. The mixture was then stirred at 23-27 ℃ for 15.5 hours and then filtered. Hexane (115g) was added to the filter cake and the resulting mixture was heated to reflux and stirred for 2-2.5 hours. The mixture was then cooled to 3-7 ℃, stirred for an additional 1-1.5 hours, and filtered to give compound 31 as a pale yellow solid.
Method for preparing 5-amino-2, 4-di-tert-butyl phenyl methyl carbonate (32)
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% based on dry weight, Johnson-Matthey Type 37). MeOH (15.0 volumes) was added to the reactor and the system was sealed. Charging N into the system2(g) Then with H2(g) The pressure was increased to 2.0 bar. The reaction was carried out at 25 ℃ C. +/-5 ℃ C. When complete, the reaction was filtered and the reactor/cake was washed with MeOH (4.00 volumes). The resulting filtrate was distilled under vacuum at a temperature not exceeding 50 ℃ to 8.00 times by volume. Water (2.00 volumes) was added at 45 ℃ +/-5 ℃. The resulting slurry was cooled to 0 deg.C +/-5. The slurry was maintained at 0 deg.C +/-5 deg.C for not less than 1 hour and filtered. With a mixture of 0 ℃ C. +/-5 ℃ MeOH/H2The O (8: 2) (2.00 volumes) cake was washed 1 time. The cake was dried under vacuum (-0.90 bar and-0.86 bar) at 35 deg.C-40 deg.C to give compound 32.1H NMR(400MHz,DMSO-d6)7.05(s,1H),6.39(s,1H),4.80(s,2H),3.82(s,3H),1.33(s,9H),1.23(s,9H).
Once the reaction is complete, the resulting mixture is diluted with about 5 to 10 volumes of MeOH (e.g., about 6 to about 9 volumes of MeOH, about 7 to about 8.5 volumes of MeOH, about 7.5 to about 8 volumes of MeOH, or about 7.7 volumes of MeOH), heated to a temperature of about 35 ± 5 ℃, filtered as described above, washed, and dried.
Preparation of N- (2, 4-di-tert-butyl-5-hydroxyphenyl) -4-oxo-1, 4-dihydroquinoline-3-carboxamide (Compound 2).
4-oxo-1, 4-dihydroquinoline-3-carboxylic acid 26(1.0 eq) and 5-amino-2, 4-di-tert-butylphenyl methylcarbonate to 32(1.1 eq) were added to the reactor. 2-MeTHF (4.0 volumes relative to acid) was added followed by a
Another preparation of N- (2, 4-di-tert-butyl-5-hydroxyphenyl) -4-oxo-1, 4-dihydroquinoline-3-carboxamide (Compound 2).
4-oxo-1, 4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and 5-amino-2, 4-di-tert-butylphenyl methylcarbonate 32(1.1 eq) are charged to the reactor. 2-MeTHF (4.0 volumes relative to acid) was added followed by a
The filtered solution was concentrated to 20 volumes under reduced pressure at not more than 35 ℃ (jacket temperature) and not less than 8.0 ℃ (internal reaction temperature). Adding CH3CN to 40 times by volume, and concentrated to 20 times by volume under reduced pressure at not more than 35 ℃ at a jacket temperature and not less than 8.0 ℃ (internal reaction temperature). Will add CH3CN and concentration cycle are repeated more than 2 times, and CH is added for 3 times in total3CN and 4 times concentrated to 20 volumes. After final concentration to 20 volumes, 16.0 volumes of CH were added3CN, then 4.0 times of H2O, to a final concentration of 10% H of 40 volumes relative to the starting acid2O/CH3And (C) CN. The slurry was heated to 78.0 deg.C +/-5.0 deg.C (reflux). The slurry was then stirred for not less than 5 hours. The slurry was cooled to 20-25 ℃ over 5 hours and filtered. With CH heated to 20-25
Recrystallization method of N- (2, 4-di-tert-butyl-5-hydroxyphenyl) -4-oxo-1, 4-dihydroquinoline-3-formamide (compound 2)
Compound 2(1.0 equivalent) was added to the reactor. 2-MeTHF (20.0 volumes) was added followed by 0.1N HCl (5.0 volumes). The biphasic solution was stirred, separated and the upper organic phase was washed 2 times with 0.1n hcl (5.0 volumes). The organic solution was fine filtered to remove any particulate matter and placed in a second reactor. The filtered solution was concentrated under reduced pressure to 10 volumes at not more than 35 ℃ (jacket temperature) and not more than 8.0 ℃ (internal reaction temperature). Isopropyl acetate (IPAc) (10 volumes) was added, and the solution was concentrated under reduced pressure to 10 volumes at not more than 35 ℃ (jacket temperature) and not more than 8.0 ℃ (internal reaction temperature). The addition of IPAc and concentration was repeated 2 more times for a total of 3 IPAc additions and 4 concentrations to 10 volumes. After final concentration, 10 volumes of IPAc were added and the slurry was heated to reflux and maintained at this temperature for 5 hours. The slurry was cooled to 0.0 ℃ +/-5 ℃ over 5 hours, filtered and the cake was washed 1 time with IPAc (5 volumes). The resulting solid was dried in a vacuum oven at 50.0 deg.C +/-5.0 deg.C.
Preparation of solid Dispersion comprising substantially
A solvent system of MEK and DI water, formulated according to a ratio of 90 wt% MEK/10 wt% DI water, was heated to a temperature of 20-30 ℃ in a reactor equipped with a magnetic stirrer and a thermodynamic cycle. Hypromellose acetate succinate polymer (HPMCAS) (HG grade), SLS and
table 5: ingredients for solid spray dispersions of intermediate F
Unit of
Batch size
Compound 2Kg
70.0
HPMCAS Kg
17.1
SLS Kg
0.438
Total solids Kg
87.5
MEK Kg
671
Water Kg
74.6
Total solvent Kg
746
Total spray solution weight Kg
833
The mixture is adjusted to a temperature in the range of 20-45 ℃ and mixed until it is substantially homogeneous and all ingredients are substantially dissolved.
In normal Spray drying mode, a Spray Dryer Niro PSD4 Commercial Spray Dryer equipped with a pressure nozzle fitted with an anti-reflecting cap (Spray systems Maximum Passage series SK-MFP with orifice/core size 54/21) was used according to the dry Spray process parameters shown in table 6 below.
Table 6: dry spray process parameters for production of intermediate F
The high efficiency cyclone separates the wet product from the spray gas and solvent vapors. The wet product comprises 8.5-9.7% MEK and 0.56-0.83% water and has an average 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 solvent to a level below about 5000ppm and to produce a spray dried dispersion of
Tablet formation by a fully continuous wet granulation process
apparatus/Process
Device
A full Continuous deployment and Launch kit (DLR) or similar type of device.
Sieving
Blending of
The
Wet granulation
The granulation liquid may be prepared by dissolving 48g of sodium lauryl sulfate and 159g of polyvinylpyrrolidone in 1, 626g of water using an overhead stirrer with a stirring speed of 700 RPM. The granulation liquid may be placed in a container from which it is pumped into a twin screw granulator using a peristaltic pump having a mass flow meter and a controller, using a flow rate suitable for the process. The blend may be pelletized using a twin screw pelletizer, such as a pelletizer that is part of the DLR. The blend can be fed into a twin screw granulator using a loss in weight feeder, such as a K-Tron feeder on DLR, at a feed rate of 8kg/hr to 24 kg/hr. The twin screw granulator was operated at a barrel temperature of 25 ℃ and a screw speed of 200 and 950 RPM. For small batch sizes, the granulation process may be performed for 3 minutes; or for large batch sizes, several hours.
Drying
The wet granulation may be fed directly into a fluid bed dryer, such as a staged fluid bed dryer on DLR. The end point of drying may be selected at a product temperature during discharge of 40-55 c, at which point the moisture content of the granules may be 2.1% w/w ("loss on drying, LOD") or less. The drying time may be 12 minutes or may be short or long to reach the desired drying end point.
Grinding
The dried particles may be milled to reduce particle size. A conical mill, such as an integrated Quadro U10 coim, can be used for this purpose.
Blending of
The granules can be blended with extragranular excipients such as fillers and lubricants using a weight loss feeder and a continuous blender. The blending speed may be 80-300 RPM.
Pressing
The compressed blend can be compressed into tablets using a single or rotary tablet press, such as a Courtoy Modul P press (which is an integral part of the DLR system), using a cutter of appropriate size. The tablet weight for a dose of 200mg of
Film coating
Tablets can be film coated using an innovative omega film coater that is part of the DLR system. The coating machine can rapidly coat film for 1-4 kg small batches for continuous manufacturing.
Printing
Film-coated tablets are printed with letter designs on one or both surfaces of the tablet using, for example, an Ackley variable speed printer.
Tablet formation by twin screw wet granulation process
apparatus/Process
Device
A double-screw wet granulator: ConsiGma-1, ConsiGma-25 or Leistritz nano.
Sieving/weighing
Blending of
Wet granulation
The granulation liquid may be prepared by dissolving 48g of sodium lauryl sulfate and 159g of polyvinylpyrrolidone in 1, 626g of water using an overhead stirrer with a stirring speed of 700 RPM. The blend may be pelletized using a twin screw pelletizer such as ConsiGma-1. The granulation liquid may be fed into the twin-screw granulator using a pump on a peristaltic pump Consi Gma-1 at a feed rate of 67 g/min. The blend may be fed into a twin screw granulator using a loss-in-weight feeder such as the Brabender feeder on ConsiGma-1 at a feed rate of 10 kg/hr. The twin screw granulator can be run at a barrel temperature of 25 ℃ and a screw speed of 400 RPM. The granulation process may be performed for 4 minutes. The granulation process may be carried out for shorter or longer periods of time to produce smaller or larger quantities of wet granulation.
Drying
The wet granules may be fed directly into a fluid bed dryer, such as a drying chamber on ConsiGma-1 or a staged fluid bed dryer on CTL-25. The end point of drying may be selected to be at a product temperature of 43 c, at which point the moisture content of the granules may be 1.6% w/w ("loss on drying, LOD"). The drying time may be 12 minutes or may be short or long to reach the desired drying end point. The drying may be at 59m3Air flow/min and inlet temperature of 60 ℃. Alternatively, the wet granulation from the twin screw granulator may be collected into a bin or container for a defined period of time, after which the wet granulation is transferred to a separate stand alone fluid bed dryer, such as a
Grinding
The dried particles may be milled to reduce particle size. A conical mill such as Quadro 194CoMil may be used for this purpose.
Blending of
The pellets can be blended with extragranular excipients such as fillers and lubricants using a V-shell blender or bin blender. The blending time may be 5, 3 or 1 minute.
Pressing
The compressed blend can be compressed into tablets using a single or rotary tablet press, such as a Courtoy Modul P press, using 0.55 'x 0.33' oval knives. The tablet weight for a dose of 200mg of
Film coating
The tablets may be film coated using a pan coater such as a Thomas Engineering Comu-Lab coater. Trace amounts of carnauba wax may be added to improve tablet appearance and processability.
Printing
Film-coated tablets are printed with a letter pattern on one or both surfaces of the tablet by, for example, a Hartnett Delta printer.
Tablet formation by continuous twin screw wet granulation process
apparatus/Process
Device
A granulator: ConsiGma or Leistritz or Thermo Fisher twin screw granulator.
Sieving/weighing
Blending of
Granulation operation
Granulation liquid-SLS and binder are added to purified water and mixed until dissolved. A suitable ratio is 2.5% w/w aqueous SLS solution and 10.0% w/w aqueous PVP K30 solution.
Granulation-the
Grinding
The particle size is reduced with a screen mill or a conical mill before or after drying or both.
Drying
The granules may be dried in a vacuum oven, tray dryer, double cone dryer or fluidized bed dryer.
Blending of
The granules may be blended with extra-granular excipients. The pellets were blended for 60 revolutions using a 300 liter bin blender.
Pressing
The compressed blend was compressed into tablets using a Courtoy module P rotary tablet press.
Film coating
The tablets are film coated with a coating pan (e.g., O' Hara Labcoat).
Printing
Film-coated tablets are printed with a letter pattern on one or both surfaces of the tablet by, for example, a Hartnett Delta printer.
Assay method
Assays for detecting and measuring the synergistic properties of a compound
Membrane potential optical assay for determining Δ F508-CFTR modulating properties of compounds
The present assay employs a fluorescent voltage sensing dye to determine changes in membrane potential as a readout of increased functional Δ F508-CFTR in NIH3T3 cells using a fluorescent culture plate reader (e.g., FLIPR III, Molecular Devices, Inc.). The driving force for this response is the chloride ion gradient generation and binding channel activation caused by a single liquid addition step after pre-treatment with a compound and subsequent loading of the voltage sensing dye.
Identification of potentiator compounds
To identify potentiators of Δ F508-CFTR, a dual addition HTS assay format was developed. This HTS assay uses a fluorescent voltage sensing dye to determine the membrane potential on FLIPR III as a measure of the increase in gated (conductance) af 508CFTR in temperature corrected af 508CFTR NIH3T3 cells. The driving force for this response is Cl in a single liquid addition step using a fluorescent plate reader such as FLIPR III after pre-treatment with the potentiator compound (or DMSO vehicle control) and subsequent loading of the redistribution dye-Ion and channel activation using forskolin in combination.
Solutions of
Warm bath liquid # 1: NaCl 160, KCl 4.5, CaCl (unit mM)22、
Chloride-free warm bath: the chloride salt in warm bath #1 (above) was replaced by gluconate.
Cell culture
NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for optical measurement of membrane potential. Cells were maintained at 37 ℃ with 5% CO2And 175cm at 90% humidity2Dulbecco's modified eagle (Eagl) in culture flaskse) Medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β -ME, 1X penicillin/streptomycin and 25mM hepes for all optical assays, cells were seeded at-20,000/well on 384-well matrigel-coated plates and cultured at 37 ℃ for 2hrs, then at 27 ℃ for potentiator assays for correction assays, cells were incubated with and without compounds at 27 ℃ or 37 ℃ for 16-24 hours.
An electrophysiological assay for determining Δ F508-CFTR modulating properties of a compound.
Ussing Chamber assay
Ussing laboratory experiments were performed on polarized epithelial cells expressing Δ F508-CFTR to further characterize the Δ F508-CFTR potentiators or inducers identified in the optical assay. non-CF and CF airway epithelia were isolated from bronchial tissue cultured as described above (Galietta, l.j.v., Lantero, s., Gazzolo, a., Sacco, o., Romano, l., Rossi, g.a.,&Zegarra-Moran, O. (1998) In Vitro cell. Dev. biol.34, 478-481) and placed In pre-coating with NIH3T 3-conditioned mediumSnapwellTMOn the filter. After 4 days, the apical medium was removed and the cells were allowed to grow at an air-liquid interface>This results in a fully differentiated columnar cell monolayer, ciliated, characterized by features of airway epithelium, isolating non-CF HBE from non-smokers who do not have any known lung disease CF-HBE was isolated from patients homozygous for △ F508.
Will grow in
SnapwellTMHBE on cell culture cannulae was mounted in a Ussing chamber (physiologic instruments, Inc., San Diego, Calif.) and the basal lateral to apical Cl was measured using a voltage clamp system (Deparatment of Bioengineering, university of Iowa, Iowa)-Gradient (I)SC) StoreTransepithelial resistance and short circuit current below. Briefly, at 37 ℃ under voltage clamp system recording conditions (V)Holding0mV) was examined for HBE. The solution on the outside of the substrate contained (mM units) 145NaCl, 0.83K2HPO4、3.3KH2PO4、1.2MgCl2、1.2CaCl210 glucose, 10HEPES (pH adjusted to 7.35 with NaOH), and the apical solution comprised (in mM units) 145 sodium gluconate, 1.2MgCl2、1.2CaCl210 glucose, 10HEPES (pH adjusted to 7.35 with NaOH).Identification of potentiator compounds
Typical protocols employ Cl from the outside of the substrate to the top membrane-A concentration gradient. To establish this gradient, a normal loop was used on the basal outer membrane, while the apical NaCl was replaced with equimolar sodium gluconate (titrated to pH7.4 with NaOH) to give large Cl across the epithelium-A concentration gradient. Forskolin (10 μm) and all test compounds were added to the apical side of the cell culture cannulae. The performance of the putative af 508-CFTR potentiator was compared to the potency of the known potentiator genistein.
Patch clamp record
Monitoring of total Cl in Δ F508-NIH3T3 cells using perforated film recording conformation as described above-Electric current (Rae, j., Cooper, k., Gates, p.,&watsky, m. (1991) j. neurosci. methods 37, 15-26). Voltage clamp recordings were performed at 22 ℃ using an Axoatch 200B patch clamp amplifier (Axon Instruments Inc., Foster City, Calif.). Pipette solutions containing (in mM) 150N-methyl-D-glucosamine (NMDG) -Cl, 2MgCl2、2CaCl210EGTA, 10HEPES and 240. mu.g/mL amphotericin-B (pH adjusted to 7.35 with HCl). The extracellular medium contains (in mM)150NMDG-Cl, 2MgCl2、2CaCl210HEPES (pH adjusted to 7.35 with HCl). Pulsing, data acquisition and analysis were performed using a PC equipped with a Digidata 1320A/D interface in conjunction with Clampex 8(Axon Instruments Inc.). To activate Δ F508-CFTR, 10 μ M forskolin and 20 μ M genistein were added to the bath, and each timeThe current-voltage relationship was monitored every 30 seconds.
Identification of potentiator compounds
Also, the use of a perforated-patch-recording technique to study the macroscopic Δ F508-CFTR potentiator in increasing NIH3T3 cells stably expressing Δ F508-CFTR-Electric current (I)△F508) The ability of the cell to perform. Potentiating agents identified from optical assays cause I.DELTA.F508With efficacy and potency similar to those observed in optical assays. In all cells tested, the reversal potential before and during the application of the potentiator was about-30 mV, which is the calculated ECl(-28mV)。
Cell culture
NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for whole cell recordings. Cells were maintained at 37 ℃ with 5% CO2And 175cm at 90% humidity2In Dulbecco's modified eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β -ME, 1X penicillin/streptomycin and 25mM HEPES in culture flasks for whole cell recording, 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and cultured at 27 ℃ for 24-48hrs, then used to test for potentiator activity, and incubated with or without a correcting compound at 37 ℃ to determine correcting agent activity.
Single channel recording
As described above (Dalemans, W., Barbry, P., Champgny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R.G., Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-2、2MgCl2And 10HEPES (pH adjusted to 7.35 with Tris base). The warm bath solution contained (in mM): 150NMDG-Cl, 2MgCl25EGTA, 10TES and 14Tris base (pH adjusted to 7.35 with HCl). After cutting offMicroelectrode was maintained at 80 mV. to analyze channel activity from patches comprising ≤ 2 active channels, while the maximum of open determined the number of active channels during the experiment, data recorded from △ F508-CFTR activity of 120s were filtered "off-line" at 100Hz to determine single channel current amplitude, and then used to construct an amplitude histogram of all points fitted with a Bio-Patch analysis software (Bio-Logic company, france) by multiple gauss functions, total microscopic current and open probability (P) was determined by channel activity of 120s (p.s)o). Using Bio-Patch software or from the relation PoMeasurement of P ═ I/I (N)oWhere I is the average current, I is the single channel current amplitude, and N is the number of active channels in the membrane.
Cell culture
NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for incised patch clamp recordings. Cells were maintained at 37 ℃ with 5% CO2And 175cm at 90% humidity2In Dulbecco's modified eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β -ME, 1X penicillin/streptomycin, and 25mM HEPES in culture flasks 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and cultured at 27 ℃ for 24-48hrs for single channel recording, and then used.
Assays for detecting and measuring Δ F508-CFTR correction properties of compounds
Membrane potential optical methods for determining Δ F508-CFTR modulating properties of a compound.
Optical Membrane potential assay Using Voltage-sensitive FRET sensors as described by Gonzalez and Tsien (see Gonzalez, J.E.and R.Y.Tsien (1995) "Voltage sensing by fluorescence sensitivity transfer in single cells"Biophys J69(4):1272-80(Gonzalez, J.E. and R.Y.Tsien, 1995 "-in single cellsVoltage "is sensed by fluorescence resonance energy transfer," journal of biophysics,
These voltage-sensitive assays are based on the membrane-soluble, voltage-sensitive dye DiSBAC2(3) Change in Fluorescence Resonance Energy Transfer (FRET) with the fluorescent phospholipid CC2-DMPE, which is attached to the outer leaflet of the plasma membrane and acts as a FRET donor. Membrane potential (V)m) Resulting in a negatively charged DiSBAC2(3) Redistributed across the plasma membrane and the amount of energy transferred from the CC2-DMPE changed accordingly. VIPR for change of fluorescence emissionTMII is monitored as an integrated liquid processor and fluorescence detector designed for cell-based screening in 96 or 384 well microtiter plates.
Identification of the correction Compound
To identify small molecules that correct for transport defects associated with Δ F508-CFTR, a single-addition HTS assay format was developed.cells were incubated in serum-free medium for 16 hours at 37 ℃ in the presence or absence (negative control) of test compounds.as a positive control, cells plated in 384-well plates were incubated at 27 ℃ for 16 hours to "temperature-corrected" △ F508-CFTR. then the cells were washed 3 times with Krebs Ringers solution and loaded with voltage-sensitive dye in order to activate △ F508-CFTR,10 μ M forskolin and CFTR potentiator genistein (20 μ M) with Cl-free-The medium of (2) is added to each well. Addition of Cl-free-The medium of (a) promotes Cl in response to activation of Δ F508-CFTR-Outflowing, and the resulting membrane depolarization optically monitored using FRET-based voltage sensor dyes.
Identification of potentiator compounds
To identify potentiators of Δ F508-CFTR, a dual addition HTS assay format was developed. In the first addition, Cl-free additive was added to each well in the presence or absence of test compound-The medium of (1). After 22 seconds, a second addition of Cl-free solution-2-10. mu.M forskolin-containing medium to activate Δ F508-CFTR. Extracellular Cl after two additions-Concentration 28mM, which promotes Cl in response to AF 508-CFTR activation-Outflowing, and the resulting membrane depolarization optically monitored using FRET-based voltage sensor dyes.
Solutions of
Warm bath liquid # 1: NaCl 160, KCl 4.5, CaCl (unit mM)22、
Chloride-free warm bath: the chloride salt in warm bath #1 (above) was replaced by gluconate.
CC 2-DMPE: stock solutions of 10mM DMSO were prepared and stored at-20 ℃.
DiSBAC2(3): stock solutions of 10mM DMSO were prepared and stored at-20 ℃.
Cell culture
NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for optical measurement of membrane potential. Cells were maintained at 37 ℃ with 5% CO2And 175cm at 90% humidity2Dulbecco's modified eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β -ME, 1X penicillin/streptomycin, and 25mM HEPES in culture flasks cells were seeded at 30,000/well on 384-well matrigel-coated plates and cultured at 37 ℃ for 2hrs for all optical assays, followed by incubation atIncubated at 27 ℃ for 24hrs for booster assays. For the correction assay, cells were incubated with and without compound at 27 ℃ or 37 ℃ for 16-24 hours.
Electrophysiological assay for determining the Δ F508-CFTR modulating properties of a compound
Ussing Chamber assay
Using laboratory experiments were performed on polarized epithelial cells expressing Δ F508-CFTR to further characterize the Δ F508-CFTR potentiators or inducers identified in the optical assay. FRT to be grown on Costar Snapwell cell culture cannulaeΔF508-CFTREpithelial cells were fixed in a Ussing chamber (physiologic instruments, inc., San Diego, CA)) and monolayers were continuously shorted using a voltage clamp system (Department of biotechnology, University of Iowa, IA) and San Diego, california. Transepithelial resistance was measured by applying a 2mV pulse. Under these conditions, FRT epithelium exhibits 4K Ω/cm2Or a higher resistance. The solution was maintained at 27 ℃ and air was bubbled. Correction of electrode offset potential and fluid resistance using a cell-free cannula. Under these conditions, the current reflects Cl-Flow through Δ F508-CFTR expressed in apical membrane. I was obtained digitally using the MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.)SC。
Identification of the correction Compound
Typical protocols employ Cl from the outside of the substrate to the top membrane-A concentration gradient. To establish this gradient, a normal loop was used on the basal outer membrane, while the apical NaCl was replaced with equimolar sodium gluconate (titrated to pH7.4 with NaOH) to give large Cl across the epithelium-A concentration gradient. All experiments were performed with the entire monolayer. To fully activate af 508-CFTR forskolin (10 μ Μ) and the PDE inhibitor IBMX (100 μ Μ) were applied, then the CFTR potentiator genistein (50 μ Μ) was added.
As observed in other cell types, at lowIncubation of FRT cells stably expressing AF 508-CFTR at room temperature increases the functional density of CFTR in the plasma membrane. To determine the activity of the correction compound, cells were incubated with 10 μ M test compound at 37 ℃ for 24 hours, followed by 3 washes and then recorded. Treatment of cAMP-and genistein-mediated Compounds in cells ISCNormalized to 27 ℃ and 37 ℃ controls and expressed as percent activity. Pre-incubation of cells with correction compounds significantly increased cAMP-and genistein-mediated I compared to 37 ℃ controlsSC。
Identification of potentiator compounds
Typical protocols employ Cl from the outside of the substrate to the top membrane-A concentration gradient. To establish this gradient, a normal loop was used on the basolateral membrane and permeabilized with nystatin (360. mu.g/mL) while the apical NaCl was replaced with equimolar sodium gluconate (titrated to pH7.4 with NaOH) to give large Cl spanning the epithelium-Concentration gradient all experiments were performed 30min after nystatin permeabilization forskolin (10 μ M) and all test compounds were applied to both sides of the cell culture cannula the potency of the putative △ F508-CFTR potentiator was compared to the potency of the known potentiator genistein.
Solutions of
Substrate-outside solution (unit mM): NaCl (135), CaCl2(1.2)、MgCl2(1.2)、K2HPO4(2.4)、KHPO4(0.6), N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) (10), and dextrose (10). The solution was titrated with NaOH up to p Η 7.4.
Apical solution (in mM): same as the solution outside the substrate, but NaCl was replaced by sodium gluconate (135).
Cell culture
Will express Δ F508-CFTR (FRT)ΔF508-CFTR) Fisher rat epithelial (FRT) cells of (a) were used in a Ussing laboratory experiment for putative Δ F508-CFTR modulators identified from our optical assay. Cells were cultured on Costar Snapwell cell culture cannulae and incubated at 37 ℃ and 5% CO2Next, the cells were cultured in Coon's modified Ham's F-12 medium supplemented with 5% fetal Bovine Serum (BSA), 100U/mL) for five daysCells were incubated at 27 ℃ for 16-48h to correct △ F508-CFTR before use to characterize the potentiator activity of a compound, cells were incubated with and without compound for 24 hours at 27 ℃ or 37 ℃ in order to determine the activity of the correcting compound.
Whole cell recording
Monitoring of macroscopic △ F508-CFTR Current (I) in temperature and test Compound-corrected NIH3T3 cells stably expressing Δ F508-CFTR using a perforated patch whole cell recordΔF508). Briefly, I was performed at room temperature using an Axoatch 200B patch clamp amplifier (Axon Instruments Inc., FosterCity, Calif.)ΔF508Voltage clamp record of (1). All recordings were taken at a sampling frequency of 10kHz and low pass filtered at 1 kHz. The microelectrodes have a resistance of 5-6 M.OMEGA.when filled with intracellular solution. Cl calculated at room temperature under these recording conditions-Reversal potential (E)Cl) Was-28 mV. All records have>A sealing resistance of 20 G.OMEGA.<A series resistance of 15M omega. Pulsing, data acquisition and analysis were performed using a PC equipped with a Digidata 1320A/D interface and Clampex 8(Axon instruments, Inc.). The warm bath liquid comprises<250 μ L of saline was continuously perfused at a rate of 2mL/min using a gravity-driven perfusion system.
Identification of the correction Compound
To determine the activity of the correcting compound to increase the density of functional af 508-CFTR in plasma membranes, we measured the current density 24 hours after treatment with the correcting compound using the open-cell patch recording technique described above. To fully activate Δ F508-CFTR, 10 μ M forskolin and 20 μ M genistein were added to the cells. Under our recording conditions, the current density after 24 hours of incubation at 27 ℃ was higher than that observed after 24 hours of incubation at 37 ℃. These results are consistent with the known effect of low temperature incubation on Δ F508-CFTR density in plasma membranes. To determine the effect of the correction compound on the CFTR current density, cells were incubated with 10 μ M of test compound at 37 ℃ for 24 hours and the current density (% activity) was compared to 27 ℃ and 37 ℃ controls. Prior to recording, cells were washed 3 times with extracellular recording medium to remove any remaining test compound. Preincubation with 10 μ M correction compound significantly increased cAMP-and genistein-dependent current compared to 37 ℃ control.
Identification of potentiator compounds
The use of perforated patch recording technology to study the enhancement of the synergist of AF 508-CFTR in macroscopic AF 508-CFTR Cl in NIH3T3 cells stably expressing AF 508-CFTR-Electric current (I)ΔF508) The ability of the cell to perform. The potentiators identified from the optical assay give rise to I with similar potency and potency as observed in the optical assayΔF508The dose dependence increases. In all cells studied, the reversal potential before and during the application of the synergist was about-30 mV, which is the calculated ECl(-28mV)。
Solutions of
Intracellular solution (in mM): cesium aspartate (90), CsCl (50), MgCl2(1) HEPES (10) and 240. mu.g/mL amphotericin-B (pH adjusted to 7.35 with CsOH).
Extracellular solution (in mM): N-methyl-D-glucamine (NMDG) -Cl (150), MgCl2(2)、CaCl2(2) HEPES (10) (pH 7.35 with HCl).
Cell culture
NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for whole cell recordings. Cells were maintained at 37 ℃ with 5% CO2And 175cm at 90% humidity2In Dulbecco's modified eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β -ME, 1X penicillin/streptomycin and 25mM HEPES in culture flasks for whole cell recording, 2,500-5,000 cells were seeded on poly-L-lysine coated coverslips and cultured at 27 ℃ for 24-48h, then used to test for potentiator activity, and incubated with or without a correction compound at 37 ℃ to measure the activity of the correction agent.
Single channel recording
The single channel activity of temperature-corrected af 508-CFTR stably expressed in NIH3T3 cells and the activity of the potentiator compound were observed using excised membrane-inside-out membranes. Simple and convenientFor this, single channel active voltage clamp recordings were performed at room temperature using an Axopatch 200B patch clamp amplifier (Axon instruments). All recordings were taken at a sampling frequency of 10kHz and low pass filtered at 400 Hz. The patch microelectrode was made of Corning Kovar Sealing #7052 glass (World Precision Instruments, inc., Sarasota, FL) and had a resistance of 5-8M Ω when filled with extracellular solution. After excision Δ F508-CFTR was activated by addition of 1mM Mg-ATP and 75nM cAMP-dependent protein kinase catalytic subunit (PKA; Promega Corp. Madison, Wis.). After channel activity stabilized, the membranes were perfused using a gravity-driven micro perfusion system. The internal flow was brought close to the membrane, resulting in complete exchange of solution within 1-2 s. To maintain Δ F508-CFTR activity during rapid perfusion, non-specific phosphatase inhibitor F was added-(10mM Na F) was added to the warm bath. Under these recording conditions, the channel activity remained constant (up to 60min) throughout the patch recording. The current generated by the positive charge moving from the intracellular solution to the extracellular solution (the anions moving in the opposite direction) appears as a positive current. Will micro electrode potential (V)p) The concentration is maintained at 80 mV.
Channel activity was analyzed from a patch containing ≤ 2 active channels. While the maximum open value determines the number of active channels during the experiment. To determine single channel current amplitudes, data recorded from Δ F508-CFTR activity at 120s was filtered "off-line" at 100Hz and then used to construct an amplitude histogram for all points, which was fitted by a multiple gaussian function using Bio-Patch analysis software (Bio-Logic comp. Total microscopic Current and open probability (P) determined by 120s channel Activityo). Using Bio-Patch software or from the relation PoMeasurement of P ═ I/I (N)oWhere I is the average current, I is the single channel current amplitude, and N is the number of active channels in the membrane.
Solutions of
Extracellular solution (in mM): NMDG (150), aspartic acid (150), CaCl2(5)、MgCl2(2) And HEPES (10) (adjusted to pH 7.35 with Tris base).
Intracellular solution (in mM): NMDG-Cl (150), MgCl2(2) EGTA (5), TES (10) and Tris base (14) (pH adjusted to 7.35 with HCl).
Cell culture
NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for patch clamp recordings of incised membranes. Cells were maintained at 37 ℃ with 5% CO2And 175cm at 90% humidity2In Dulbecco's modified eagle's medium in culture flasks, supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β -ME, 1X penicillin/streptomycin, and 25mM HEPES for single channel recording, 2,500-5,000 cells were seeded on poly-L-lysine coated coverslips and used after 24-48h of culture at 27 ℃.
Table 5.
Other embodiments
All publications and patents mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the meaning of a term in any patent or publication incorporated by reference conflicts with the meaning of the term used in the present disclosure, the meaning of the term in the present disclosure is intended to control. Moreover, the foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that: various changes, modifications and alterations can be made without departing from the spirit and scope of the invention as defined in the following claims.