Patent Description:
CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cells types, including absorptive and secretory epithelia cells, where it regulates anion flux across the membrane, as well as the activity of other ion channels and proteins. In epithelia cells, normal functioning of CFTR is critical for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. CFTR is composed of approximately <NUM> amino acids that encode a protein made up of a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are linked by a large, polar, regulatory (R)-domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See <NPL>; <NPL>), (<NPL>). A defect in this gene causes mutations in CFTR resulting in cystic fibrosis ("CF"), the most common fatal genetic disease in humans. Cystic fibrosis affects approximately one in every <NUM>,<NUM> infants in the United States. Within the general United States population, up to <NUM> million people carry a single copy of the defective gene without apparent ill effects. In contrast, individuals with two copies of the CF associated gene suffer from the debilitating and fatal effects of CF, including chronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenously expressed in respiratory epithelia leads to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to enhanced mucus accumulation in the lung and the accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, results in death. In addition, the majority of males with cystic fibrosis are infertile and fertility is decreased among females with cystic fibrosis. In contrast to the severe effects of two copies of the CF associated gene, individuals with a single copy of the CF associated gene exhibit increased resistance to cholera and to dehydration resulting from diarrhea - perhaps explaining the relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of disease causing mutations (<NPL>;<NPL>; and<NPL>; <NPL>). To date, > <NUM> disease causing mutations in the CF gene have been identified (http://www. The most prevalent mutation is a deletion of phenylalanine at position <NUM> of the CFTR amino acid sequence, and is commonly referred to as ΔF508-CFTR. This mutation occurs in approximately <NUM>% of the cases of cystic fibrosis and is associated with a severe disease.

The deletion of residue <NUM> in ΔF508-CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit the ER, and traffic to the plasma membrane. As a result, the number of channels present in the membrane is far less than observed in cells expressing wild-type CFTR. In addition to impaired trafficking, the mutation results in defective channel gating. Together, the reduced number of channels in the membrane and the defective gating lead to reduced anion transport across epithelia leading to defective ion and fluid transport. Studies have shown, however, that the reduced numbers of ΔF508-CFTR in the membrane are functional, albeit less than wild-type CFTR. (<NPL>; Denning et al. , supra; <NPL>). In addition to ΔF508-CFTR, other disease causing mutations in CFTR that result in defective trafficking, synthesis, and/or channel gating could be up- or down-regulated to alter anion secretion and modify disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions, it is clear that this role (the transport of anions) represents one element in an important mechanism of transporting ions and water across the epithelium. The other elements include the epithelial Na+ channel, ENaC, Na+/2Cl-/K+ co-transporter, Na+-K+-ATPase pump and the basolateral membrane K+ channels, that are responsible for the uptake of chloride into the cell.

These elements work together to achieve directional transport across the epithelium via their selective expression and localization within the cell. Chloride absorption takes place by the coordinated activity of ENaC and CFTR present on the apical membrane and the Na+-K+-ATPase pump and Cl- channels expressed on the basolateral surface of the cell. Secondary active transport of chloride from the luminal side leads to the accumulation of intracellular chloride, which can then passively leave the cell via Cl- channels, resulting in a vectorial transport. Arrangement of Na+/2Cl-/K+ co-transporter, Na+-K+-ATPase pump and the basolateral membrane K+ channels on the basolateral surface and CFTR on the luminal side coordinate the secretion of chloride via CFTR on the luminal side. Because water is probably never actively transported itself, its flow across epithelia depends on tiny transepithelial osmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue <NUM> in ΔF508-CFTR prevents the nascent protein from folding correctly, resulting in the inability of this mutant protein to exit the ER, and traffic to the plasma membrane. As a result, insufficient amounts of the mature protein are present at the plasma membrane and chloride transport within epithelial tissues is significantly reduced. In fact, this cellular phenomenon of defective ER processing of ABC transporters by the ER machinery, has been shown to be the underlying basis not only for CF disease, but for a wide range of other isolated and inherited diseases. The two ways that the ER machinery can malfunction is either by loss of coupling to ER export of the proteins leading to degradation, or by the ER accumulation of these defective/misfolded proteins [<NPL>); <NPL>); <NPL>);<NPL>); <NPL>)].

<NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-Difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid in salt form is disclosed in International <CIT> as a modulator of CFTR activity and thus useful in treating CFTR-mediated diseases such as cystic fibrosis. However, there is a need for stable solid forms of said compound that can be used readily in pharmaceutical compositions suitable for use as therapeutics.

The present invention provides a solid dosage form for oral administration comprising <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid characterized as Form I and at least one inert, pharmaceutically acceptable excipient or carrier, wherein Form I is characterized as a crystal form having a monoclinic crystal system, a P2<NUM>/n space group, and the following unit cell dimensions:.

wherein Form I is characterized by one or more peaks at <NUM> ± <NUM> degrees, <NUM> ± <NUM> degrees, and <NUM> ± <NUM> degrees in an X-ray powder diffraction obtained using Cu K alpha radiation at <NUM> kV, <NUM> mA.

The present invention also provides a solid dosage form of the invention for use in a method of treating cystic fibrosis in a human.

The present invention also provides the use of a solid dosage form of the invention in the manufacture of a medicament for treating cystic fibrosis in a human.

<NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid (herein after "Compound <NUM>") has the structure below:
<CHM>.

Compound <NUM> and pharmaceutically acceptable compositions thereof are useful for treating or lessening the severity of cystic fibrosis. In the present invention, Compound <NUM> is in a substantially crystalline and salt free form referred to as Form I as described and characterized herein.

Processes described herein can be used to prepare the compositions of this invention comprising Form I. The amounts and the features of the components used in the processes would be as described herein.

As used herein, the following definitions shall apply unless otherwise indicated.

The term "CFTR" as used herein means cystic fibrosis transmembrane conductance regulator or a mutation thereof capable of regulator activity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see, e.g., http://www. ca/cftr/, for CFTR mutations).

As used herein "crystalline" refers to compounds or compositions where the structural units are arranged in fixed geometric patterns or lattices, so that crystalline solids have rigid long range order. The structural units that constitute the crystal structure can be atoms, molecules, or ions. Crystalline solids show definite melting points.

The term "modulating" as used herein means increasing or decreasing, e.g. activity, by a measurable amount.

The present invention provides a solid dosage form for oral administration comprising <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl)cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid characterized as Form I and at least one inert, pharmaceutically acceptable excipient or carrier, wherein Form I is characterized as a crystal form having a monoclinic crystal system, a P2<NUM>/n space group, and the following unit cell dimensions:.

In an embodiment, Form I is characterized by one or more peaks at <NUM> to <NUM> degrees, <NUM> to <NUM> degrees, and <NUM> to <NUM> degrees in an X-ray powder diffraction obtained using Cu K alpha radiation.

In another embodiment, Form I is characterized by one or more peaks at <NUM>, <NUM>, and <NUM> degrees.

In another embodiment, Form I is further characterized by a peak at <NUM> to <NUM> degrees.

In another embodiment, Form I is further characterized by a peak at <NUM> degrees.

In some embodiments, Form I is characterized by a diffraction pattern substantially similar to that of <FIG>.

The particle size distribution of D90 may be about <NUM> or less for Form I.

The particle size distribution of D50 may be about <NUM> or less for Form I.

The present invention provides solid dosage forms for oral administration comprising Form I and at least one inert, pharmaceutically acceptable excipient or carrier.

In one aspect, the present invention features the solid dosage form of the invention for use in a method of treating cystic fibrosis in a human comprising administering to the human an effective amount of Form I.

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

In one aspect, the invention features a crystal form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid having a monoclinic crystal system, a P21/n space group, and the following unit cell dimensions: a = <NUM> (<NUM>) Å , b = <NUM> (<NUM>) Å, c = <NUM> (<NUM>) Å, α = <NUM>°, β = <NUM> (<NUM>)°, and γ = <NUM>°.

Form I may be prepared from dispersing or dissolving a salt form, such as HCL, of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid in an appropriate solvent for an effective amount of time. Form I may be prepared from dispersing a salt form, such as HCL, of <NUM>-(<NUM>-(<NUM>- (<NUM>,<NUM>-difluorobenzo[d] [<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid in an appropriate solvent for an effective amount of time. Form I may be formed directly from <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate and an appropriate acid, such as formic acid. The HCl salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid may be the starting point and may be prepared by coupling an acid chloride moiety with an amine moiety according to Schemes <NUM>-<NUM>. <CHM>
<CHM>
<CHM>.

Using the HCI, for example, salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid as a starting point, Form I can be formed in high yields by dispersing or dissolving the HCI salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid in an appropriate solvent for an effective amount of time. Other salt forms of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid may be used such as, for example, other mineral or organic acid forms. The other salt forms result from hydrolysis of the t-butyl ester with the corresponding acid. Other acids/salt forms include nitric, sulfuric, phosphoric, boric, acetic, benzoic, malonic, and the like. The salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid may or may not be soluble depending upon the solvent used, but lack of solubility does not hinder formation of Form I. For example, the appropriate solvent may be water or an alcohol/water mixture such as <NUM>% methanol/water mixture, even though the HCI salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid is only sparingly soluble in water. The appropriate solvent may be water.

The effective amount of time for formation of Form I from the salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid can be any time between <NUM> to <NUM> hours or greater. Generally, greater than <NUM> hours is not needed to obtain high yields (-<NUM>%), but certain solvents may require greater amounts of time. It is also recognized that the amount of time needed is inversely proportional to the temperature. That is, the higher the temperature the less time needed to affect dissociation of acid to Form I. When the solvent is water, stirring the dispersion for approximately <NUM> hours at room temperature gives Form I in an approximately <NUM>% yield. If a solution of the salt form of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>- methylpyridin-<NUM>-yl)benzoic acid is desired for process purposes, an elevated temperature may be used. After stirring the solution for an effective amount of time at the elevated temperature, recrystallization upon cooling yields substantially pure forms of Form I. Substantially pure may refer to greater than about <NUM>% purity. Substantially pure may refer to greater than about <NUM>% purity. Substantially pure may refer to greater than about <NUM>% purity. Substantially pure may refer to greater than about <NUM>% purity. The temperature selected depends in part on the solvent used and is well within the capabilities of someone of ordinary skill in the art to determine. The temperature may be between room temperature and about <NUM>. The temperature may be between room temperature and about <NUM>. The temperature may be between about <NUM> and about <NUM>. The temperature may be between about <NUM> and about <NUM>.

Form I may be further purified by recrystallization from an organic solvent. Examples of organic solvents include, but are not limited to, toluene, cumene, anisole, <NUM>-butanol, isopropylacetate, butyl acetate, isobutyl acetate, methyl t-butyl ether, methyl isobutyl ketone, or <NUM>-propanol/water (at various ratios). Temperature may be used as described above. For example, Form I may be dissolved in <NUM>-butanol at <NUM> until it is completely dissolved. Cooling down the solution to <NUM> at a rate of <NUM>/min yields crystals of Form I which may be isolated by filtration.

In another aspect of the present invention, solid dosage forms for oral administration are provided, wherein these compositions comprise Form I as described herein, and comprise at least one inert, pharmaceutically acceptable excipient or carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

As described above, the solid dosage forms of the present invention additionally comprise an inert pharmaceutically acceptable excipient or carrier. Excipient, adjuvant, or vehicle, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants, as suited to the particular dosage form desired. <NPL>) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compound of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the solid dosage form, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

In yet another aspect, the present invention provides the solid dosage form of the invention for use in a method of treating cystic fibrosis in a human.

A "CFTR-mediated disease" as used herein is a disease selected from cystic fibrosis, Hereditary emphysema, Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency, Type <NUM> hereditary angioedema, Lipid processing deficiencies, such as Familial hypercholesterolemia, Type <NUM> chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, such as I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism, Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma, Glycanosis CDG type <NUM>, Hereditary emphysema, Congenital hyperthyroidism, Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer' s disease, Parkinson' s disease, Amyotrophic lateral sclerosis, Progressive supranuclear plasy, Pick's disease, several polyglutamine neurological disorders such as Huntington, Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry- eye disease, and Sjogren's disease.

According to the invention an "effective amount" of Form I or a pharmaceutically acceptable composition thereof is that amount effective for treating or lessening the severity of any of the diseases recited above.

Form I or a pharmaceutically acceptable composition thereof may be administered using any amount and any route of administration effective for treating or lessening the severity of one or more of the diseases recited above.

The solid dosage forms comprising Form I described herein are useful for treating or lessening the severity of cystic fibrosis in patients who exhibit residual CFTR activity in the apical membrane of respiratory and non-respiratory epithelia. The presence of residual CFTR activity at the epithelial surface can be readily detected using methods known in the art, e.g., standard electrophysiological, biochemical, or histochemical techniques. Such methods identify CFTR activity using in vivo or ex vivo electrophysiological techniques, measurement of sweat or salivary Cl- concentrations, or ex vivo biochemical or histochemical techniques to monitor cell surface density. Using such methods, residual CFTR activity can be readily detected in patients heterozygous or homozygous for a variety of different mutations, including patients homozygous or heterozygous for the most common mutation, ΔF508.

The solid dosage forms comprising Form I described herein are useful for treating or lessening the severity of cystic fibrosis in patients within certain genotypes exhibiting residual CFTR activity, e.g., class III mutations (impaired regulation or gating), class IV mutations (altered conductance), or class V mutations (reduced synthesis) (<NPL>). Other patient genotypes that exhibit residual CFTR activity include patients homozygous for one of these classes or heterozygous with any other class of mutations, including class I mutations, class II mutations, or a mutation that lacks classification.

The solid dosage forms comprising Form I described herein are useful for treating or lessening the severity of cystic fibrosis in patients within certain clinical phenotypes, e.g., a moderate to mild clinical phenotype that typically correlates with the amount of residual CFTR activity in the apical membrane of epithelia. Such phenotypes include patients exhibiting pancreatic insufficiency or patients diagnosed with idiopathic pancreatitis and congenital bilateral absence of the vas deferens, or mild lung disease.

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, and its mode of administration. The expression "dosage unit form" as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the solid dosage form of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts. The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

The solid dosage forms of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, the compound of the invention may be administered orally at dosage levels of about <NUM>/kg to about <NUM>/kg and preferably from about <NUM>/kg to about <NUM>/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

In certain embodiments, the dosage amount of Form I in the solid dosage form is from <NUM> to <NUM>,<NUM>. In another embodiment, the dosage amount of Form I is from <NUM> to <NUM>. In another embodiment, the dosage amount of Form I is from <NUM> to <NUM>. In another embodiment, the dosage amount of Form I is from <NUM> to <NUM>. In another embodiment, the dosage amount of Form I is from <NUM> to <NUM>.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in <NUM>,<NUM>-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers of extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar--agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols.

The active compounds can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

It will also be appreciated that Form I described herein or a pharmaceutically acceptable composition thereof can be employed in combination therapies, that is, Form I can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated".

In one embodiment, the additional agent is selected from a mucolytic agent, bronchodialator, an anti-biotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than a compound of the present invention, or a nutritional agent.

In another embodiment, the additional agent is a compound selected from gentamicin, curcumin, cyclophosphamide, <NUM>-phenylbutyrate, miglustat, felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMP modulators such as rolipram, sildenafil, milrinone, tadalafil, amrinone, isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP <NUM> inhibitors, HSP <NUM> inhibitors, proteosome inhibitors such as epoxomicin, lactacystin, etc..

In another embodiment, the additional agent is a compound disclosed in <CIT>, <CIT>, <CIT>, <CIT>, or <CIT>.

In another embodiment, the additional agent is a benzo(c)quinolizinium derivative that exhibits CFTR modulation activity or a benzopyran derivative that exhibits CFTR modulation activity.

In another embodiment, the additional agent is a compound disclosed in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, or <CIT>.

In another embodiment, the additional agent is a compound disclosed in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, or <CIT>.

In another embodiment, the an additional agent selected from compounds disclosed in <CIT>, published as <CIT>. In another embodiment, the additional agent is N-(<NUM>-hydroxy-<NUM>,<NUM>-ditert-butyl-phenyl)-<NUM>-oxo-<NUM>-quinoline-<NUM>-carboxamide. These combinations are useful for treating the diseases described herein including cystic fibrosis. These combinations are also useful in the kits described herein.

The amount of additional therapeutic agent present in the solid dosage forms of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about <NUM>% to <NUM>% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

Form I described herein or a pharmaceutically acceptable composition thereof may also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Suitable coatings and the general preparation of coated implantable devices are described in <CIT>; <CIT>; and <CIT>. The coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccarides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

The Differential scanning calorimetry (DSC) data of Form I were collected using a DSC Q100 V9. <NUM> Build <NUM> (TA Instruments, New Castle, DE). Temperature was calibrated with indium and heat capacity was calibrated with sapphire. Samples of <NUM>-<NUM> were weighed into aluminum pans that were crimped using lids with <NUM> pin hole. The samples were scanned from <NUM> to <NUM> at a heating rate of <NUM>/min and with a nitrogen gas purge of <NUM>/min. Data were collected by Thermal Advantage Q SeriesTM version <NUM>. <NUM> software and analyzed by Universal Analysis software version <NUM>. 1D (TA Instruments, New Castle, DE). The reported numbers represent single analyses.

The X-Ray diffraction (XRD) data of Form <NUM> were collected on a Bruker D8 DISCOVER powder diffractometer with HI-STAR <NUM>-dimensional detector and a flat graphite monochromator. Cu sealed tube with Kα radiation was used at <NUM> kV, 35mA. The samples were placed on zero-background silicon wafers at <NUM>. For each sample, two data frames were collected at <NUM> seconds each at <NUM> different θ<NUM> angles: <NUM>° and <NUM>°. The data were integrated with GADDS software and merged with DIFFRACTplusEVA software. Uncertainties for the reported peak positions are ± <NUM> degrees.

Vitride® (sodium bis(<NUM>-methoxyethoxy)aluminum hydride [or NaAlH<NUM>(OCH<NUM>CH<NUM>OCH<NUM>)<NUM>], <NUM> wgt% solution in toluene) was purchased from Aldrich Chemicals.

<NUM>,<NUM>-Difluoro-<NUM>,<NUM>-benzodioxole-<NUM>-carboxylic acid was purchased from Saltigo (an affiliate of the Lanxess Corporation).

Anywhere in the present application where a name of a compound may not correctly describe the structure of the compound, the structure supersedes the name and governs.

Commercially available <NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxole-<NUM>-carboxylic acid (<NUM> eq) is slurried in toluene (<NUM> vol). Vitride® (<NUM> eq) is added via addition funnel at a rate to maintain the temperature at <NUM>-<NUM>. At the end of addition the temperature is increased to <NUM> for <NUM> then <NUM>% (w/w) aq. NaOH (<NUM> eq) is carefully added via addition funnel maintaining the temperature at <NUM>-<NUM>. After stirring for an additional <NUM> minutes, the layers are allowed to separate at <NUM>. The organic phase is cooled to <NUM> then washed with water (<NUM> x <NUM> vol), dried (Na<NUM>SO<NUM>), filtered, and concentrated to afford crude (<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-methanol that is used directly in the next step.

(<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-methanol (<NUM> eq) is dissolved in MTBE (<NUM> vol). A catalytic amount of DMAP (<NUM> mol %) is added and SOCl<NUM> (<NUM> eq) is added via addition funnel. The SOCl<NUM> is added at a rate to maintain the temperature in the reactor at <NUM>-<NUM>. The temperature is increased to <NUM> for <NUM> hour then cooled to <NUM> then water (<NUM> vol) is added via addition funnel maintaining the temperature at less than <NUM>. After stirring for an additional <NUM> minutes, the layers are allowed to separate. The organic layer is stirred and <NUM>% (w/v) aq. NaOH (<NUM> vol) is added. After stirring for <NUM> to <NUM> minutes, the layers are allowed to separate. The organic phase is then dried (Na<NUM>SO<NUM>), filtered, and concentrated to afford crude <NUM>-chloromethyl-<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxole that is used directly in the next step.

A solution of <NUM>-chloromethyl-<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxole (<NUM> eq) in DMSO (<NUM> vol) is added to a slurry of NaCN (<NUM> eq) in DMSO (<NUM> vol) maintaining the temperature between <NUM>-<NUM>. The mixture is stirred for <NUM> hour then water (<NUM> vol) is added followed by MTBE (<NUM> vol). After stirring for <NUM>, the layers are separated. The aqueous layer is extracted with MTBE (<NUM> vol). The combined organic layers are washed with water (<NUM> vol), dried (Na<NUM>SO<NUM>), filtered, and concentrated to afford crude (<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-acetonitrile (<NUM>%) that is used directly in the next step.

A mixture of (<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-acetonitrile (<NUM> eq), <NUM> wt % aqueous KOH (<NUM> eq) <NUM>-bromo-<NUM>-chloroethane (<NUM> eq), and Oct<NUM>NBr (<NUM> eq) is heated at <NUM> for <NUM>. The reaction mixture is cooled then worked up with MTBE and water. The organic phase is washed with water and brine then the solvent is removed to afford (<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-cyclopropanecarbonitrile.

(<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-cyclopropanecarbonitrile is hydrolyzed using <NUM> NaOH (<NUM> equiv) in ethanol (<NUM> vol) at <NUM> overnight. The mixture is cooled to room temperature and ethanol is evaporated under vacuum. The residue is taken into water and MTBE, <NUM> HCl was added and the layers are separated. The MTBE layer was then treated with dicyclohexylamine (<NUM> equiv). The slurry is cooled to <NUM>, filtered and washed with heptane to give the corresponding DCHA salt. The salt is taken into MTBE and <NUM>% citric acid and stirred until all solids dissolve. The layers are separated and the MTBE layer was washed with water and brine. Solvent swap to heptane followed by filtration gives <NUM>-(<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-cyclopropanecarboxylic acid after drying in a vacuum oven at <NUM> overnight.

<NUM>-(<NUM>,<NUM>-difluoro-<NUM>,<NUM>-benzodioxol-<NUM>-yl)-cyclopropanecarboxylic acid (<NUM> eq) is slurried in toluene (<NUM> vol) and the mixture heated to <NUM>. SOCl<NUM> (<NUM> eq) is added via addition funnel. The toluene and SOCl<NUM> are distilled from the reaction mixture after <NUM> minutes. Additional toluene (<NUM> vol) is added and distilled again.

<NUM>-Bromo-<NUM>-methylpyridine (<NUM> eq) is dissolved in toluene (<NUM> vol). K<NUM>CO<NUM> (<NUM> eq) is added followed by water (<NUM> vol) and the mixture heated to <NUM> under a stream of N<NUM> for <NUM> hour. <NUM>-(t-Butoxycarbonyl)phenylboronic acid (<NUM> eq) and Pd(dppf)Cl<NUM>·CH<NUM>Cl<NUM> (<NUM> eq) are then added and the mixture is heated to <NUM>. After <NUM> hours, the heat is turned off, water is added (<NUM> vol) and the layers are allowed to separate. The organic phase is then washed with water (<NUM> vol) and extracted with <NUM>% aqueous methanesulfonic acid (<NUM> eq MsOH, <NUM> vol). The aqueous phase is made basic with <NUM>% aqueous NaOH (<NUM> eq) and extracted with EtOAc (<NUM> vol). The organic layer is concentrated to afford crude tert-butyl-<NUM>-(<NUM>-methylpyridin-<NUM>-yl)benzoate (<NUM>%) that is used directly in the next step.

tert-Butyl-<NUM>-(<NUM>-mcthylpyridin-<NUM>-yl)bcnzoatc (<NUM> eq) is dissolved in EtOAc (<NUM> vol). Water (<NUM>. <NUM> vol) is added followed by urea-hydrogen peroxide (<NUM> eq). The phthalic anhydride (<NUM> eq) is added portion-wise as a solid to maintain the temperature in the reactor below <NUM>. After completion of phthalic anhydride addition, the mixture is heated to <NUM>. After stirring for an additional <NUM> hours, the heat is turned off. <NUM>% w/w aqueous Na<NUM>SO<NUM> (<NUM> eq) is added via addition funnel. After completion of Na<NUM>SO<NUM> addition, the mixture is stirred for an additional <NUM> minutes and the layers separated. The organic layer is stirred and <NUM>% w/w aq. Na<NUM>CO<NUM> (<NUM> eq) is added. After stirring for <NUM> minutes, the layers are allowed to separate. The organic phase is washed <NUM>% w/v aq NaCl. The organic phase is then filtered and concentrated to afford crude <NUM>-(<NUM>-(tert-butoxycarbonyl)phenyl)-<NUM>-methylpyridine-<NUM>-oxide (<NUM>%) that is used directly in the next step.

A solution of <NUM>-(<NUM>-(tert-butoxycarbonyl)phenyl)-<NUM>-methylpyridine-<NUM>-oxide (<NUM> eq) and pyridine (<NUM> eq) in MeCN (<NUM> vol) is heated to <NUM>. A solution of methanesulfonic anhydride (<NUM> eq) in MeCN (<NUM> vol) is added over <NUM> via addition funnel maintaining the temperature at less than <NUM>. The mixture is stirred for an additional <NUM> hours after complete addition. The mixture is then allowed to cool to ambient. Ethanolamine (<NUM> eq) is added via addition funnel. After stirring for <NUM> hours, water (<NUM> vol) is added and the mixture is cooled to <NUM>. After stirring for NLT <NUM> hours, the solid is collected by filtration and washed with water (<NUM> vol), <NUM>:<NUM> MeCN/water (<NUM> vol), and MeCN (<NUM> x <NUM> vol). The solid is dried to constant weight (<<NUM>% difference) in a vacuum oven at <NUM> with a slight N<NUM> bleed to afford tert-butyl-<NUM>-(<NUM>-amino-<NUM>-methylpyridin-<NUM>-yl)benzoate as a red-yellow solid (<NUM>% yield).

The crude acid chloride is dissolved in toluene (<NUM> vol based on acid chloride) and added via addition funnel to a mixture of tert-butyl-<NUM>-(<NUM>-amino-<NUM>-methylpyridin-<NUM>-yl)benzoate (<NUM> eq), dimethylaminopyridine (DMAP, <NUM> eq), and triethylamine (<NUM> eq) in toluene (<NUM> vol based on tert-butyl-<NUM>-(<NUM>-amino-<NUM>-methylpyridin-<NUM>-yl)benzoate). After <NUM> hours, water (<NUM> vol based on tert-butyl-<NUM>-(<NUM>-amino-<NUM>-methylpyridin-<NUM>-yl)benzoate) is added to the reaction mixture. After stirring for <NUM> minutes, the layers are separated. The organic phase is then filtered and concentrated to afford a thick oil of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate (quantitative crude yield). MeCN (<NUM> vol based on crude product) is added and distilled until crystallization occurs. Water (<NUM> vol based on crude product) is added and the mixture stirred for <NUM>. The solid is collected by filtration, washed with <NUM>:<NUM> (by volume) MeCN/water (<NUM> x <NUM> vol based on crude product), and partially dried on the filter under vacuum. The solid is dried to constant weight (<<NUM>% difference) in a vacuum oven at <NUM> with a slight N<NUM> bleed to afford <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate as a brown solid.

To a slurry of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate (<NUM> eq) in MeCN (<NUM> vol) is added water (<NUM> vol) followed by concentrated aqueous HCl (<NUM> vol). The mixture is heated to <NUM> ± <NUM>. After stirring for <NUM> to <NUM> hours the reaction is complete and the mixture is allowed to cool to ambient. Water (<NUM> vol) is added and the mixture stirred. The solid is collected by filtration, washed with water (<NUM> x <NUM> vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (<<NUM>% difference) in a vacuum oven at <NUM> with a slight N<NUM> bleed to afford <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d] [<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid • HCl as an off-white solid.

A slurry of <NUM>-(<NUM>-(I-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid • HCl (<NUM> eq) in water (<NUM> vol) is stirred at ambient temperature. A sample is taken after stirring for <NUM> hours. The sample is filtered and the solid washed with water (<NUM> x). The solid sample is submitted for DSC analysis. When DSC analysis indicates complete conversion to Form I, the solid is collected by filtration, washed with water (<NUM> x <NUM> vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (<<NUM>% difference) in a vacuum oven at <NUM> with a slight N<NUM> bleed to afford Form I as an off-white solid (<NUM>% yield). <NUM>H NMR (<NUM>, DMSO-d6) <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

To a slurry of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid • HCl (<NUM> eq) in water (<NUM> vol) stirred at ambient temperature is added <NUM>% w/w aq. NaOH (<NUM> eq). The mixture is stirred for NLT <NUM> or until a homogeneous solution. Concentrated HCl (<NUM> eq) is added to crystallize Form I. The mixture is heated to <NUM> or <NUM> if needed to reduce the level of the t-butylbenzoate ester. The mixture is heated until HPLC analysis indicates NMT <NUM>% (AUC) t-butylbenzoate ester. The mixture is then cooled to ambient and the solid is collected by filtration, washed with water (<NUM> x <NUM> vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (<<NUM>% difference) in a vacuum oven at <NUM> with a slight N<NUM> bleed to afford Form I as an off-white solid (<NUM>% yield).

A solution of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate (<NUM> eq) in formic acid (<NUM> vol) is heated to <NUM> ± <NUM>. The reaction is continued until the reaction is complete (NMT <NUM>% AUC <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate) or heating for NMT <NUM>. The mixture is allowed to cool to ambient. The solution is added to water (<NUM> vol) heated at <NUM> and the mixture stirred. The mixture is then heated to <NUM> ± <NUM> until the level of <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)-t-butylbenzoate is NMT <NUM>% (AUC). The solid is collected by filtration, washed with water (<NUM> x <NUM> vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (<<NUM>% difference) in a vacuum oven at <NUM> with a slight N<NUM> bleed to afford Compound <NUM> in Form I as an off-white solid.

An X-ray diffraction pattern calculated from a single crystal structure of Compound <NUM> in Form <NUM> is shown in <FIG>. Table <NUM> lists the calculated peaks for <FIG>.

An actual X-ray powder diffraction pattern of Compound <NUM> in Form I is shown in <FIG>. Table <NUM> lists the actual peaks for <FIG>.

An overlay of an X-ray diffraction pattern calculated from a single crystal structure of Compound <NUM> in Form I, and an actual X-ray powder diffraction pattern of Compound <NUM> in Form <NUM> is shown in <FIG>. The overlay shows good agreement between the calculated and actual peak positions, the difference being only about <NUM> degrees.

The DSC trace of Compound <NUM> in Form I is shown in <FIG>. Melting for Compound <NUM> in Form I occurs at about <NUM>.

Conformational pictures of Compound <NUM> in Form I based on single crystal X-ray analysis are shown in <FIG>. <FIG> show hydrogen bonding between carboxylic acid groups of a dimer and the resulting stacking that occurs in the crystal. The crystal structure reveals a dense packing of the molecules. Compound <NUM> in Form I is monoclinic, P2<NUM>/n, with the following unit cell dimensions: a = <NUM>(<NUM>) Å, b = <NUM>(<NUM>) Å, c = <NUM> (<NUM>) Å, β = <NUM>(<NUM>)°, V = <NUM>Å<NUM>, Z = <NUM>. Density of Compound <NUM> in Form I calculated from structural data is <NUM>/cm<NUM> at <NUM>.

<NUM>HNMR spectra of Compound <NUM> are shown in <FIG> (<FIG> and <FIG> depict Compound <NUM> in Form I in a <NUM>/mL, <NUM> methyl cellulose-polysorbate <NUM> suspension, and <FIG> depicts Compound <NUM> as an HCl salt).

Table <NUM> below recites additional analytical data for Compound <NUM>.

The optical membrane potential assay utilized voltage-sensitive FRET sensors described by Gonzalez and Tsien (See, <NPL>, and <NPL>) in combination with instrumentation for measuring fluorescence changes such as the Voltage/Ion Probe Reader (VIPR) (See, <NPL>).

These voltage sensitive assays are based on the change in fluorescence resonant energy transfer (FRET) between the membrane-soluble, voltage-sensitive dye, DiSBAC<NUM>(<NUM>), and a fluorescent phospholipid, CC2-DMPE, which is attached to the outer leaflet of the plasma membrane and acts as a FRET donor. Changes in membrane potential (Vm) cause the negatively charged DiSBAC<NUM>(<NUM>) to redistribute across the plasma membrane and the amount of energy transfer from CC2-DMPE changes accordingly. The changes in fluorescence emission were monitored using VIPR™ II, which is an integrated liquid handler and fluorescent detector designed to conduct cell-based screens in <NUM>- or <NUM>-well microtiter plates.

To identify small molecules that correct the trafficking defect associated with ΔF508-CFTR; a single-addition HTS assay format was developed. The cells were incubated in serum-free medium for <NUM> hrs at <NUM> in the presence or absence (negative control) of test compound. As a positive control, cells plated in <NUM>-well plates were incubated for <NUM> hrs at <NUM> to "temperature-correct" ΔF508-CFTR. The cells were subsequently rinsed 3X with Krebs Ringers solution and loaded with the voltage-sensitive dyes. To activate ΔF508-CFTR, <NUM> forskolin and the CFTR potentiator, genistein (<NUM>), were added along with Cl--free medium to each well. The addition of Cl--free medium promoted Cl- efflux in response to ΔF508-CFTR activation and the resulting membrane depolarization was optically monitored using the FRET-based voltage-sensor dyes.

To identify potentiators of ΔF508-CFTR, a double-addition HTS assay format was developed. During the first addition, a Cl--free medium with or without test compound was added to each well. After <NUM> sec, a second addition of Cl--free medium containing <NUM>-<NUM> forskolin was added to activate ΔF508-CFTR. The extracellular Cl- concentration following both additions was <NUM>, which promoted Cl- efflux in response to ΔF508-CFTR activation and the resulting membrane depolarization was optically monitored using the FRET-based voltage-sensor dyes.

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used for optical measurements of membrane potential. The cells are maintained at <NUM> in <NUM>% CO<NUM> and <NUM> % humidity in Dulbecco's modified Eagle's medium supplemented with <NUM> glutamine, <NUM> % fetal bovine serum, <NUM> X NEAA, β-ME, <NUM> X pen/strep, and <NUM> HEPES in <NUM><NUM> culture flasks. For all optical assays, the cells were seeded at <NUM>,<NUM>/well in <NUM>-well matrigel-coated plates and cultured for <NUM> hrs at <NUM> before culturing at <NUM> for <NUM> hrs for the potentiator assay. For the correction assays, the cells are cultured at <NUM> or <NUM> with and without compounds for <NUM> - <NUM> hours.

Using chamber experiments were performed on polarized epithelial cells expressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulators identified in the optical assays. FRTΔF508-CFTR epithelial cells grown on Costar Snapwell cell culture inserts were mounted in an Ussing chamber (Physiologic Instruments, Inc. , San Diego, CA), and the monolayers were continuously short-circuited using a Voltage-clamp System (Department of Bioengineering, University of Iowa, IA, and, Physiologic Instruments, Inc. , San Diego, CA). Transepithelial resistance was measured by applying a <NUM>-mV pulse. Under these conditions, the FRT epithelia demonstrated resistances of <NUM> KΩ/ cm<NUM> or more. The solutions were maintained at <NUM> and bubbled with air. The electrode offset potential and fluid resistance were corrected using a cell-free insert. Under these conditions, the current reflects the flow of Cl- through ΔF508-CFTR expressed in the apical membrane. The ISC was digitally acquired using an MP100A-CE interface and AcqKnowledge software (v3. <NUM>; BIOPAC Systems, Santa Barbara, CA).

Typical protocol utilized a basolateral to apical membrane Cl- concentration gradient. To set up this gradient, normal ringer was used on the basolateral membrane, whereas apical NaCl was replaced by equimolar sodium gluconate (titrated to pH <NUM> with NaOH) to give a large Cl- concentration gradient across the epithelium. All experiments were performed with intact monolayers. To fully activate ΔF508-CFTR, forskolin (<NUM>) and the PDE inhibitor, IBMX (<NUM>), were applied followed by the addition of the CFTR potentiator, genistein (<NUM>).

As observed in other cell types, incubation at low temperatures of FRT cells stably expressing ΔF508-CFTR increases the functional density of CFTR in the plasma membrane. To determine the activity of correction compounds, the cells were incubated with <NUM> of the test compound for <NUM> hours at <NUM> and were subsequently washed 3X prior to recording. The cAMP- and genistein-mediated ISC in compound-treated cells was normalized to the <NUM> and <NUM> controls and expressed as percentage activity. Preincubation of the cells with the correction compound significantly increased the cAMP- and genistein-mediated ISC compared to the <NUM> controls.

Typical protocol utilized a basolateral to apical membrane Cl- concentration gradient. To set up this gradient, normal ringers was used on the basolateral membrane and was permeabilized with nystatin (<NUM>µg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate (titrated to pH <NUM> with NaOH) to give a large Cl- concentration gradient across the epithelium. All experiments were performed <NUM> after nystatin permeabilization. Forskolin (<NUM>) and all test compounds were added to both sides of the cell culture inserts. The efficacy of the putative ΔF508-CFTR potentiators was compared to that of the known potentiator, genistein.

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR (FRTΔF508-CFTR) were used for Ussing chamber experiments for the putative ΔF508-CFTR modulators identified from our optical assays. The cells were cultured on Costar Snapwell cell culture inserts and cultured for five days at <NUM> and <NUM>% CO<NUM> in Coon's modified Ham's F-<NUM> medium supplemented with <NUM>% fetal calf serum, <NUM> U/ml penicillin, and <NUM>µg/ml streptomycin. Prior to use for characterizing the potentiator activity of compounds, the cells were incubated at <NUM> for <NUM>-<NUM> hrs to correct for the ΔF508-CFTR. To determine the activity of corrections compounds, the cells were incubated at <NUM> or <NUM> with and without the compounds for <NUM> hours.

The macroscopic ΔF508-CFTR current (IΔF508) in temperature- and test compound-corrected NIH3T3 cells stably expressing ΔF508-CFTR were monitored using the perforated-patch, whole-cell recording. Briefly, voltage-clamp recordings of IΔF508 were performed at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc. , Foster City, CA). All recordings were acquired at a sampling frequency of <NUM> and low-pass filtered at <NUM>. Pipettes had a resistance of <NUM> - <NUM> MΩ when filled with the intracellular solution. Under these recording conditions, the calculated reversal potential for Cl- (ECl) at room temperature was -<NUM> mV. All recordings had a seal resistance > <NUM> GQ and a series resistance < <NUM> MΩ. Pulse generation, data acquisition, and analysis were performed using a PC equipped with a Digidata <NUM> A/D interface in conjunction with Clampex <NUM> (Axon Instruments Inc. The bath contained < <NUM>µl of saline and was continuously perifused at a rate of <NUM>/min using a gravity-driven perfusion system.

To determine the activity of correction compounds for increasing the density of functional ΔF508-CFTR in the plasma membrane, we used the above-described perforated-patch-recording techniques to measure the current density following <NUM>-hr treatment with the correction compounds. To fully activate ΔF508-CFTR, <NUM> forskolin and <NUM> genistein were added to the cells. Under our recording conditions, the current density following <NUM>-hr incubation at <NUM> was higher than that observed following <NUM>-hr incubation at <NUM>. These results are consistent with the known effects of low-temperature incubation on the density of ΔF508-CFTR in the plasma membrane. To determine the effects of correction compounds on CFTR current density, the cells were incubated with <NUM> of the test compound for <NUM> hours at <NUM> and the current density was compared to the <NUM> and <NUM> controls (% activity). Prior to recording, the cells were washed 3X with extracellular recording medium to remove any remaining test compound. Preincubation with <NUM> of correction compounds significantly increased the cAMP- and genistein-dependent current compared to the <NUM> controls.

The ability of ΔF508-CFTR potentiators to increase the macroscopic ΔF508-CFTR Cl- current (IΔF508) in NIH3T3 cells stably expressing ΔF508-CFTR was also investigated using perforated-patch-recording techniques. The potentiators identified from the optical assays evoked a dose-dependent increase in IΔF508 with similar potency and efficacy observed in the optical assays. In all cells examined, the reversal potential before and during potentiator application was around -<NUM> mV, which is the calculated ECl (-<NUM> mV).

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used for whole-cell recordings. The cells are maintained at <NUM> in <NUM>% CO<NUM> and <NUM> % humidity in Dulbecco's modified Eagle's medium supplemented with <NUM> glutamine, <NUM> % fetal bovine serum, <NUM> X NEAA, β-ME, <NUM> X pen/strep, and <NUM> HEPES in <NUM><NUM> culture flasks. For whole-cell recordings, <NUM>,<NUM> - <NUM>,<NUM> cells were seeded on poly-L-lysine-coated glass coverslips and cultured for <NUM> - <NUM> hrs at <NUM> before use to test the activity of potentiators; and incubated with or without the correction compound at <NUM> for measuring the activity of correctors.

The single-channel actdivities of temperature-corrected ΔF508-CFTR stably expressed in NIH3T3 cells and activities of potentiator compounds were observed using excised inside-out membrane patch. Briefly, voltage-clamp recordings of single-channel activity were performed at room temperature with an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc. All recordings were acquired at a sampling frequency of <NUM> and low-pass filtered at <NUM>. Patch pipettes were fabricated from Corning Kovar Sealing #<NUM> glass (World Precision Instruments, Inc. , Sarasota, FL) and had a resistance of <NUM> - <NUM> MΩ when filled with the extracellular solution. The ΔF508-CFTR was activated after excision, by adding <NUM> Mg-ATP, and <NUM> of the cAMP-dependent protein kinase, catalytic subunit (PKA; Promega Corp. Madison, WI). After channel activity stabilized, the patch was perifused using a gravity-driven microperfusion system. The inflow was placed adjacent to the patch, resulting in complete solution exchange within <NUM>-<NUM> sec. To maintain ΔF508-CFTR activity during the rapid perifusion, the nonspecific phosphatase inhibitor F- (<NUM> NaF) was added to the bath solution. Under these recording conditions, channel activity remained constant throughout the duration of the patch recording (up to <NUM>). Currents produced by positive charge moving from the intra-to extracellular solutions (anions moving in the opposite direction) are shown as positive currents. The pipette potential (Vp) was maintained at <NUM> mV.

Channel activity was analyzed from membrane patches containing ≤ <NUM> active channels. The maximum number of simultaneous openings determined the number of active channels during the course of an experiment. To determine the single-channel current amplitude, the data recorded from <NUM> sec of ΔF508-CFTR activity was filtered "off-line" at <NUM> and then used to construct all-point amplitude histograms that were fitted with multigaussian functions using Bio-Patch Analysis software (Bio-Logic Comp. The total microscopic current and open probability (Po) were determined from <NUM> sec of channel activity. The Po was determined using the Bio-Patch software or from the relationship Po = I/i(N), where I = mean current, i = single-channel current amplitude, and N = number of active channels in patch.

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used for excised-membrane patch-clamp recordings. The cells are maintained at <NUM> in <NUM>% CO<NUM> and <NUM> % humidity in Dulbecco's modified Eagle's medium supplemented with <NUM> glutamine, <NUM> % fetal bovine serum, <NUM> X NEAA, β-ME, <NUM> X pen/strep, and <NUM> HEPES in <NUM><NUM> culture flasks. For single channel recordings, <NUM>,<NUM> - <NUM>,<NUM> cells were seeded on poly-L-lysine-coated glass coverslips and cultured for <NUM> - <NUM> hrs at <NUM> before use.

Claim 1:
A solid dosage form for oral administration comprising <NUM>-(<NUM>-(<NUM>-(<NUM>,<NUM>-difluorobenzo[d][<NUM>,<NUM>]dioxol-<NUM>-yl) cyclopropanecarboxamido)-<NUM>-methylpyridin-<NUM>-yl)benzoic acid characterized as Form I and at least one inert, pharmaceutically acceptable excipient or carrier, wherein Form I is characterized as a crystal form having a monoclinic crystal system, a P2<NUM>/n space group, and the following unit cell dimensions:

<TAB>

wherein Form I is characterized by one or more peaks at <NUM> ± <NUM> degrees, <NUM> ± <NUM> degrees, and <NUM> ± <NUM> degrees in an X-ray powder diffraction obtained using Cu K alpha radiation at <NUM> kV, <NUM> mA.