Methods of Treatment for Cystic Fibrosis

[0001] The instant application claims priority to U.S. Provisional Application No. 62/533,388, filed 7/17/2017; U.S. Provisional Application No. 62/623,734, filed

1/30/2018; and U.S. Provisional Application No. 62/633,167, filed 2/21/2018, the entire contents of each of which are expressly incorporated herein by reference in their respective entireties.

[0002] Disclosed herein is a modulator of Cystic Fibrosis Transmembrane

Conductance Regulator (CFTR), pharmaceutical compositions containing the modulator, methods of treatment of cystic fibrosis, and a process for making the modulator.

[0003] Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 70,000 children and adults worldwide. Despite progress in the treatment of CF, there is no cure.

[0004] In patients with CF, mutations in CFTR endogenously expressed in respiratory epithelia lead to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to enhanced mucus accumulation in the lung and 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, result in death. In addition, the majority of males with cystic fibrosis are infertile, and fertility is reduced among females with cystic fibrosis.

[0005] Sequence analysis of the CFTR gene has revealed a variety of disease causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B-S. et al. (1989) Science 245: 1073-1080; Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 2000 mutations in the CF gene have been identified; currently, the CFTR2 database contains information on only 322 of these identified mutations, with sufficient evidence to define 281 mutations as disease causing. The most prevalent disease-causing mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence, and is commonly referred to as the F508del mutation. This mutation occurs in approximately 70% of the cases of cystic fibrosis and is associated with severe disease. [0006] The deletion of residue 508 in CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit the endoplasmic reticulum (ER) and traffic to the plasma membrane. As a result, the number of CFTR channels for anion transport present in the membrane is far less than observed in cells expressing wild-type CFTR, i.e., CFTR having no mutations. 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 and fluid transport across epithelia. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). The channels that are defective because of the F508del mutation are still functional, albeit less functional than wild-type CFTR channels. (Dalemans et al. (1991), Nature Lond. 354: 526-528; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to F508del, other disease causing mutations in CFTR that result in defective trafficking, synthesis, and/or channel gating could be up- or down-regulated to alter anion secretion and modify disease progression and/or severity.

[0007] CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cell types, including absorptive and secretory epithelia cells, where it regulates anion flux across the membrane, as well as the activity of other ion channels and proteins. In epithelial cells, normal functioning of CFTR is critical for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. CFTR is composed of approximately 1480 amino acids that encode a protein which is 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.

[0008] Chloride transport takes place by the coordinated activity of ENaC and CFTR present on the apical membrane and the Na ⁺ -K ⁺ - ATPase pump and CI- 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 CI ^" channels, resulting in a vectorial transport. Arrangement of Na ⁺ /2C1 ^" /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.

[0009] Accordingly, there is a need for novel treatments of CFTR mediated diseases.

[0010] Disclosed herein is Compound I and pharmaceutically acceptable salts thereof. Compound I can be depicted as having the following structure:

[0011] A chemical name for Compound I is N-[(6-amino-2-pyridyl)sulfonyl]-6-(3- fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin -l-yl]pyridine-3- carboxamide. PCT Publication No. WO 2016/057572, incorporated herein by reference, discloses Compound I, a method of making Compound I, and that Compound I is a CFTR modulator with an EC30 of < 3 μΜ.

[0012] Disclosed herein are pharmaceutical compositions wherein the properties of one therapeutic agent are improved by the presence of two therapeutic agents, kits, and methods of treatment thereof. In one embodiment, the disclosure features pharmaceutical compositions comprising N-[(6-amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy- phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin- l-yl]pyridine-3-carboxamide (Compound I), ( ?)-l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-N-(l-(2,3-dihydr oxypropyl)-6-fluoro-2-(l- hydroxy-2-methylpropan-2-yl)- lH-indol-5-yl)cyclopropanecarboxamide (Compound II), and N-[2,4-bis( 1 , 1 -dimethylethyl)-5-hydroxyphenyl] - 1 ,4-dihydro-4-oxoquinoline-3- carboxamide (Compound III), wherein the composition has improved properties.

[0013] Also disclosed herein is a solid dispersion of N-[(6-amino-2-pyridyl)sulfonyl]- 6-(3-fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrro lidin-l-yl]pyridine-3- carboxamide (Compound I) in a polymer. In one embodiment, the solid dispersion is prepared by spray drying, and is referred to a spray-dried dispersion (SDD). In one embodiment, the spray dried dispersion has a Tg of from 80 °C to 180 °C. In one embodiment, Compound I in the spray dried dispersion is substantially amorphous. Brief Description of the Drawings

[0014] FIG. 1 is a representative list of CFTR genetic mutations.

[0015] As stated above, disclosed herein is Compound I, which can be depicted as having the following structure:

[0016] A chemical name for Compound I is N-[(6-amino-2-pyridyl)sulfonyl]-6-(3- fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin - l-yl]pyridine-3- carboxamide. Compound I may be in the form of a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, Compound I (and/or at least one pharmaceutically acceptable salt thereof) can be administered in combination with at least one additional active pharmaceutical ingredient. In some embodiments, the at least one additional active pharmaceutical ingredient is chosen from:

(a) Compound II:

which has the following chemical name: ( ?)- l-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)-N- (l-(2,3-dihydroxypropyl)-6-fluoro-2-(l-hydroxy-2-methylpropa n-2-yl)- lH-indol-5- yl)cyclopropanecarboxamide, and pharmaceutically acceptable salts thereof; and (b) Compound III:

which has the following chemical name: N-(5-hydroxy-2,4-di-ie/t-butyl-phenyl)-4-oxo- lH-quinoline-3-carboxamide, and pharmaceutically acceptable salts thereof, or

Compound ΙΙΓ :

and pharmaceutically acceptable salts thereof.

Definitions

[0018] As used herein, "CFTR" means cystic fibrosis transmembrane conductance regulator.

[0019] As used herein, "mutations" can refer to mutations in the CFTR gene or the CFTR protein. A "CFTR gene mutation" refers to a mutation in the CFTR gene, and a "CFTR protein mutation" refers to a mutation in the CFTR protein. A genetic defect or mutation, or a change in the nucleotides in a gene in general results in a mutation in the CFTR protein translated from that gene, or a frame shift(s).

[0020] The term "F508del" refers to a mutant CFTR protein which is lacking the amino acid phenylalanine at position 508.

[0021] As used herein, a patient who is "homozygous" for a particular gene mutation has the same mutation on each allele.

[0022] As used herein, a patient who is "heterozygous" for a particular gene mutation has this mutation on one allele, and a different mutation on the other allele. [0023] As used herein, the term "modulator" refers to a compound that increases the activity of a biological compound such as a protein. For example, a CFTR modulator is a compound that increases the activity of CFTR. The increase in activity resulting from a CFTR modulator includes but is not limited to compounds that correct, potentiate, stabilize and/or amplify CFTR.

[0024] As used herein, the term "CFTR corrector" refers to a compound that facilitates the processing and trafficking of CFTR to increase the amount of CFTR at the cell surface. Compounds I and II disclosed herein are CFTR correctors.

[0025] As used herein, the term "CFTR potentiator" refers to a compound that increases the channel activity of CFTR protein located at the cell surface, resulting in enhanced ion transport. Compound III disclosed herein is a CFTR potentiator.

[0026] As used herein, the term "active pharmaceutical ingredient" or "therapeutic agent" ("API") refers to a biologically active compound.

[0027] As used herein, the term "pharmaceutically acceptable salt" refers to a salt form of a compound of this disclosure wherein the salt is nontoxic. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0028] Suitable pharmaceutically acceptable salts are, for example, those disclosed in S. M. Berge, et al. J. Pharmaceutical Sciences, 1977, 66, 1-19. For example, Table 1 of that article provides the following pharmaceutically acceptable salts:

Table 1:

Dihydrochloride Mucate Potassium

Edetate Napsylate Sodium

Edisylate Nitrate Zinc

Estolate Pamoate (Embonate)

Esylate Pantothenate

Fumarate Pho sphate/dipho sphate

Gluceptate Polygalacturonate

Gluconate Salicylate

Glutamate Stearate

Glycollylar s anilate Subacetate

Hexylresorcinate Succinate

Hydrabamine Sulfate

Hydrobromide Tannate

Hydrochloride Tartrate

Hydroxynaphthoate Teociate

Triethiodide

[0029] Non-limiting examples of pharmaceutically acceptable acid addition salts include: salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, or perchloric acid; salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid; and salts formed by using other methods used in the art, such as ion exchange. Non- limiting examples of pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N ⁺ (Ci^ alkyl) ₄ salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Suitable non-limiting examples of alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further non-limiting examples of pharmaceutically acceptable salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Other suitable, non-limiting examples of pharmaceutically acceptable salts include besylate and glucosamine salts.

[0030] The terms "patient" and "subject" are used interchangeably and refer to an animal including humans.

[0031] The terms "effective dose" and "effective amount" are used interchangeably herein and refer to that amount of a compound that produces the desired effect for which it is administered (e.g., improvement in CF or a symptom of CF, or lessening the severity of CF or a symptom of CF). The exact amount of an effective dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0032] As used herein, the terms "treatment," "treating," and the like generally mean the improvement of CF or its symptoms or lessening the severity of CF or its symptoms in a subject. "Treatment," as used herein, includes, but is not limited to, the following:

increased growth of the subject, increased weight gain, reduction of mucus in the lungs, improved pancreatic and/or liver function, reduction of chest infections, and/or reductions in coughing or shortness of breath. Improvements in or lessening the severity of any of these symptoms can be readily assessed according to standard methods and techniques known in the art.

[0033] As used herein, the term "in combination with," when referring to two or more compounds, agents, or additional active pharmaceutical ingredients, means the

administration of two or more compounds, agents, or active pharmaceutical ingredients to the patient prior to, concurrent with, or subsequent to each other.

[0034] The term "approximately", when used in connection with doses, amounts, or weight percent of ingredients of a composition or a dosage form, include the value of a specified dose, amount, or weight percent or a range of the dose, amount, or weight percent that is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percent.

[0035] Each of Compounds I, II, and III, and their pharmaceutically acceptable salts thereof independently can be administered once daily, twice daily, or three times daily. In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereofthereof is administered once daily. In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereofthereof are administered twice daily. In some embodiments, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof are administered twice daily. In some embodiments, at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered once daily. In some embodiments, at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are administered twice daily.

[0036] The term "daily" means per day. For example, 100 mg of Compound I is administered daily means total of 100 mg of Compound I per day is administered, which can be administered, for example, once daily, twice daily, or three times daily. For example, 100 mg of Compound I is administered once daily (qd) means 100 mg of Compound I per dosing is administered once per day. For example, 50 mg of Compound I is administered twice daily (bid) means 50 mg of Compound I per dosing is administered twice per day. In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered once daily. In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered twice daily. In some embodiments, Compound II or its pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, Compound II or its pharmaceutically acceptable salts thereof are

administered twice daily. In some embodiments, Compound III or its pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, Compound III or its pharmaceutically acceptable salts thereof are administered twice daily. In some embodiments, Compound Ill-d or its pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, Compound Ill-d or its pharmaceutically acceptable salts thereof are administered twice daily. In some embodiments, Compound IV or its pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, Compound IV or its pharmaceutically acceptable salts thereof are administered twice daily. [0037] In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered in an amount from 50 mg to 1000 mg, 100 mg to 800 mg, 100 mg to 700 mg, 100 mg to 600 mg, 200 mg to 600 mg, 300 mg to 600 mg, 400 mg to 600 mg, 500 mg to 700 mg, or 500 mg to 600 mg, daily. In some embodiments, at least one compound chosen from Compound I and

pharmaceutically acceptable salts thereof is administered in an amount of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg, daily. In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof are administered in an amount of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, once daily. In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered in an amount of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg, twice daily.

[0038] One of ordinary skill in the art would recognize that, when an amount of "a compound or a pharmaceutically acceptable salt thereof is disclosed, the amount of the pharmaceutically acceptable salt form of the compound is the amount equivalent to the concentration of the free base of the compound. It is noted that the disclosed amounts of the compounds or their pharmaceutically acceptable salts thereof herein are based upon their free base form. For example, "100 mg of at least one compound chosen from

Compound I and pharmaceutically acceptable salts thereof includes 100 mg of

Compound I and a concentration of pharmaceutically acceptable salt of Compound I equivalent to 100 mg of Compound I.

[0039] Compounds I, II, and III, and their pharmaceutically acceptable salts thereof can be comprised in a single pharmaceutical composition or separate pharmaceutical compositions. Such pharmaceutical compositions can be administered once daily or multiple times daily, such as twice daily.

[0040] In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is comprised in a second pharmaceutical composition; and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is comprised in a third pharmaceutical composition. [0041] In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; and at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in a second pharmaceutical composition. In some embodiments, the second pharmaceutical composition comprises a half of the daily dose of said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, and the other half of the daily dose of said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered in a third pharmaceutical composition.

[0042] In some embodiments, at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof; and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in a first pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered to the patient twice daily.

[0043] In some embodiments, the disclosure features a pharmaceutical composition comprising at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier.

[0044] In some embodiments, the disclosure features a pharmaceutical composition comprising at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and

pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier.

[0045] In some embodiments, the disclosure features a pharmaceutical composition comprising at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier. [0046] In some embodiments, the disclosure features a pharmaceutical composition comprising at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and

pharmaceutically acceptable salts thereof, at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier.

[0047] In some embodiments, pharmaceutical compositions disclosed herein comprise at least one additional active pharmaceutical ingredient. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR modulator. In some

embodiments, the at least one additional active pharmaceutical ingredient is a CFTR corrector. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR potentiator. In some embodiments, the pharmaceutical composition comprises Compound I and at least two additional active pharmaceutical ingredients, one of which is a CFTR corrector and one of which is a CFTR potentiator.

[0048] In some embodiments, at least one additional active pharmaceutical ingredient is selected from mucolytic agents, bronchodilators, antibiotics, anti-infective agents, and anti-inflammatory agents.

[0049] A pharmaceutical composition may further comprise at least one

pharmaceutically acceptable carrier. In some embodiments, the at least one

pharmaceutically acceptable carrier is chosen from pharmaceutically acceptable vehicles and pharmaceutically acceptable adjuvants. In some embodiments, the at least one pharmaceutically acceptable is chosen from pharmaceutically acceptable fillers, disintegrants, surfactants, binders, lubricants.

[0050] It will also be appreciated that a pharmaceutical composition of this disclosure, including a pharmaceutical composition comprising combinations described previously, can be employed in combination therapies; that is, the compositions can be administered concurrently with, prior to, or subsequent to, at least one additional active pharmaceutical ingredient or medical procedures.

[0051] In some embodiments, a pharmaceutical composition disclosed herein comprises at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a polymer. In some embodiments, the pharmaceutically acceptable carrier is HPMCAS. In some

embodiments, the pharmaceutically acceptable carrier is HPMCAS-H. In some embodiments, the pharmaceutical composition comprises a solid dispersion of compound I in HPMCAS-H.

[0052] As described above, pharmaceutical compositions disclosed herein may optionally further comprise at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier may be chosen from adjuvants and vehicles. The at least one pharmaceutically acceptable carrier, as used herein, includes any and all solvents, diluents, other liquid vehicles, dispersion aids, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, solid binders, and lubricants, as suited to the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D.B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier is incompatible with the compounds of this disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure. Non-limiting examples of suitable 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, and potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts, and electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and 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 as propylene glycol and 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, phosphate buffer solutions, non-toxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants.

[0053] It will also be appreciated that a pharmaceutical composition of this disclosure, including a pharmaceutical composition comprising any of the combinations described previously, can be employed in combination therapies; that is, the compositions can be administered concurrently with, prior to, or subsequent to, at least one active

pharmaceutical ingredients or medical procedures.

[0054] In some embodiments, the methods disclosed herein employ administering to a patient in need thereof at least one compound chosen from Compound I and

pharmaceutically acceptable salts thereof; and at least one selected from Compound II, Compound III, and pharmaceutically acceptable salts thereof.

[0055] Any suitable pharmaceutical compositions known in the art can be used for Compound I, Compound II, Compound III, and pharmaceutically acceptable salts thereof. Some exemplary pharmaceutical compositions for Compound I and its pharmaceutically acceptable salts are described in the Examples. Some exemplary pharmaceutical compositions for Compound II and its pharmaceutically acceptable salts can be found in WO 2011/119984 and WO 2014/015841, both of which are incorporated herein by reference. Some exemplary pharmaceutical compositions for Compound III and its pharmaceutically acceptable salts can be found in WO 2007/134279, WO 2010/019239, WO 2011/019413, WO 2012/027731, and WO 2013/130669, all of which are incorporated herein by reference.

[0056] In some embodiments, a pharmaceutical composition comprising at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered with a pharmaceutical composition comprising Compound II and Compound III. Pharmaceutical compositions comprising Compound II and Compound III are disclosed in PCT Publication No. WO 2015/160787, incorporated herein by reference. An exemplary embodiment is shown in the following Table: Table 2. Exemplary Tablet Comprising 100 mg of Compound II and 150

Compound III.

[0057] In some embodiments, a pharmaceutical composition comprising Compound I is administered with a pharmaceutical composition comprising Compound III.

Pharmaceutical compositions comprising Compound III are disclosed in PCT Publication No. WO 2010/019239, incorporated herein by reference. An exemplary embodiment is shown in the following Table:

Table 3: Ingredients for Exemplary Tablet of Compound III.

Microcrystalline cellulose 30.51% 167.8 21.36

Lactose 30.40% 167.2 21.28

Sodium croscarmellose 3.000% 16.50 2.100

SLS 0.500% 2.750 0.3500

Colloidal silicon dioxide 0.500% 2.750 0.3500

Magnesium stearate 1.000% 5.500 0.7000

Total 100% 550 70

[0058] Additional pharmaceutical compositions comprising Compound III are disclosed in PCT Publication No. WO 2013/130669, incorporated herein by reference. Exemplary mini-tablets (~2 mm diameter, ~2 mm thickness, each mini-tablet weighing about 6.9 mg) was formulated to have approximately 50 mg of Compound III per 26 mini- tablets and approximately 75 mg of Compound III per 39 mini-tablets using the amounts of ingredients recited in Table 4, below.

Table 4: Ingredients for mini-tablets for 50 mg and 75 mg potency

[0059] In some embodiments, the pharmaceutical compositions are a tablet. In some embodiments, the tablets are suitable for oral administration.

[0060] These combinations are useful for treating cystic fibrosis.

[0061] A CFTR mutation may affect the CFTR quantity, i.e., the number of CFTR channels at the cell surface, or it may impact CFTR function, i.e., the functional ability of each channel to open and transport ions. Mutations affecting CFTR quantity include mutations that cause defective synthesis (Class I defect), mutations that cause defective processing and trafficking (Class II defect), mutations that cause reduced synthesis of CFTR (Class V defect), and mutations that reduce the surface stability of CFTR (Class VI defect). Mutations that affect CFTR function include mutations that cause defective gating (Class III defect) and mutations that cause defective conductance (Class IV defect).

[0062] In some embodiments, disclosed herein methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis in a patient comprising

administering an effective amount of a compound, pharmaceutically acceptable salt thereof, or a deuterated analog of any of the foregoing; or a pharmaceutical composition, of this disclosure to a patient, such as a human, wherein said patient has cystic fibrosis. In some embodiments, the patient has F508del/minimal function (MF) genotypes,

F508del/F508del genotypes, F508del/gating genotypes, or F508del/residual function (RF) genotypes.

[0063] As used herein, "minimal function (MF) mutations" refer to CFTR gene mutations associated with minimal CFTR function (little-to-no functioning CFTR protein) and include, for example, mutations associated with severe defects in ability of the CFTR channel to open and close, known as defective channel gating or "gating mutations"; mutations associated with severe defects in the cellular processing of CFTR and its delivery to the cell surface; mutations associated with no (or minimal) CFTR synthesis; and mutations associated with severe defects in channel conductance. Table C below includes a non-exclusive list of CFTR minimal function mutations, which are detectable by an FDA-cleared genotyping assay. In some embodiments, a mutation is considered a MF mutation if it meets at least 1 of the following 2 criteria:

(1) biological plausibility of no translated protein (genetic sequence predicts the complete absence of CFTR protein), or

(2) in vitro testing that supports lack of responsiveness to Compound II,

Compound III or the combination of Compound II and Compound III, and evidence of clinical severity on a population basis (as reported in large patient registries).

[0064] In some embodiments, the minimal function mutations are those that result in little-to-no functioning CFTR protein and are not responsive in vitro to Compound II, Compound III, or the combination of Compound II and Compound III. [0065] In some embodiments, the minimal function mutations are those that are not responsive in vitro to Compound II, Compound III, or the combination of Compound II and Compound III. In some embodiments, the minimal function mutations are mutations based on in vitro testing met the following criteria in in vitro experiments:

• baseline chloride transport that was <10% of wildtype CFTR, and

• an increase in chloride transport of <10% over baseline following the addition of TEZ, IVA, or TEZ/IVA in the assay.

In some embodiments, patients with at least one minimal function mutation exhibit evidence of clinical severity as defined as:

• average sweat chloride >86 mmol/L, and

• prevalence of pancreatic insufficiency (PI) >50%.

[0066] Patients with an F508del/minimal function genotype are defined as patients that are heterozygous F508del-CFTR with a second CFTR allele containing a a minimal function mutation. In some embodiments, patients with an F508del/minimal function genotype are patients that are heterozygous F508del-CFTR with a second CFTR allele containing a mutation that results in a CFTR protein with minimal CFTR function (little- to-no functioning CFTR protein) and that is responsive in vitro to Compound II,

Compound III, or the combination of Compound II and Compound III.

[0067] In some embodiemtns, minimal function mutations can be using 3 major sources:

• biological plausibility for the mutation to respond (i.e., mutation class)

• evidence of clinical severity on a population basis (per CFTR2 patient registry; accessed on 15 February 2016)

o average sweat chloride >86 mmol/L, and

o prevalence of pancreatic insufficiency (PI) >50%

• in vitro testing

o mutations resulting in baseline chloride transport <10% of wild-type CFTR were considered minimal function

o mutations resulting in chloride transport <10% of wild-type CFTR following the addition of Compound II and/or Compound III were considered nonresponsive.

[0068] As used herein, a "residual function mutations" refer to are Class II through V mutations that have some residual chloride transport and result in a less severe clinical phenotype. Residual function mutations are mutation in the CFTR gene that result in reduced protein quantity or function at the cell surface which can produce partial CFTR activity.

[0069] Non-limiting examples of CFTR gene mutations known to result in a residual function phenotype include a CFTR residual function mutation selected from

2789+5G^A, 3849+1 OkbC^T, 3272-26A^G, 711+3A^G, E56K, P67L, R74W, DllOE, Dl lOH, R117C, L206W, R347H, R352Q, A455E, D579G, E831X, S945L, S977F, F1052V, R1070W, F1074L, Dl 152H, D1270N, E193K, and K1060T. For example, CFTR mutations that cause defective mRNA splicing, such as 2789+507 A, result in reduced protein synthesis, but deliver some functional CFTR to the surface of the cell to provide residual function. Other CFTR mutations that reduce conductance and/or gating, such as Rl 17H, result in a normal quantity of CFTR channels at the surface of the cell, but the functional level is low, resulting in residual function. In some embodiments, the CFTR residual function mutation is selected from R117H, S1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, A1067T, E193K, and K1060T. In some embodiments, the CFTR residual function mutation is selected from R117H, S 1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, and A1067T.

[0070] Residual CFTR function can be characterized at the cellular (in vitro) level using cell based assays, such as an FRT assay (Van Goar, F. et al. (2009) PNAS Vol. 106, No. 44, 18825-18830; and Van Goor, F. et al. (2011) PNAS Vol. 108, No. 46, 18843- 18846), to measure the amount of chloride transport through the mutated CFTR channels. Residual function mutations result in a reduction but not complete elimination of CFTR dependent ion transport. In some embodiments, residual function mutations result in at least about 10% reduction of CFTR activity in an FRT assay. In some embodiments, the residual function mutations result in up to about 90% reduction in CFTR activity in an FRT assay.

[0071] Patients with an F508del/residual function genotype are defined as patients that are heterozygous F508del-CFTR with a second CFTR allele that contains a mutation that results in reduced protein quantity or function at the cell surface which can produce partial CFTR activity.

[0072] Patients with an F508del/gating mutation genotype are defined as patients that are heterozygous F508del-CFTR with a second CFTR allele that contains a mutation associated with a gating defect and clinically demonstrated to be responsive to Compound III. Examples of such mutations include: G178R, S549N, S549R, G551D, G551S, G1244E, S 125 IN, S 1255P, and G1349D.

[0073] In some embodiments, the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein are each independently produces an increase in chloride transport above the baseline chloride transport of the patient.

[0074] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation. In some embodiments, the paitent is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation, and is expected to be and/or is responsive to any of the compounds disclosed herein, such as Compound 1, Compound II, and/or Compound III genotypes based on in vitro and/or clinical data. In some embodiments, the paitent is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation, and is expected to be and/or is responsive to any combinations of (i) Compound 1, and (ii) Compound II, and/or Compound III and/or Compound IV genotypes based on in vitro and/or clinicCompound IVal data.

[0075] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation selected from any of the mutations listed in Table A.

Table A. CF Mutations

1341+1G->A 1717-lG^A

078delT 1342-2A->C 1717-8G^A

1078delT 1461ins4 1782delA

11234V 1471delA 1811+1.6kbA->G

1154insTC 1497delGG 1811+1G->C

1161delC 1507del 1811+1.6kbA^G

1213delT 1525-lG^A 1811+lG^C

1248+lG^A 1525-2A^G 1812-1G->A

1249-lG^A 1548delG 1898+1G->A

124del23bp 1577delTA 1812-lG^A

1259insA 1609del CA 1824delA

1288insTA 1677delTA 182delT 1119delA

1716G/A 185+lG^T 1898+1G->T 3028delA 405+IG->A

1898+lG^A 3040G^C 406-lG^A

1898+lG^C 306insA 406-IG->A

1898+3A->G 306insA 1138insG 4209TGTT->A

1898+5G->T 3120G^A 4209TGTT^AA

1924del7 3121-lG^A 4279insA

1949del84 3121-2A^G 4326delTC

2043delG 3121-977_3499+248 4374+lG^T

2055del9^A de!2515 4374+IG->T

2105- 3132delTG 4382delA

2117dell3insAGAAA 3141del9 4428insGA

2118dell4 3171delC 442delA

2143delT 3195del6 457TAT^G

2183AA->G+ 3199del6 541delC

2183AA^G 3272-26A->G 574delA

2183AA^G ^a 3500-2A^G 5T

2183delAA->G# 3600+2insT 621+lG^T

2183delAA^G 365-366insT 621+3A->G

2184delA 3659delC 663delT

2184insA 3667ins4 663delT 1548delG

2307insA 3737delA 675del4

2347delG 3791delC 711+1G->T

2556insAT 3821delT 711+3A->G

2585delT 3849+lOkbC^T 711+lG^T

2594delGT 3849+IOkbC->T 711+3A^G

2622+lG->A 3850-lG^A 711+5G^A

2622+IG->A 3850-3T->G 712-1G->T

7T

2659delC 3850-IG->A

852del22

2711delT 3876delA

935delA

271delT 3878delG

991del5

2721delll 3905lnsT

A1006E

2732insA 3905insT

A120T

2789+2insA 394delTT

A234D

2789+5G^A 4005+lG->A

A349V

4005+2T->C

2790-lG^C A455E

4005+lG^A

2790-IG->C A613T

4005+IG->A

2869insG A46D

4010del4

2896insAG A46Db

2942insT 4015delA A559T

2957delT 4016insT A559Tb

296+lG^A 4021dupT A561E

2991del32 4040delA C276X

3007delG 405+lG^A C524R

405+3A^C C524X G1249R I3336K

CFTRdel2,3 G126D I502T

CFTRdele22-23 G1349D I506S

D110E G149R I506T

D110H G178R I507del

D1152H G194R I507del

D1270N G194V I601F

D192G G27R I618T

D443Y G27X I807M

D513G G314E I980K

D579G G330X IVS14b+5G->A

D614G G458V K710X

D836Y G463V K710X

D924N G480C K710X

D979V G542X L102R

E1104X G550X L1065P

E116K G551D L1077P

E1371X G551S L1077Pb

E193K G576A L1254X

E193X G622D L1324P

E403D G628R L1335P

E474K G628R(G->A) L138ins

E56K G970D L1480P

E585X G673X L15P

E588V G85E L165S

E60K G91R L206W

E822K G970R L218X

E822X G970R L227R

E831X H1054D L320V

E92K H1085P L346P

E92X H1085R L453S

F1016S H1375P L467P

F1052V H139R L467Pb

F1074L H199R L558S

F1099L H199Y L571S

F191V H609R L732X

F311del H939R L927P

F311L 11005 R L967S

F508C I1027T L997F

F508del 11234V M1101K

F575Y I1269N M1101R

G1061R I1366N M152V G1069R I148T MIT

G1244E 1175V M1V M265R R1162X S1255X

M470V R117C S13F

M952I R117G S341P

M952T R117H S434X

N1303K R117L S466X

P205S R117P S489X

P574H R1283M S492F

P5L R1283S S4X

P67L R170H S549N

P750L R258G S549R

P99L R31C S549R(A->C)

Q1100P R31L S549R(T->G)

Q1291H R334L S589N

Q1291R R334Q S737F

Q1313X R334W S912L

Q1382X R347H S912X

Q1411X R347L S945L

Q1412X R347P S977F

Q220X R352Q T1036N

Q237E R352W T1053I

Q237H R516G T1246I

Q452P R553Q T338I

Q290X R553X T604I

Q359K/T360K R560K V1153E

Q39X R560S V1240G

Q414 R560T V1293G

Q414X R668C V201M

E585X R709X V232D

Q493X R74W V456A Q525X R751L V456F Q552X R75Q V520F Q685X R75X V562I Q890X R764X V754M Q890X R792G W1089X

Q98R R792X W1098C

Q98X R851X W1098R

R1066C R933G W1098X

R1066H S1118F W1204X

R1066M S1159F W1282R

R1070Q S1159P W1282X

R1070W S1196X W361R

R1102X S1235R W401X

R1158X S1251N W496X

W57G

R1162L S1255P W57R Y109N Y569C

W57X Y122X Y569D

W846X Y161D Y569Db

Y1014C Y161S Y849X

Y1032C Y563D Y913C

Y1092X Y563N Y913X

[0076] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R, S 1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S 1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 405+lG->A, 406-lG->A, 4005+lG->A, 1812-1G->A, 1525-1G->A, 712- 1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 3850-lG->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, IVS 14b+5G->A, 1898+1G->T, 4005+2T->C, 621+3A- >G, 1949del84, 3141del9, 3195del6, 3199del6, 3905InsT, 4209TGTT->A, A1006E, A120T, A234D, A349V, A613T, C524R, D192G, D443Y, D513G, D836Y, D924N, D979V, E116K, E403D, E474K, E588V, E60K, E822K, F1016S, F1099L, F191V, F311del, F311L, F508C, F575Y, G1061R, G1249R, G126D, G149R, G194R, G194V, G27R, G314E, G458V, G463V, G480C, G622D, G628R, G628R(G->A), G91R, G970D, H1054D, H1085P, H1085R, H1375P, H139R, H199R, H609R, H939R, I1005R, I1234V, I1269N, I1366N, I175V, I502T, I506S, I506T, I601F, I618T, I807M, I980K, L102R, L1324P, L1335P, L138ins, L1480P, L15P, L165S, L320V, L346P, L453S, L571S, L967S, M1101R, M152V, MIT, M1V, M265R, M952I, M952T, P574H, P5L, P750L, P99L, Q1100P, Q1291H, Q1291R, Q237E, Q237H, Q452P, Q98R, R1066C, R1066H, R117G, R117L, R117P, R1283M, R1283S, R170H, R258G, R31L, R334L, R334Q, R347L, R352W, R516G, R553Q, R751L, R792G, R933G, S 1118F, S 1159F, S 1159P, S 13F, S549R(A->C), S549R(T->G), S589N, S737F, S912L, T1036N, T1053I, T1246I, T604I, V1153E, V1240G, V1293G, V201M, V232D, V456A, V456F, V562I, W1098C,

W1098R, W1282R, W361R, W57G, W57R, Y1014C, Y1032C, Y109N, Y161D, Y161S, Y563D, Y563N, Y569C, and Y913C. [0077] In some embodiments, the patient has at least one combination mutation chosen from: G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R, S 125 IN, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S 1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717- 1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 405+1G- >A, 406-lG->A, 4005+lG->A, 1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 3850-lG->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, rVS 14b+5G->A, 1898+1G->T, 4005+2T->C, and 621+3A->G.

[0078] In some embodiments, the patient has at least one combination mutation chosen from: 1949del84, 3141del9, 3195del6, 3199del6, 3905InsT, 4209TGTT->A, A1006E, A120T, A234D, A349V, A613T, C524R, D192G, D443Y, D513G, D836Y, D924N, D979V, E116K, E403D, E474K, E588V, E60K, E822K, F1016S, F1099L, F191V, F311del, F311L, F508C, F575Y, G1061R, G1249R, G126D, G149R, G194R, G194V, G27R, G314E, G458V, G463V, G480C, G622D, G628R, G628R(G->A), G91R, G970D, H1054D, H1085P, H1085R, H1375P, H139R, H199R, H609R, H939R, I1005R, I1234V, I1269N, I1366N, I175V, I502T, I506S, I506T, I601F, I618T, I807M, I980K, L102R, L1324P, L1335P, L138ins, L1480P, L15P, L165S, L320V, L346P, L453S, L571S, L967S, M1101R, M152V, MIT, M1V, M265R, M952I, M952T, P574H, P5L, P750L, P99L, Q1100P, Q1291H, Q1291R, Q237E, Q237H, Q452P, Q98R, R1066C, R1066H, R117G, R117L, R117P, R1283M, R1283S, R170H, R258G, R31L, R334L, R334Q, R347L, R352W, R516G, R553Q, R751L, R792G, R933G, S 1118F, S 1159F, S 1159P, S 13F, S549R(A->C), S549R(T->G), S589N, S737F, S912L, T1036N, T1053I, T1246I, T604I, V1153E, V1240G, V1293G, V201M, V232D, V456A, V456F, V562I, W1098C,

W1098R, W1282R, W361R, W57G, W57R, Y1014C, Y1032C, Y109N, Y161D, Y161S, Y563D, Y563N, Y569C, and Y913C.

[0079] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation G551D. In some embodiments, the patient is homozygous for the G551D genetic mutation. In some embodiments, the patient is heterozygous for the G551D genetic mutation. In some embodiments, the patient is heterozygous for the G551D genetic mutation, having the G551D mutation on one allele and any other CF- causing mutation on the other allele. In some embodiments, the patient is heterozygous for the G551D genetic mutation on one allele and the other CF-causing genetic mutation on the other allele is any one of F508del, G542X, N1303K, W1282X, R117H, R553X, 1717-1G->A, 621+1G->T, 2789+5G->A, 3849+10kbC->T, R1162X, G85E, 3120+1G- >A, ΔΙ507, 1898+1G->A, 3659delC, R347P, R560T, R334W, A455E, 2184delA, or 711+1G->T. In some embodiments, the patient is heterozygous for the G551D genetic mutation, and the other CFTR genetic mutation is F508del. In some embodiments, the patient is heterozygous for the G551D genetic mutation, and the other CFTR genetic mutation is Rl 17H.

[0080] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation F508del. In some embodiments, the patient is homozygous for the F508del genetic mutation. In some embodiments, the patient is heterozygous for the F508del genetic mutation wherein the patient has the F508del genetic mutation on one allele and any CF-causing genetic mutation on the other allele. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any CF- causing mutation, including, but not limited to G551D, G542X, N1303K, W1282X, R117H, R553X, 1717-1G->A, 621+1G->T, 2789+5G->A, 3849+10kbC->T, R1162X, G85E, 3120+1G->A, ΔΙ507, 1898+1G->A, 3659delC, R347P, R560T, R334W, A455E, 2184delA, or 711+1G->T. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is G551D. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is Rl 17H.

[0081] In some embodiments, the patient has at least one combination mutation chosen from:

D443Y;G576A;R668C, F508C;S 1251N, G576A; R668C, G970R; M470V, R74W;D1270N, R74W;V201M, and R74W;V201M;D1270N.

[0082] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R, S 125 IN, E193K, F1052V and G1069R. In some embodiments, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R and S 125 IN. In some embodiments, the patient possesses a CFTR genetic mutation selected from E193K, F1052V and G1069R. In some embodiments, the method produces an increase in chloride transport relative to baseline chloride transport of the patient of the patient.

[0083] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation selected from R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S 1235R, S945L, R1070W, F1074L, D110E, D1270N and D1152H.

[0084] In some embodiments, the patient possesses a CFTR genetic mutation selected from 1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 405+lG->A, 406-lG->A, 4005+lG->A, 1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 3850-lG->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, IVS 14b+5G->A, 1898+1G->T, 4005+2T->C and 621+3A- >G. In some embodiments, the patient possesses a CFTR genetic mutation selected from 1717-1G->A, 1811+1.6kbA->G, 2789+5G->A, 3272-26A->G and 3849+10kbC->T. In some embodiments, the patient possesses a CFTR genetic mutation selected from

2789+5G->A and 3272-26A->G.

[0085] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S 1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S 1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 405+lG->A, 406-lG->A, 4005+lG->A, 1812-1G->A, 1525-1G->A, 712- 1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 3850-lG->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, IVS 14b+5G->A, 1898+1G->T, 4005+2T->C and

621+3 A->G, and human CFTR mutations selected from F508del, R117H, and G551D.

[0086] In some embodiments, in the methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis disclosed herein, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R, S 1251N, E193K, F1052V, G1069R, R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S 1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 405+lG->A, 406-lG->A, 4005+lG->A, 1812-1G->A, 1525-1G->A, 712- 1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 3850-lG->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, IVS 14b+5G->A, 1898+1G->T, 4005+2T->C, 621+3A- >G, and a CFTR mutation selected from F508del, Rl 17H, and G551D; and a CFTR mutations selected from F508del, R117H, and G551D.

[0087] In some embodiments, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R, S 125 IN, E193K, F 1052V and G1069R, and a human CFTR mutation selected from F508del, R117H, and G551D. In some embodiments, the patient possesses a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S 1255P, G1349D, S549N, S549R and S 125 IN, and a human CFTR mutation selected from F508del, R117H, and G551D. In some embodiments, the patient possesses a CFTR genetic mutation selected from E193K, F 1052V and G1069R, and a human CFTR mutation selected from F508del, R117H, and G551D.

[0088] In some embodiments, the patient possesses a CFTR genetic mutation selected from R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S 1235R, S945L, R1070W, F1074L, D110E, D1270N and D1152H, and a human CFTR mutation selected from F508del, R117H, and G551D.

[0089] In some embodiments, the patient possesses a CFTR genetic mutation selected from 1717-1G->A, 621+1G->T, 3120+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 405+lG->A, 406-lG->A, 4005+lG->A, 1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 3850-lG->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, IVS 14b+5G->A, 1898+1G->T, 4005+2T->C and 621+3A- >G, and a human CFTR mutation selected from F508del, Rl 17H, and G55 ID. In some embodiments, the patient possesses a CFTR genetic mutation selected from 1717-1G->A, 1811+1.6kbA->G, 2789+5G->A, 3272-26A->G and 3849+10kbC->T, and a human CFTR mutation selected from F508del, Rl 17H, and G55 ID. In some embodiments, the patient possesses a CFTR genetic mutation selected from 2789+5G->A and 3272-26A->G, and a human CFTR mutation selected from F508del, Rl 17H.

[0090] In some embodiments, the patient is heterozygous having a CF-causing mutation on one allele and a CF-causing mutation on the other allele. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation, including, but not limited to F508del on one CFTR allele and a CFTR mutation on the second CFTR allele that is associated with minimal CFTR function, residual CFTR function, or a defect in CFTR channel gating activity.

[0091] In some embodiments, the CF-causing mutation is selected from Table A. In some embodiments, the CF-causing mutation is selected from Table B. In some embodiments, the CF-causing mutation is selected from Table C. In some embodiments, the CF-causing mutation is selected from FIG. 1. In some embodiments, the patient is heterozygous having a CF-causing mutation on one CFTR allele selected from the mutations listed in the table from FIG. 1 and a CF- causing mutation on the other CFTR allele is selected from the CFTR mutations listed in Table B:

Table B: CFTR Mutations

Q39X Q414X K710X

W57X S434X L732X

E60X S466X R764X

R75X S489X R785X

E92X Q493X R792X

Q98X W496X E822X

Y122X Q525X W846X

L218X G542X R851X

Q220X Q552X Q890X

C276X R553X S912X

Q290X E585X W1089X

G330X G673X Y1092X

W401X R709X E1104X R1158X 1154insTC R1066M

R1162X 2183delAA→G L1077P

S1196X 2143delT H1085R

W1204X 1677delTA M1101K

S1255X 3876delA N1303K

W1282X 2307insA 3849+10kbC→T

Q1313X 4382delA 3272-26A→G

621+1G→T 4016insT 711+3A→G

711+1G→T 2347delG E56K

711+5G→A 3007delG P67L

712-1G→T 574delA R74W

405+lG→A 2711delT D110E

405+3A→C 3791delC D110H

406-lG→A CFTRdele22-23

R117C

621+1G→T 457TAT→G L206W

1248+1G→A 2043delG R347H

1341+1G→A 2869insG R352Q

1717-1G→A 3600+2insT A455E

1811+1.6kbA→G 3737delA D579G

1811+1G→C 4040delA E831X

541delC

1812-1G→A S945L

1898+1G→A A46D S977F

2622+ 1G→ A T338I F1052V

R347P

3120+1G→A R1070W

L927P

3120G→A F1074L

G85E

3850-lG→A D1152H

S341P

4005+ 1G→ A D1270N

L467P

4374+ 1G→T G178R

I507del

663delT S549N

V520F

2183AA→G S549R

A559T

CFTRdel2,3 G551D

R560T

3659delC G551S

R560S

394delTT G1244E

A561E

2184insA S1251N

Y569D

3905insT S1255P

L1065P

2184delA G1349D

R1066C

1078delT

Table C: CFTR Mutations

Criteria Mutation

Truncation mutations Q2X L218X Q525X R792X E1104X

• %PI >50% and/or S4X Q220X G542X E822X W1145X

SwCl ^" >86 mmol L W19X Y275X G550X W882X R1158X

• No full-length G27X C276X Q552X W846X R1162X protein

Q39X Q290X R553X Y849X S 1196X

W57X G330X E585X R851X W1204X

E60X W401X G673X Q890X L1254X

R75X Q414X Q685X S912X S 1255X

L88X S434X R709X Y913X W1282X

E92X S466X K710X Q1042X Q1313X

Q98X S489X Q715X W1089X Q1330X

Y122X Q493X L732X Y1092X E1371X

E193X W496X R764X W1098X Q1382X

W216X C524X R785X R1102X Q1411X

Splice mutations 185+1G→T 711+5G→A 1717-8G→A 2622+lG→A 3121-1G→A

• %PI >50% and/or 296+lG→A 712-1G→T 1717-1G→A 2790- 1G→C 3500-2A→G

SwCl ^" >86 mmol L 296+lG→T 1248+1G→A 1811+1G→C 3040G→C 3600+2insT

• No or little mature 405+lG→A 1249- 1G→A 1811+1.6kbA→G (G970R) 3850- 1G→A mRNA

405+3A→C 1341+1G→A 1811+1643G→T 3120G→A 4005+ 1G→A

406-lG→A 1525-2A→G 1812-1G→A 3120+1G→A 4374+ 1G→T

621+1G→T 1525-1G→A 1898+1G→A 3121-2A→G

711+1G→T 1898+1G→C

Small (<3 nucleotide) 182delT 1078delT 1677delTA 2711delT 3737delA insertion/deletion 306insA 1119delA 1782delA 2732insA 3791delC

(ins/del) frameshift 306delTAGA 1138insG 1824delA 2869insG 3821delT mutations

365-366insT 1154insTC 1833delT 2896insAG 3876delA

• %PI >50% and/or

394delTT 2942insT

SwCl ^" >86 mmol/L 1161delC 2043delG 3878delG

• Garbled and/or 442delA 1213delT 2143delT 2957delT 3905insT truncated protein 444delA 1259insA 2183AA→G ^a 3007delG 4016insT

457TAT→G 1288insTA 2184delA 3028delA 4021dupT

541delC 1343delG 2184insA 3171delC 4022insT

574delA 1471delA 2307insA 3171insC 4040delA

663delT 1497delGG 2347delG 3271delGG 4279insA

849delG 1548delG 2585delT 3349insT 4326delTC

935delA 1609del CA 2594delGT 3659delC

Non-small (>3 CFTRdelel CFTRdelel 6- 17b 1461ins4

nucleotide) CFTRdele2 CFTRdelel 7a, 17b 1924del7

insertion/deletion CFTRdele2,3 CFTRdelel 7a- 18 2055del9→A

(ins/del) frameshift

mutations CFTRdele2-4 CFTRdelel 9 2105-2117dell3insAGAAA

• %PI >50% and/or CFTRdele3-10,14b-16 CFTRdelel9-21 2372del8

SwCl- >86 mmol/L CFTRdele4-7 CFTRdele21 2721dell l

• Garbled and/or CFTRdele4-l l CFTRdele22-24 2991del32

truncated protein CFTR50kbdel CFTRdele22,23 3121-977_3499+248del2515

CFTRdup6b-10 124del23bp 3667ins4

CFTRdelel l 602dell4 4010del4

CFTRdelel3,14a 852del22 4209TGTT→ AA

CFTRdelel4b-17b 991del5 Criteria Mutation

Class II, III, IV A46D ^b V520F Y569D ^b N1303K

mutations not responsive G85E A559T ^b L1065P

to Compound II, R347P R560T R1066C

Compound III, or

L467P ^b

Compound II Compound R560S L1077P ^b

III I507del A561E M1101K

• %PI>50% and/or

SwCr >86 mmol L

AND

• Not responsive in

vitro to Compound

II, Compound III,

or Compound

II/Compound III

CFTR: cystic fibrosis transmembrane conductance regulator; SwCl: sweat chloride

Source: CFTR2.org [Internet]. Baltimore (MD): Clinical and functional translation of CFTR. The Clinical and Functional Translation of CFTR (CFTR2), US Cystic Fibrosis Foundation, Johns Hopkins University, the Hospital for Sick Children. Available at: http://www.cftr2.org/. Accessed 15 February 2016.

Notes: %PI: percentage of F508del-CFTR heterozygous patients in the CFTR2 patient registry who are pancreatic insufficient; SwCl: mean sweat chloride of F508del-CFTR heterozygous patients in the CFTR2 patient registry.

Also known as 2183delAA→G.

b Unpublished data.

[0092] In some embodiments, the patient is: with F508del/MF (F/MF) genotypes (heterozygous for F508del and an MF mutation not expected to respond to CFTR modulators, such as Compound III); with F508del/F508del (F/F) genotype (homozygous for F508del); and/or with F508del/ gating (F/G) genotypes (heterozygous for F508del and a gating mutation known to be CFTR modulator-responsive (e.g., Compound III- responsive). In some embodiments, the patient with F508del/MF (F/MF) genotypes has a MF mutation that is not expected to respond to Compound II, Compound III, and both of Compound II and Compound III. In some embodiments, the patient with F508del/M (F/MF) genotypes has any one of the MF mutations in Table C.

[0093] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation, including truncation mutations, splice mutations, small (<3 nucleotide) insertion or deletion (ins/del) frameshift mutations; non- small (>3 nucleotide) insertion or deletion (ins/del) frameshift mutations; and Class II, III, IV mutations not responsive to Compound III alone or in combination with Compound II or Compound IV.

[0094] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is a truncation mutation. In some specific embodiments, the truncation mutation is a truncation mutation listed in Table C. [0095] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is a splice mutation. In some specific embodiments, the splice mutation is a splice mutation listed in Table C.

[0096] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is a small (<3 nucleotide) insertion or deletion (ins/del) frameshift mutation. In some specific embodiments, the small (<3 nucleotide) insertion or deletion (ins/del) frameshift mutation is a small (<3 nucleotide) insertion or deletion (ins/del) frameshift mutation listed in Table C.

[0097] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation expected to be and/or is responsive to, based on in vitro and/or clinical data, any combination of Compounds (I), (II), (III), (IIP), and pharmaceutically acceptable salts thereof, and their deuterated derivatives).

[0098] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any CF-causing mutation expected to be and/or is responsive, based on in vitro and/or clinical data, to the triple combination of Compounds (I), (II), (III), (IIP), and pharmaceutically acceptable salts thereof, and their deuterated

derivatives).

[0099] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is a non-small (>3 nucleotide) insertion or deletion (ins/del) frameshift mutation. In some specific embodiments, the non-small (>3 nucleotide) insertion or deletion (ins/del) frameshift mutation is a non-small (>3 nucleotide) insertion or deletion (ins/del) frameshift mutation listed in Table C.

[00100] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is a Class II, III, IV mutations not responsive to Compound III alone or in combination with Compound II or Compound IV. In some specific

embodiments, the Class II, III, IV mutations not responsive to Compound III alone or in combination with Compound II or Compound IV is a Class II, III, IV mutations not responsive to Compound III alone or in combination with Compound II or Compound IV listed in Table C.

[00101] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any mutation listed in Table C. [00102] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any mutation, but other than F508del, listed in Table A, B, C, and FIG. 1.

[00103] In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any mutation listed in Table A. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any mutation listed in Table B. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any mutation listed in Table C. In some embodiments, the patient is heterozygous for F508del, and the other CFTR genetic mutation is any mutation listed in FIG. 1.

[00104] In some embodiments, the patient is homozygous for F508del.

[00105] In some embodiments, the patient is heterozygous having one CF-causing mutation on one CFTR allele selected from the mutations listed in the table from FIG. 1 and another CF-causing mutation on the other CFTR allele is selected from the CFTR mutations listed in Table C.

[00106] In some embodiments, the composition disclosed herein is useful for treating, lessening the severity of, or symptomatically treating cystic fibrosis in patients who exhibit residual CFTR activity in the apical membrane of respiratory and non-respiratory epithelia. The presence of residual CFTR activity at the epithelial surface can be readily detected using methods known in the art, e.g., standard electrophysiological, biochemical, or histochemical techniques. Such methods identify CFTR activity using in vivo or ex vivo electrophysiological techniques, measurement of sweat or salivary CI ^" concentrations, or ex vivo biochemical or histochemical techniques to monitor cell surface density. Using such methods, residual CFTR activity can be readily detected for patients that are heterozygous or homozygous for a variety of different mutations, including patients heterozygous for the most common mutation, F508del, as well as other mutations such as the G551D mutation, or the Rl 17H mutation. In some embodiments, compositions disclosed herein are useful for treating, lessening the severity of, or symptomatically treating cystic fibrosis in patients who exhibit little to no residual CFTR activity. In some embodiments, compositions disclosed herein are useful for treating, lessening the severity of, or symptomatically treating cystic fibrosis in patients who exhibit little to no residual CFTR activity in the apical membrane of respiratory epithelia. [00107] In some embodiments, the compositions disclosed herein are useful for treating or lessening the severity of cystic fibrosis in patients who exhibit residual CFTR activity using pharmacological methods. Such methods increase the amount of CFTR present at the cell surface, thereby inducing a hitherto absent CFTR activity in a patient or augmenting the existing level of residual CFTR activity in a patient.

[00108] In some embodiments, the compositions disclosed herein are useful for treating or lessening the severity of cystic fibrosis in patients with certain genotypes exhibiting residual CFTR activity.

[00109] In some embodiments, compositions disclosed herein are useful for treating, lessening the severity of, or symptomatically treating cystic fibrosis in patients within certain clinical phenotypes, e.g., a mild to moderate clinical phenotype that typically correlates with the amount of residual CFTR activity in the apical membrane of epithelia. Such phenotypes include patients exhibiting pancreatic sufficiency.

[00110] In some embodiments, the compositions disclosed herein are useful for treating, lessening the severity of, or symptomatically treating patients diagnosed with pancreatic sufficiency, idiopathic pancreatitis and congenital bilateral absence of the vas deferens, or mild lung disease wherein the patient exhibits residual CFTR activity.

[00111] In some embodiments, this disclosure relates to a method of augmenting or inducing anion channel activity in vitro or in vivo, comprising contacting the channel with a composition disclosed herein. In some embodiments, the anion channel is a chloride channel or a bicarbonate channel. In some embodiments, the anion channel is a chloride channel.

[00112] In some embodiments of the methods of treating cystic fibrosis disclosed herein, the absolute change in the patient's percent predicted forced expiratory volume in one second (ppFEVi) after 15 days of administration of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 40% relative to the ppFEVi of the patient prior to said administration.

[00113] In some embodiments of the methods of treating cystic fibrosis disclosed herein, the absolute change in ppFEVi after 29 days of administration of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 40% relative to the ppFEVl of the patient prior to said administration. In some embodiments of the methods of treating cystic fibrosis disclosed herein, the absolute change in ppFEVi after 29 days ranges from 3% to 20% relative to the ppFEVl of the patient prior to said administration.

[00114] In some embodiments of the methods of treating cystic fibrosis disclosed herein, the absolute change in the patient's sweat chloride after 15 days of administration of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from -2 to -65 mmol/L from baseline, i.e., relative to the sweat chloride of the patient prior to said administration. In some embodiments, the absolute change in sweat chloride of said patient ranges from -5 to -65 mmol/L. In some embodiments, the absolute change in sweat chloride of said patient ranges from -10 to -65 mmol/L. In some embodiments, the absolute change in sweat chloride of said patient ranges from -10 to -45 mmol/L.

[00115] In some embodiments of the methods of treating cystic fibrosis disclosed herein, the absolute change in the patient's sweat chloride after 29 days of administration of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from -2 to -65 mmol/L from baseline, i.e., relative to the sweat chloride of the patient prior to said administration. In some embodiments, the absolute change in sweat chloride of said patient ranges from -5 to -65 mmol/L. In some embodiments, the absolute change in sweat chloride of said patient ranges from -10 to -65 mmol/L. In some embodiments, the absolute change in sweat chloride of said patient ranges from -10 to -45 mmol/L. In some embodiments, the absolute change in sweat chloride of said patient ranges from -15 to -30 mmol/L.

[00116] In some embodiments, the triple combinations are administered to a patient who has one F508del mutation and one minimal function mutation, and who has not taken any of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof.

[00117] In some embodiments, the triple combinations are administered to a patient has two copies of F508del mutation, and wherein patient has taken at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, but not any of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof.

[00118] In some embodiments, the absolute change in patient's ppFEVi after 15 days of administration of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and

pharmaceutically acceptable salts thereof, and at least one compound chosen from

Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 35% relative to the ppFEVi of the patient prior to said administration.

[00119] In some embodiments, the absolute change in patient's ppFEVi after 29 days of administration of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and

pharmaceutically acceptable salts thereof, and at least one compound chosen from

Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 35% relative to the ppFEVi of the patient prior to said administration.

[00120] In some embodiments, the absolute change in a patient's ppFEVi relative to the ppFEVi of the patient prior to such administration of the triple combinations can be calculated as (postbaseline value- baseline value). The baseline value is defined as the most recent non-missing measurement collected before the first dose of study drug in the Treatment Period (Dayl).

[00121] The exact amount of a pharmaceutical composition required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular agent, its mode of administration, and the like. The compounds of this disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of this disclosure 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, such as a mammal, and even further such as a human.

[00122] In some embodiments, the disclosure also is directed to methods of treatment using isotope-labelled embodiments of the afore-mentioned compounds I, II, and III, which, in some embodiments, are referred to as Compound Γ, Compound ΙΓ, or

Compound ΙΙΓ . In some embodiments, Compound Γ, Compound ΙΓ, Compound IIP, or pharmaceutically acceptable salts thereof, wherein the formula and variables of such compounds and salts are each and independently as described above or any other embodiments described above, provided that one or more atoms therein have been replaced by an atom or atoms having an atomic mass or mass number which differs from the atomic mass or mass number of the atom which usually occurs naturally (isotope labelled). Examples of isotopes which are commercially available and suitable for the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, for example ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ 0, ¹⁷ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ C1, respectively.

[00123] The isotope-labelled compounds and salts can be used in a number of beneficial ways. They can be suitable for medicaments and/or various types of assays, such as substrate tissue distribution assays. For example, tritium ( ³ H)- and/or carbon-14 ( ¹⁴ C)- labelled compounds are particularly useful for various types of assays, such as substrate tissue distribution assays, due to relatively simple preparation and excellent detectability. For example, deuterium ( ² H)-labelled ones are therapeutically useful with potential therapeutic advantages over the non- ² H-labelled compounds. In general, deuterium (¾)- labelled compounds and salts can have higher metabolic stability as compared to those that are not isotope-labelled owing to the kinetic isotope effect described below. Higher metabolic stability translates directly into an increased in vivo half-life or lower dosages, which could be desired. The isotope-labelled compounds and salts can usually be prepared by carrying out the procedures disclosed in the synthesis schemes and the related description, in the example part and in the preparation part in the present text, replacing a non-isotope-labelled reactant by a readily available isotope-labelled reactant.

[00124] In some embodiments, the isotope-labelled compounds and salts are deuterium ( ² H)-labelled ones. In some specific embodiments, the isotope-labelled compounds and salts are deuterium ( ² H)-labelled, wherein one or more hydrogen atoms therein have been replaced by deuterium. In chemical structures, deuterium is represented as "D."

[00125] The deuterium ( ² H)-labelled compounds and salts can manipulate the oxidative metabolism of the compound by way of the primary kinetic isotope effect. The primary kinetic isotope effect is a change of the rate for a chemical reaction that results from exchange of isotopic nuclei, which in turn is caused by the change in ground state energies necessary for covalent bond formation after this isotopic exchange. Exchange of a heavier isotope usually results in a lowering of the ground state energy for a chemical bond and thus causes a reduction in the rate-limiting bond breakage. If the bond breakage occurs in or in the vicinity of a saddle-point region along the coordinate of a multi-product reaction, the product distribution ratios can be altered substantially. For explanation: if deuterium is bonded to a carbon atom at a non-exchangeable position, rate differences of i/ko = 2-7 are typical. For a further discussion, see S. L. Harbeson and R. D. Tung, Deuterium In Drug Discovery and Development, Ann. Rep. Med. Chem. 2011, 46, 403-417,

incorporated in its entirety herein by reference.

[00126] The concentration of the isotope(s) (e.g., deuterium) incorporated into the isotope-labelled compounds and salt of the disclosure may be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. In some embodiments, if a substituent in a compound of the disclosure is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium

incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

[00127] When discovering and developing therapeutic agents, the person skilled in the art attempts to optimize pharmacokinetic parameters while retaining desirable in vitro properties. It may be reasonable to assume that many compounds with poor

pharmacokinetic profiles are susceptible to oxidative metabolism.

[00128] In some embodiments, Compound ΙΙΓ as used herein includes the deuterated compound disclosed in U.S. Patent No. 8,865,902 (which is incorporated herein by reference), and CTP-656.

[00129] In some embodiments, Compound ΙΙΓ is:

[00130] Exemplary embodiments of the disclosure include:

1. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 50 mg to 1000 mg of at least one compound chosen from Compound I

and pharmaceutically acceptable salts thereof daily; and

(B) 25 mg to 200 mg of at least one compound chosen from Compound II:

and pharmaceutically acceptable salts thereof daily; and

(C) 50 mg to 600 mg of at least one compound chosen from Compound III:

and pharmaceutically acceptable salts thereof daily.

2. The method according to embodiment 1, wherein 100 mg to 800 mg, 100 mg to 700 mg, 200 mg to 700 mg, 200 mg to 600 mg, 300 mg to 600 mg, 400 mg to 600 mg, 500 mg to 700 mg, or 500 mg to 600 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

3. The method according to embodiment 1, wherein 100 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

4. The method according to embodiment 1, wherein 200 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

5. The method according to embodiment 1, wherein 300 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

6. The method according to embodiment 1, wherein 400 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

7. The method according to embodiment 1, wherein 500 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

8. The method according to embodiment 1, wherein 600 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily. 9. The method according to embodiment 1, wherein 700 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

10. The method according to embodiment 1, wherein 800 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily.

11. The method according to any one of embodiments 1-10, wherein at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered once daily.

12. The method according to any one of embodiments 1-10, wherein at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered twice daily.

13. The method according to any one of embodiments 1-12, wherein 50 mg to 150 mg or from 75 mg to 200 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered daily.

14. The method according to embodiment 13, wherein 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered daily.

15. The method according to embodiment 13, wherein 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered daily.

16. The method according to any one of embodiments 1-15, wherein at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered once daily.

17. The method according to any one of embodiments 1-15, wherein at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered in twice daily.

18. The method according to any one of embodiments 1-17, wherein 50 mg to 450 mg, from 100 mg to 400 mg, 125 mg to 300 mg, or 150 mg to 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered daily. 19. The method according to embodiment 18, wherein 150 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered daily.

20. The method according embodiment 18, wherein 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered daily.

21. The method according to any one of embodiments 1-20, wherein at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered once daily.

22. The method according to any one of embodiments 1-20, wherein the dose of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered twice daily.

23. The method according to embodiment 1, wherein 100 mg to 600 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily; 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered once daily; and 150 mg or 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered twice daily.

24. The method according to embodiment 1, wherein 100 mg to 600 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered daily; 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered twice daily; and 150 mg or 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered twice daily.

25. The method according to embodiment 1, wherein 100 mg, 200 mg, or 300 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered twice daily; 100 mg of Compound II is administered once daily; and 150 mg or 300 mg of Compound III is administered twice daily.

26. The method according to embodiment 1, wherein 100 mg, 200 mg, or 300 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is administered twice daily; 50 mg of Compound II is administered twice daily; and 150 mg or 300 mg of Compound III is administered twice daily. 27. The method according to any one of embodiments 1-26, wherein said patient has cystic fibrosis is chosen from patients with F508del/mimmal function genotypes, patients with F508del/F508del genotypes, patients with F508dell gating genotypes, patients with F508de //residual function genotypes, and patients with F508dell another CFTR genetic mutation that is expected to be and/or is responsive to the triple combination of Compound I, Compound II, and/or Compound III genotypes based on in vitro and/or clinical data.

28. The method according to embodiment 27, wherein the patient with a

F508dellm imaX function genotype has a minimal function mutation selected from:

E1I04X

W40t ΜΙβ

KSS3

∞ Q414X

L?$X S$4X

mm S <i¾X u SI255X pix S4I4X mm WOI2

QI¾S

BB4X « X L 23£ B_3?IX oiix CS3X mm& Q!IS

<¾mx mmm QI4ll: 11^~*& n ^m→A

mmm 3 &*K*~*A m~ → *m→A *-\c→r

i G→f

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∞ T im& i iM i!#« u ?mun

V520F mmm

o A559 U0S5P

RS47P 1560T RKWSC

IM7≠ L107?p ^¾

I587de! 29. The method according to embodiment 27, wherein the patient with a F508del/gating genotype has a gating mutation selected from G178R, S549N, S549R, G551D, G551S, G1244E, S 1251N, S 1255P, and G1349D.

30. The method according to embodiment 27, wherein the patient with a F508dell residual function genotype has a residual function mutation selected from 2789+5G- A, 3849+lOkbC^T, 3272-26A^ G, 711+3A^ G, E56K, P67L, R74W, D110E, D110H, R117C, L206W, R347H, R352Q, A455E, D579G, E831X, S945L, S977F, F1052V, R1070W, F1074L, D1152H, D1270N, E193K, K1060T, R117H, S 1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, A1067T, E193K, and K1060T.

31. The method according to any one of embodiments 1-30, wherein the absolute change in said patient' s percent predicted forced expiratory volume in one second (ppFEVi) after 15 days of administration of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 40% relative to the ppFEVl of the patient prior to said administration.

32. The method according to embodiment 31, wherein said patient has one F508del mutation and one minimal function mutation, and wherein patient has not taken any of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof.

33. The method according to embodiment 31, wherein said patient has two copies of F508del mutation, and wherein patient has taken at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, but not any of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof.

34. The method according to any one of embodiments 31-33, wherein said absolute change in said patient's ppFEVi ranges from 3% to 35%. 35. The method according to any one of embodiments 1-34, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is comprised in a second pharmaceutical composition; and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is comprised in a third pharmaceutical composition.

36. The method according to any one of embodiments 1-34, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; and said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in a second pharmaceutical composition.

37. The method of embodiment 36, wherein said second pharmaceutical composition comprises 1 half of a daily dose of said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, and the other half of said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered to said patient in a third pharmaceutical composition.

38. The method according to any one of embodiments 1-34, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is comprised in a second pharmaceutical composition; and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in the first pharmaceutical composition.

39. The method according to any one of embodiments 1-34, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof; said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof; and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in a first pharmaceutical composition. 40. The method according to embodiment 39, wherein the first pharmaceutical composition is administered to the patient twice daily.

41. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 100 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof twice daily:

(B) 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof once daily or 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof twice daily:

; and

(C) 150 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof twice dail :

42. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 200 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof twice daily:

(B) 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof once daily or 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof twice daily:

; and

(C) 150 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof twice dail :

43. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 300 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof twice daily:

(B) 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof once daily or 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof twice daily:

; and

(C) 150 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof twice daily:

44. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 100 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof twice daily:

(B) 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof once daily or 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof twice daily:

(C) 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof twice dail :

45. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 200 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof twice daily:

(B) 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof once daily or 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof twice daily:

; and

(C) 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof twice daily:

46. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 300 mg of at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof twice daily:

(B) 100 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof once daily or 50 mg of at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof twice daily:

; and

(C) 300 mg of at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof twice dail :

47. The method according to any one of embodiments 40-45, wherein said patient has cystic fibrosis is chosen from patients with F508del/mimmal function genotypes, patients with F508del/F508del genotypes, patients with F508dell gating genotypes, patients with F508de //residual function genotypes, and patients with F508dell another CFTR genetic mutation that is expected to be and/or is responsive to the triple combination of Compound I, Compound II, and/or Compound III genotypes based on in vitro and/or clinical data. 48. The method according to embodiment 46, wherein the patient with a F508del/uammal function genotype has a minimal function mutation selected from: X CM EI

m K lSS :

<$ X

W57 sum

I^S 1IMX LI25 X

StS

Q?IX w Y9 ⁾ %Z ins

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SifSX ειτπχ

liK w m.

$ . MISS

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£?1?.1C→A

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MM

3ΜΜΪΤ

ZIMMA §4¾

mm& 3§»fe

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S«K

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mm

mm

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AMS . 49. The method according to embodiment 47, wherein the patient with a F508del/gating genotype has a gating mutation selected from G178R, S549N, S549R, G551D, G551S, G1244E, S 1251N, S 1255P, and G1349D.

50. The method according to embodiment 47, wherein the patient with a F508dell residual function genotype has a residual function mutation selected from 2789+5G- A, 3849+lOkbC^T, 3272-26A^ G, 711+3A^ G, E56K, P67L, R74W, Dl lOE, Dl lOH, R117C, L206W, R347H, R352Q, A455E, D579G, E831X, S945L, S977F, F1052V, R1070W, F1074L, D1152H, D1270N, E193K, K1060T, R117H, S 1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, A1067T, E193K, and K1060T.

51. The method according to any one of embodiments 41-50, wherein the absolute change in said patient' s percent predicted forced expiratory volume in one second (ppFEVi) after 15 days of administration of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 40% relative to the ppFEVl of the patient prior to said administration.

52. The method according to embodiment 51, wherein said patient has one F508del mutation and one minimal function mutation, and wherein patient has not taken any of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof.

53. The method according to embodiment 51, wherein said patient has two copies of F508del mutation, and wherein patient has taken at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, but not any of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof.

54. The method according to any one of embodiments 51-53, wherein said absolute change in said patient's ppFEVi ranges from 3% to 35%. 55. The method according to any one of embodiments 41-54, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is comprised in a second pharmaceutical composition; and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is comprised in a third pharmaceutical composition.

56. The method according to any one of embodiments 41-54, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; and said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in a second pharmaceutical composition.

57. The method of embodiment 56, wherein said second pharmaceutical composition comprises a half of a daily dose of said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof, and the other half of said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is administered to said patient in a third pharmaceutical composition.

58. The method according to any one of embodiments 41-54, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof is comprised in a first pharmaceutical composition; said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is comprised in a second pharmaceutical composition; and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof is comprised in the first pharmaceutical composition.

59. The method according to any one of embodiments 41-54, wherein said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof; said at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof; and said at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof are comprised in a first pharmaceutical composition. 60. The method according to embodiment 58, wherein the first pharmaceutical composition is administered to the patient twice daily.

61. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 100 mg of Compound I twice daily:

(B) 100 mg of Compound II once daily or 50 mg of Compound II twice daily:

62. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 200 mg of Compound I twice daily:

(B) 100 mg of Compound II once daily or 50 mg of Compound II twice daily:

63. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 300 mg of Compound I twice daily:

(B) 100 mg of Compound II once daily or 50 mg of Compound II twice daily:

64. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 100 mg of Compound I twice daily:

(B) 100 mg of Compound II once daily or 50 mg of Compound II twice daily:

65. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 200 mg of Compound I twice daily:

(B) 100 mg of Compound II once daily or 50 mg of Compound II twice daily:

66. A method of treating cystic fibrosis comprising administering to a patient in need thereof:

(A) 300 mg of Compound I twice daily:

(B) 100 mg of Compound II once daily or 50 mg of Compound II twice daily:

(C) 300 m of Compound III twice daily:

67. The method according to any one of embodiments 61-66, wherein said patient has cystic fibrosis is chosen from patients with F508del/mimmal function genotypes, patients with F508del/F508del genotypes, patients with F508dell gating genotypes, and patients with F508de //residual function genotypes.

68. The method according to embodiment 67, wherein the patient with a

F508del/mimmal function genotype has a minimal function mutation selected from:

GS4:2X Ell§ £

S EMSH

14®

itSSS T14X SUM

S4¾X

Sii S12SSH q x S4l∑ IJ2 oix

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QS2SX mm . llll*! →€:

m?m

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llMmiK- miMC mtmt

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ms

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mm nam

mm

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AMI! rnmrn 69. The method according to embodiment 67, wherein the patient with a F508del/gating genotype has a gating mutation selected from G178R, S549N, S549R, G551D, G551S, G1244E, S 1251N, S 1255P, and G1349D.

70. The method according to embodiment 67, wherein the patient with a F508dell residual function genotype has a residual function mutation selected from 2789+5G- A, 3849+lOkbC^T, 3272-26A^ G, 711+3A^ G, E56K, P67L, R74W, D110E, D110H, R117C, L206W, R347H, R352Q, A455E, D579G, E831X, S945L, S977F, F1052V, R1070W, F1074L, D1152H, D1270N, E193K, K1060T, R117H, S 1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, A1067T, E193K, and K1060T.

71. The method according to any one of embodiments 61-70, wherein the absolute change in said patient' s percent predicted forced expiratory volume in one second

(ppFEVi) after 15 days of administration of said Compound I, Compound II, and

Compound III ranges from 3% to 40% relative to the ppFEVi of the patient prior to said administration.

72. The method according to embodiment 71, wherein said patient has one F508del mutation and one minimal function mutation, and wherein patient has not taken any of said Compound I, Compound II, and Compound III.

73. The method according to embodiment 71, wherein said patient has two copies of F508del mutation, and wherein patient has taken Compound II and Compound III, but not said Compound I.

74. The method according to any one of embodiments 61-73, wherein said absolute change in said patient's ppFEVi ranges from 3% to 35%.

75. The method according to any one of embodiments 61-73, wherein Compound I is comprised in a first pharmaceutical composition; Compound II is comprised in a second pharmaceutical composition; and Compound III is comprised in a third pharmaceutical composition.

76. The method according to any one of embodiments 61-73, wherein Compound I is comprised in a first pharmaceutical composition; and Compound II and Compound III are comprised in a second pharmaceutical composition. 77. The method of embodiment 76, wherein said second pharmaceutical composition comprises one half of the daily dose of Compound III, and the other half of the daily dose of Compound III is administered to said patient in a third pharmaceutical composition.

78. The method according to any one of embodiments 61-73, wherein Compound I is comprised in a first pharmaceutical composition; Compound II is comprised in a second pharmaceutical composition; and Compound III is comprised in the first pharmaceutical composition.

79. The method according to any one of embodiments 61-73, wherein said Compound I, Compound II, and Compound III are comprised in a first pharmaceutical composition.

80. The method according to embodiment 79, wherein the first pharmaceutical composition is administered to the patient twice daily.

81. The method according to any one of embodiments 1-30 and 31, wherein the absolute change in said patient's ppFEVi after 29 days of administration of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 40% relative to the ppFEVl of the patient prior to said administration.

82. The method according to any one of embodiments 31-33 and 81, wherein said absolute change in said patient's ppFEVi ranges from 3% to 35%.

83. The method according to any one of embodiment 41-50 and 51, wherein the absolute change in said patient's percent predicted forced expiratory volume in one second (ppFEVi) after 15 days of administration of said at least one compound chosen from Compound I and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound III and pharmaceutically acceptable salts thereof ranges from 3% to 40% relative to the ppFEVl of the patient prior to said administration.

84. The method according to any one of embodiments 51-53 and 83, wherein said absolute change in said patient's ppFEVi ranges from 3% to 35%.

85. The method according to any one of embodiments 61-70 and 71, wherein the absolute change in said patient's percent predicted forced expiratory volume in one second (ppFEVi) after 15 days of administration of said Compound I, Compound II, and

Compound III ranges from 3% to 40% relative to the ppFEVi of the patient prior to said administration.

86. The method according to any one of embodiments 61-73 and 85, wherein said absolute change in said patient's ppFEVi ranges from 3% to 35%.

87. The method according to any of the foregoing embodiments, wherein Compound III is replaced by Compound ΙΙΓ .

88. The method according to embodiment 87, wherein the daily dose of Compound IIP is 150 mg or 200 mg.

EXAMPLES

I. Methods of Preparing Compounds

[00131] General Experimental Procedures

[00132] Reagents and starting materials were obtained by commercial sources unless otherwise stated and were used without purification. Proton and carbon NMR spectra were acquired on either of a Bruker Biospin DRX 400 MHz FTNMR spectrometer operating at a ^l H and ¹³ C resonant frequency of 400 and 100 MHz respectively, or on a 300 MHz NMR spectrometer. One dimensional proton and carbon spectra were acquired using a broadband observe (BBFO) probe with 20 Hz sample rotation at 0.1834 and 0.9083 Hz/Pt digital resolution respectively. Proton and carbon spectra were either acquired with temperature control at 30°C or ambient temperature using standard, previously published pulse sequences and routine processing parameters. Final purity of compounds was determined by reversed phase UPLC using an Acquity UPLC BEH Cis column (50 x 2.1 mm, 1.7 μιη particle) made by Waters (pn: 186002350), and a dual gradient run from 1-99% mobile phase B over 3.0 minutes. Mobile phase A = H ₂ 0 (0.05 % CF ₃ C0 ₂ H). Mobile phase B = CH ₃ CN (0.035 % CF ₃ C0 ₂ H). Flow rate = 1.2 niL/min, injection volume = 1.5 μί, and column temperature = 60 °C. Final purity was calculated by averaging the area under the curve (AUC) of two UV traces (220 nm, 254 nm). Low- resolution mass spectra were obtained using a single quadrupole mass spectrometer with a mass accuracy of 0.1 Da and a minimum resolution of 1000 amu across the detection range using electrospray ionization (ESI) using the hydrogen ion (H ⁺ ). [00133] Compounds I, II and III can be prepared by any suitable method in the art, for example, PCT Publication Nos. WO 2011/133751 and WO 2015/160787.

Example 1. Synthesis of Compound I: N-[(6-amino-2-pyridyl)sulfonyl]-6-(3- fluoro-5-isobutoxy-phenyl)-2-[(45)-2,2,4-trimethylpyrrolidin -l- yl]pyridine-3-carboxamide

Step 1: tert-butyl 2-chloro-6-(3-fluoro-5-isobutoxy-phenyl)pyridine-3-carboxyla te

[00134] ie/t-Butyl 2,6-dichloropyridine-3-carboxylate (15.0 g, 60.5 mmol) and (3- fluoro-5-isobutoxy-phenyl)boronic acid (13.46 g, 63.48 mmol) were combined and fully dissolved in ethanol (150 mL) and toluene (150 mL). A suspsension of sodium carbonate (19.23 g, 181.4 mmol) in water (30 mL) was added.

Tetrakis(triphenylphosphine)palladium (0) (2.096 g, 1.814 mmol) was added under nitrogen. The reaction mixture was allowed to stir at 60 °C for 16 hours. Volatiles were removed under reduced pressure. The remaining solids were partitioned between water (100 mL) and ethyl acetate (100 mL). The organic layer was washed with brine (lx 100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The material was subjected silica gel column chromatography on a 330 gram silica gel column, 0 to 20% ethyl acetate in hexanes gradient. The material was repurified on a 220 gram silica gel column, isocratic 100% hexane for 10 minutes, then a 0 to 5% ethyl acetate in hexanes gradient to yield ieri-butyl 2-chloro-6-(3-fluoro-5-isobutoxy-phenyl)pyridine-3- carboxylate (18.87 g, 49.68 mmol, 82.2%) as a colorless oil. ^X H NMR (400 MHz, DMSO- de) δ 8.24 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 9.4, 2.0 Hz, 2H), 6.99 (dt, = 10.8, 2.2 Hz, 1H), 3.86 (d, = 6.5 Hz, 2H), 2.05 (dt, = 13.3, 6.6 Hz, 1H), 1.57 (d, J = 9.3 Hz, 9H), 1.00 (t, J = 5.5 Hz, 6H). ESI-MS m/z calc. 379.13504, found 380.2 (M+l) ⁺ ; Retention time: 2.57 minutes.

Step 2: 2-chloro-6-(3-fluoro-5-isobutoxy-phenyl)pyridine-3-carboxyli c acid

[00135] ie/t-Butyl 2-chloro-6-(3-fluoro-5-isobutoxy-phenyl)pyridine-3-carboxyla te (18.57 g, 48.89 mmol) was dissolved in dichloromethane (200 mL). Trifluoroacetic acid (60 mL, 780 mmol) was added and the reaction mixture was allowed to stir at room temperature for 1 hour. The reaction mixture was stirred at 40 °C for 2 hours. The reaction mixture was concentrated under reduced pressure and taken up in ethyl acetate (100 mL). It was washed with a saturated aqueous sodium bicarbonate solution (lx 100 mL) and brine (lx 100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was suspended in ethyl acetate (75 mL) and washed with aqueous HC1 (I N, lx 75 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The remaining solid (17.7 g) was stirred as a slurry in dichloromethane (35 mL) at 40 °C for 30 minutes. After cooling to room temperature, the remaining slurry was filtered, and then rinsed with cold dichloromethane to give 2-chloro-6-(3-fluoro-5-isobutoxy-phenyl)pyridine-3-carboxyli c acid (11.35 g, 35.06 mmol, 72%) as a white solid. ^l H NMR (400 MHz, DMSO-d ₆ ) δ 13.76 (s, 1H), 8.31 (d, = 8.0 Hz, 1H), 8.17 (d, = 8.1 Hz, 1H), 7.54 - 7.47 (m, 2H), 7.00 (dt, = 10.8, 2.3 Hz, 1H), 3.87 (d, J = 6.5 Hz, 2H), 2.05 (dt, 7 = 13.3, 6.6 Hz, 1H), 1.01 (d, J = 6.7 Hz, 6H). ESI-MS m/z calc. 323.1, found 324.1 (M+l) ⁺ ; Retention time: 1.96 minutes.

Step 3: N-[(6-amino-2-pyridyl)sulfonyl]-2-chloro-6-(3-fluoro-5-isobu toxy- phenyl)pyridine-3-carboxamide

[00136] 2-Chloro-6-(3-fluoro-5-isobutoxy-phenyl)pyridine-3-carboxyli c acid (3.00 g, 9.27 mmol) was dissolved in N,N-dimethylformamide (30.00 mL), and 1, 1 '- carbonyldiimidazole (2.254 g, 13.90 mmol) was added to the solution. The solution was allowed to stir at 65 °C for 1 hour. In a separate flask, sodium hydride (444.8 mg, 11.12 mmol) was added to a solution of 6-aminopyridine-2- sulfonamide (1.926 g, 11.12 mmol) in N,N-dimethylformamide (15.00 mL). This mixture was stirred for one hour before being added to the prior reaction mixture. The final reaction mixture was stirred at 65 °C for 15 minutes. Volatiles were removed under reduced pressure. The remaining oil was taken up in ethyl acetate and washed with aqueous HC1 (1 N, lx 75 mL) and brine (3x 75 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The remaining white solid (4.7 g) was fully dissolved in isopropanol (120 mL) in an 85 °C water bath. The colorless solution was allowed to slowly cool to room temperature with slow stirring over 16 hours. The crystalline solids that had formed were collected by vacuum filtration, and then rinsed with cold isopropanol (50 mL). Upon drying, N-[(6-amino-2-pyridyl)sulfonyl]-2-chloro-6-(3-fluoro-5-isobu toxy- phenyl)pyridine-3-carboxamide (3.24 g, 6.765 mmol, 73%) was obtained as a white solid. ^l H NMR (400 MHz, DMSO-d ₆ ) S 12.78 (s, 1H), 8.15 (d, = 8.0 Hz, 1H), 8.09 (d, = 7.9 Hz, 1H), 7.73 - 7.63 (m, 1H), 7.49 (dd, = 8.6, 1.9 Hz, 2H), 7.21 (d, = 7.3 Hz, 1H), 6.99 (dt, = 10.7, 2.2 Hz, 1H), 6.74 (d, = 8.4 Hz, 1H), 6.64 (s, 2H), 3.86 (d, = 6.5 Hz, 2H), 2.05 (dp, = 13.3, 6.5 Hz, 1H), 1.02 (dd, = 12.7, 6.4 Hz, 6H).

Step 4: N-[(6-amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy-phen yl)-2-[(45)- 2,2,4-trimethylpyrrolidin-l-yl]pyridine-3-carboxamide (Compound I) and N-[(6- amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy-phenyl)-2- [(4/f)-2,2,4- trimethylpyrrolidin-l-yl]pyridine-3-carboxamide

[00137] N-[(6-Amino-2-pyridyl)sulfonyl]-2-chloro-6-(3-fluoro-5-isobu toxy- phenyl)pyridine-3-carboxamide (309 mg, 0.645 mmol) was dissolved in dimethylsulfoxide (3.708 mL) and potassium carbonate (445.9 mg, 3.226 mmol) was slowly added, followed by 2,2,4-trimethylpyrrolidine (146.0 mg, 1.290 mmol). The reaction mixture was sealed and heated at 150 °C for 72 hours. The reaction was cooled down, diluted with water (50 mL), extracted 3 times with 50 mL portions of ethyl acetate, washed with brine, dried over sodium sulfate, filtered and evaporated to dryness. The crude material was dissolved in 2 mL of dichloromethane and purified by on silica gel using a gradient of 0 to 80% ethyl acetate in hexanes. The stereoisomers were separated using supercritical fluid

chromatography on a ChiralPak AD-H (250 x 4.6 mm), 5μιη column using 25%

isopropanol with 1.0% diethylamine in C0 ₂ at a flow rate of 3.0 niL/min. The separated enationmers were separately concentrated, diluted with ethyl acetate (3 mL) and washed with IN aqueous hydrochloric acid. The organic layers were dried over sodium sulfate, filtered, and evaporated to dryness to give the pure compounds as pale yellow solids.

[00138] The first compound to elute from the SFC conditions given above gave N-[(6- amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy-phenyl)-2- [(4 ?)-2,2,4- trimethylpyrrolidin-l-yl]pyridine-3-carboxamide (Hydrochloric Acid) ^l H NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H), 7.78 (d, = 8.0 Hz, 1H), 7.69 - 7.57 (m, 1H), 7.56 - 7.46 (m, 1H), 7.41 (dt, = 10.1, 1.8 Hz, 1H), 7.26 (d, = 8.0 Hz, 1H), 7.21 (d, = 7.2 Hz, 1H), 6.89 (dt, J = 10.7, 2.3 Hz, 1H), 6.69 (d, J = 8.3 Hz, 1H), 3.83 (d, J = 6.7 Hz, 2H), 2.61 (dq, J = 9.7, 4.9 Hz, 2H), 2.24 (d, J = 15.8 Hz, 1H), 2.06 (dq, J = 13.3, 6.7 Hz, 1H), 1.93 - 1.82 (m, 1H), 1.61 (s, 3H), 1.59 (s, 3H), 1.48 - 1.33 (m, 1H), 1.32 - 1.20 (m, 2H), 0.99 (d, J = 6.6 Hz, 6H), 0.88 (d, J = 6.2 Hz, 3H). ESI-MS m/z calc. 555.2, found 556.4 (M+l) ⁺ ; Retention time: 2.76 minutes.

[00139] The second compound to elute from the SFC conditions described above gave N-[(6-amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy-phen yl)-2-[(4S)-2,2,4- trimethylpyrrolidin-l-yl]pyridine-3-carboxamide (Compound I) (Hydrochloric Acid (1)) ^l H NMR (400 MHz, Chloroform-d) δ 15.49 (s, 1H), 8.49 (d, J = 8.2 Hz, 1H), 7.75 - 7.56 (m, 3H), 7.34 (t, J = 1.8 Hz, 1H), 7.30 (dt, J = 9.4, 1.9 Hz, 1H), 6.75 - 6.66 (m, 2H), 3.95 (s, 1H), 3.78 (d, J = 6.5 Hz, 2H), 3.42 (s, 1H), 2.88 - 2.74 (m, 1H), 2.23 (dd, J = 12.5, 8.0 Hz, 1H), 2.17 - 2.08 (m, 1H), 1.98 - 1.87 (m, 1H), 1.55 (s, 3H), 1.39 (s, 3H), 1.31 (d, J = 6.7 Hz, 3H), 1.05 (d, J = 6.7 Hz, 6H). ESI-MS m/z calc. 555.2, found 556.4 (M+l) ⁺ ;

Retention time: 2.77 minutes. Absolute stereochemistry was confirmed by X-ray crystallography. Example 2. Synthesis of Compound II: (R)-l-(2,2-

Difluorobenzo[d][l,3]dioxol-5-yl)-N-(l-(2,3-dihydroxyprop yl)-6 fluoro-2-(l-hydroxy-2-methylpropan-2-yl)-lH-indol-5- yl)cyclopropanecarboxamide

Step A: (R)-Benzyl 2-(l-((2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-6-fluoro-5-nit ro- lH-indol-2-yl)-2-methylpropanoate and ((S)-2,2-Dimethyl-l,3-dioxolan-4-yl)methyl 2-(l-(((R)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-6-fluoro-5 -nitro-lH-indol-2-yl)-2- methylpropanoate

[00140] Cesium carbonate (8.23 g, 25.3 mmol) was added to a mixture of benzyl 2-(6- fluoro-5-nitro-lH-indol-2-yl)-2-methylpropanoate (3.0 g, 8.4 mmol) and (S)-(2,2- dimethyl-l,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (7.23 g, 25.3 mmol) in DMF (17 mL). The reaction was stirred at 80 °C for 46 hours under a nitrogen

atmosphere. The mixture was then partitioned between ethyl acetate and water. The aqueous layer was extracted with ethyl acetate. The combined ethyl acetate layers were washed with brine, dried over MgS0 ₄ , filtered and concentrated. The crude product, a viscous brown oil which contains both of the products shown above, was taken directly to the next step without further purification. (R)-Benzyl 2-(l-((2,2-dimethyl-l,3-dioxolan-4- yl)methyl)-6-fluoro-5-nitro-lH-indol-2-yl)-2-methylpropanoat e, ESI-MS m/z calc. 470.2, found 471.5 (M+l) ⁺ . Retention time 2.20 minutes. ((S)-2,2-Dimethyl-l,3-dioxolan-4- yl)methyl 2-(l-(((R)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-6-fluoro-5 -nitro-lH-indol-2- yl)-2-methylpropanoate, ESI-MS m/z calc. 494.5, found 495.7 (M+l) ⁺ . Retention time 2.01 minutes.

Step B: (R)-2-(l-((2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-6-fluoro-5 -nitro-lH-indol- 2-yl) -2-methylpropan- 1 -ol [00141] The crude reaction mixture obtained in step (A) was dissolved in THF (42 mL) and cooled in an ice-water bath. LiAlH ₄ (16.8 mL of 1 M solution, 16.8 mmol) was added drop-wise. After the addition was complete, the mixture was stirred for an additional 5 minutes. The reaction was quenched by adding water (1 mL), 15% NaOH solution (1 mL) and then water (3 mL). The mixture was filtered over Celite, and the solids were washed with THF and ethyl acetate. The filtrate was concentrated and purified by column chromatography (30-60% ethyl acetate- hexanes) to obtain (R)-2-(l-((2,2-dimethyl-l,3- dioxolan-4-yl)methyl)-6-fluoro-5-nitro-lH-indol-2-yl)-2-meth ylpropan-l-ol as a brown oil (2.68g, 87 % over 2 steps). ESI-MS m/z calc. 366.4, found 367.3 (M+l) ⁺ . Retention time 1.68 minutes. ^l H NMR (400 MHz, DMSO- 6) δ 8.34 (d, J = 7.6 Hz, 1H), 7.65 (d, J = 13.4 Hz, 1H), 6.57 (s, 1H), 4.94 (t, J = 5.4 Hz, 1H), 4.64 - 4.60 (m, 1H), 4.52 - 4.42(m, 2H), 4.16 - 4.14 (m, 1H), 3.76 - 3.74 (m, 1H), 3.63 - 3.53 (m, 2H), 1.42 (s, 3H), 1.38 - 1.36 (m, 6H) and 1.19 (s, 3H) ppm

Step C: (R)-2-(5-amino-l-((2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-6- fluoro-lH-indol- 2-yl) -2-methylpropan- 1 -ol

[00142] (R)-2-(l-((2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-6-fluoro-5 -nitro-lH-indol-2- yl)-2-methylpropan-l-ol (2.5 g, 6.82 mmol) was dissolved in ethanol (70 mL) and the reaction was flushed with N ₂ . Then Pd-C (250 mg, 5% wt) was added. The reaction was flushed with nitrogen again and then stirred under H ₂ (atm). After 2.5 hours only partial conversion to the product was observed by LCMS. The reaction was filtered through Celite and concentrated. The residue was re-subjected to the conditions above. After 2 hours LCMS indicated complete conversion to product. The reaction mixture was filtered through Celite. The filtrate was concentrated to yield the product as a black solid (1.82 g, 79 %). ESI-MS m/z calc. 336.2, found 337.5 (M+l) ⁺ . Retention time 0.86 minutes. ^l U NMR (400 MHz, DMSO- 6) δ 7.17 (d, J = 12.6 Hz, 1H), 6.76 (d, J = 9.0 Hz, 1H), 6.03 (s, 1H), 4.79 - 4.76 (m, 1H), 4.46 (s, 2H), 4.37 - 4.31 (m, 3H),4.06 (dd, J = 6.1, 8.3 Hz, 1H), 3.70 - 3.67 (m, 1H), 3.55 - 3.52 (m, 2H), 1.41 (s, 3H), 1.32 (s, 6H) and 1.21 (s, 3H) ppm. Step D: (R)-l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-N-(l-((2,2-dime thyl-l,3-dioxolan- 4-yl)methyl)-6-fluoro-2-(l-hydroxy-2-methylpropan-2-yl)-lH-i ndol-5- yl)cyclopropanecarboxamide

[00143] DMF (3 drops) was added to a stirring mixture of l-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxylic acid (1.87 g, 7.7 mmol) and thionyl chloride (1.30 mL, 17.9 mmol). After 1 hour a clear solution had formed. The solution was concentrated under vacuum and then toluene (3 mL) was added and the mixture was concentrated again. The toluene step was repeated once more and the residue was placed on high vacuum for 10 minutes. The acid chloride was then dissolved in dichloromethane (10 mL) and added to a mixture of (R)-2-(5-amino-l-((2,2-dimethyl- l,3- dioxolan-4-yl)methyl)-6-fluoro- lH-indol-2-yl)-2-methylpropan- l-ol (1.8 g, 5.4 mmol) and triethylamine (2.24 mL, 16.1 mmol) in dichloromethane (45 mL). The reaction was stirred at room temperature for 1 hour. The reaction was washed with IN HC1 solution, saturated NaHC0 ₃ solution and brine, dried over MgS0 ₄ and concentrated to yield the product as a black foamy solid (3g, 100%). ESI-MS m/z calc. 560.6, found 561.7 (M+l) ⁺ . Retention time 2.05 minutes. ^l H NMR (400 MHz, DMSO- 6) δ 8.31 (s, 1H), 7.53 (s, 1H), 7.42 - 7.40 (m, 2H), 7.34 - 7.30 (m, 3H), 6.24 (s, 1H), 4.51 - 4.48 (m, 1H), 4.39 - 4.34 (m,2H), 4.08 (dd, J = 6.0, 8.3 Hz, 1H), 3.69 (t, J = 7.6 Hz, 1H), 3.58 - 3.51 (m, 2H), 1.48 - 1.45 (m, 2H), 1.39 (s, 3H), 1.34 - 1.33 (m, 6H), 1.18 (s, 3H) and 1.14 - 1.12 (m, 2H) ppm

Step E: (R)-l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-N-(l-(2,3-dihyd roxypropyl)-6- fluoro-2-(l-hydroxy-2-methylpropan-2-yl)-lH-indol-5-yl)cyclo propanecarboxamide

[00144] (R)- 1 -(2,2-difluorobenzo [d] [ 1 ,3]dioxol-5-yl)-N-( l-((2,2-dimethyl- 1 ,3-dioxolan- 4-yl)methyl)-6-fluoro-2-(l-hydroxy-2-methylpropan-2-yl)- lH-indol-5- yl)cyclopropanecarboxamide (3.0 g, 5.4 mmol) was dissolved in methanol (52 mL). Water (5.2 mL) was added followed by p-TsOH.H ₂ 0 (204 mg, 1.1 mmol). The reaction was heated at 80 °C for 45 minutes. The solution was concentrated and then partitioned between ethyl acetate and saturated NaHC0 ₃ solution. The ethyl acetate layer was dried over MgS0 ₄ and concentrated. The residue was purified by column chromatography (50- 100 % ethyl acetate - hexanes) to yield the product as a cream colored foamy solid. (1.3 g, 47 %, ee >98% by SFC). ESI-MS m/z calc. 520.5, found 521.7 (M+l) ⁺ . Retention time 1.69 minutes. ^l H NMR (400 MHz, DMSO- 6) δ 8.31 (s, 1H), 7.53 (s, 1H), 7.42 - 7.38 (m, 2H), 7.33 - 7.30 (m, 2H), 6.22 (s, 1H), 5.01 (d, J = 5.2 Hz, 1H), 4.90 (t, J = 5.5 Hz, 1H), 4.75 (t, J = 5.8 Hz, 1H), 4.40 (dd, J = 2.6, 15.1 Hz, 1H), 4.10 (dd, J = 8.7, 15.1 Hz, 1H), 3.90 (s, 1H), 3.65 - 3.54 (m, 2H), 3.48 - 3.33 (m, 2H), 1.48 - 1.45 (m, 2H), 1.35 (s, 3H), 1.32 (s, 3H) and 1.14 - 1.11 (m, 2H) ppm.

Example 3. Synthesis of Compound III: N-(2,4-di-tert-butyl-5- hydroxyphenyl)-4-oxo-l,4-dihydroquinoline-3-carboxamide

Part A: Preparation of 4-oxo-l,4-dihydroquinoline-3-carboxylic acid

Step A: 2-Phenylaminomethylene-malonic acid diethyl ester

[00145] A mixture of aniline (25.6 g, 0.275 mol) and diethyl 2- (ethoxymethylene)malonate (62.4 g, 0.288 mol) was heated at 140-150 °C for 2 h. The mixture was cooled to room temperature and dried under reduced pressure to afford 2- phenylaminomethylene-malonic acid diethyl ester as a solid, which was used in the next step without further purification. ^X H NMR (DMSO-ifc) δ 11.00 (d, 1H), 8.54 (d, = 13.6 Hz, 1H), 7.36-7.39 (m, 2H), 7.13-7.17 (m, 3H), 4.17-4.33 (m, 4H), 1.18-1.40 (m, 6H).

Step B: 4-Hydroxyquinoline-3-carboxylic acid ethyl ester

[00146] A I L three-necked flask fitted with a mechanical stirrer was charged with 2- phenylaminomethylene-malonic acid diethyl ester (26.3 g, 0.100 mol), polyphosphoric acid (270 g) and phosphoryl chloride (750 g). The mixture was heated to 70 °C and stirred for 4 h. The mixture was cooled to room temperature and filtered. The residue was treated with aqueous Na ₂ C03 solution, filtered, washed with water and dried. 4- Hydroxyquinoline-3-carboxylic acid ethyl ester was obtained as a pale brown solid (15.2 g, 70%). The crude product was used in next step without further purification.

Step C: 4-Oxo-l,4-dihydroquinoline-3-carboxylic acid

[00147] 4-Hydroxyquinoline-3-carboxylic acid ethyl ester (15 g, 69 mmol) was suspended in sodium hydroxide solution (2N, 150 mL) and stirred for 2 h at reflux. After cooling, the mixture was filtered, and the filtrate was acidified to pH 4 with 2N HC1. The resulting precipitate was collected via filtration, washed with water and dried under vacuum to give 4-oxo-l,4-dihydroquinoline-3-carboxylic acid as a pale white solid (10.5 g, 92 %). ^l U NMR (DMSO-4) δ 15.34 (s, 1 H), 13.42 (s, 1 H), 8.89 (s, 1H), 8.28 (d, = 8.0 Hz, 1H), 7.88 (m, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.60 (m, 1H).

Part B: N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-l,4-dihydroquino line-3- carboxamide

Step A: Carbonic acid 2,4-di-tert-butyl-phenyl ester methyl ester

[00148] Methyl chloroformate (58 mL, 750 mmol) was added dropwise to a solution of 2,4-di-ie/ -butyl-phenol (103.2 g, 500 mmol), Et ₃ N (139 mL, 1000 mmol) and DMAP (3.05 g, 25 mmol) in dichloromethane (400 mL) cooled in an ice-water bath to 0 °C. The mixture was allowed to warm to room temperature while stirring overnight, then filtered through silica gel (approx. 1L) using 10% ethyl acetate - hexanes (~ 4 L) as the eluent. The combined filtrates were concentrated to yield carbonic acid 2,4-di-ie/ -butyl-phenyl ester methyl ester as a yellow oil (132 g, quant.). ^l U NMR (400 MHz, DMSO- ₆ ) δ 7.35 (d, J = 2.4 Hz, 1H), 7.29 (dd, J = 8.5, 2.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 3.85 (s, 3H), 1.30 (s, 9H), 1.29 (s, 9H).

Step B: Carbonic acid 2,4-di-tert-butyl-5-nitro-phenyl ester methyl ester and

Carbonic acid 2,4-di-tert-butyl-6-nitro-phenyl ester methyl ester

[00149] To a stirring mixture of carbonic acid 2,4-di-ie/ -butyl-phenyl ester methyl ester (4.76 g, 180 mmol) in cone, sulfuric acid (2 mL), cooled in an ice-water bath, was added a cooled mixture of sulfuric acid (2 mL) and nitric acid (2 mL). The addition was done slowly so that the reaction temperature did not exceed 50 °C. The reaction was allowed to stir for 2 h while warming to room temperature. The reaction mixture was then added to ice-water and extracted into diethyl ether. The ether layer was dried (MgS0 ₄ ),

concentrated and purified by column chromatography (0 - 10% ethyl acetate - hexanes) to yield a mixture of carbonic acid 2,4-di-ieri-butyl-5-nitro-phenyl ester methyl ester and carbonic acid 2,4-di-ieri-butyl-6-nitro-phenyl ester methyl ester as a pale yellow solid (4.28 g), which was used directly in the next step.

Step C: 2,4-Di-tert-butyl-5-nitro-phenol and 2,4-Di-tert-butyl-6-nitro-phenol

[00150] The mixture of carbonic acid 2,4-di-ieri-butyl-5-nitro-phenyl ester methyl ester and carbonic acid 2,4-di-ieri-butyl-6-nitro-phenyl ester methyl ester (4.2 g, 14.0 mmol) was dissolved in MeOH (65 mL) before KOH (2.0 g, 36 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction mixture was then made acidic (pH 2-3) by adding cone. HCl and partitioned between water and diethyl ether. The ether layer was dried (MgS0 ₄ ), concentrated and purified by column chromatography (0 - 5 % ethyl acetate - hexanes) to provide 2,4-di-ieri-butyl-5-nitro-phenol (1.31 g, 29% over 2 steps) and 2,4-di-ieri-butyl-6-nitro-phenol. 2,4-Di-ieri-butyl-5-nitro-phenol: ^X H NMR (400 MHz, DMSO-ifc) δ 10.14 (s, 1H, OH), 7.34 (s, 1H), 6.83 (s, 1H), 1.36 (s, 9H), 1.30 (s, 9H). 2,4-Di-ieri-butyl-6-nitro-phenol: ^l H NMR (400 MHz, CDC1 ₃ ) δ 11.48 (s, 1H), 7.98 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 2.4 Hz, 1H), 1.47 (s, 9H), 1.34 (s, 9H).

Step D: 5-Amino-2,4-di-tert-butyl-phenol

[00151] To a refluxing solution of 2,4-di-ieri-butyl-5-nitro-phenol (1.86 g, 7.40 mmol) and ammonium formate (1.86 g) in ethanol (75 mL) was added Pd-5% wt. on activated carbon (900 mg). The reaction mixture was stirred at reflux for 2 h, cooled to room temperature and filtered through Celite. The Celite was washed with methanol and the combined filtrates were concentrated to yield 5-amino-2,4-di-ie/ -butyl-phenol as a grey solid (1.66 g, quant.). ^l H NMR (400 MHz, DMSO-d ₆ ) δ 8.64 (s, 1H, OH), 6.84 (s, 1H), 6.08 (s, 1H), 4.39 (s, 2H, NH ₂ ), 1.27 (m, 18H); HPLC ret. time 2.72 min, 10-99 %

CH3CN, 5 min run; ESI-MS 222.4 m/z [M+H] ⁺ .

Step E: N- 5-hydroxy-2,4-di-tert-butyl-phenyl)-4-oxo-lH-quinoline-3-car boxamide

[00152] To a suspension of 4-oxo-l,4-dihydroquinolin-3-carboxylic acid (35.5 g, 188 mmol) and HBTU (85.7 g, 226 mmol) in DMF (280 mL) was added Et ₃ N (63.0 mL, 451 mmol) at ambient temperature. The mixture became homogeneous and was allowed to stir for 10 min before 5-amino-2,4-di-ie/ -butyl-phenol (50.0 g, 226 mmol) was added in small portions. The mixture was allowed to stir overnight at ambient temperature. The mixture became heterogeneous over the course of the reaction. After all of the acid was consumed (LC-MS analysis, MH+ 190, 1.71 min), the solvent was removed in vacuo. EtOH was added to the orange solid material to produce a slurry. The mixture was stirred on a rotovap (bath temperature 65 °C) for 15 min without placing the system under vacuum. The mixture was filtered and the captured solid was washed with hexanes to provide a white solid that was the EtOH crystalate. Et ₂ 0 was added to the solid obtained above until a slurry was formed. The mixture was stirred on a rotovap (bath temperature 25 °C) for 15 min without placing the system under vacuum. The mixture was filtered and the solid captured. This procedure was performed a total of five times. The solid obtained after the fifth precipitation was placed under vacuum overnight to provide N-(5-hydroxy-2,4-di- ieri-butyl-phenyl)-4-oxo-lH-quinoline-3-carboxamide as a white powdery solid (38 g, 52%). HPLC ret. time 3.45 min, 10-99% CH ₃ CN, 5 min run; ^l H NMR (400 MHz, DMSO-ifc) δ 12.88 (s, 1H), 11.83 (s, 1H), 9.20 (s, 1H), 8.87 (s, 1H), 8.33 (dd, J = 8.2, 1.0 Hz, 1H), 7.83-7.79 (m, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.54-7.50 (m, 1H), 7.17 (s, 1H), 7.10 (s, 1H), 1.38 (s, 9H), 1.37 (s, 9H); ESI-MS m z calc'd 392.21; found 393.3 [M+H] ⁺ .

Example 4: Studies to Evaluate the Safety, Tolerability, and Bioavailability of Compound I

[00153] A randomized, double-blind, placebo-controlled, single- and multiple-dose, dose-escalation study was conduced in healty volunteer subjects. Subjects were randomized to receive Compound I or placebo (Part A and Part B), and triple combination of Compound I, Compound II, and Compound III, or triple placebo (Part C).

[00154] In summary, Compound I was well tolerated as single doses from 50 mg up to 2000 mg and as multiple doses up to 400 mg ql2h for 14 days and up to 300 mg ql2h in triple combination with Compound II (100 mg qd) and Compound III (150 mg ql2h) for 13 days. Dose-limiting adverse events were observed with multiple doses of 800 mg ql2h. All of the adverse events were mild or moderate. There were no deaths or serious or severe adverse events. Example 5: Study to Evaluate the Safety and Efficacy of Compound I in Combination Therapy

[00155] The safety of Compound I in triple combination with Compounds II and III is evaluated in CF subjects in a 2-part, randomized, double-blind, placebo- and Compound II/III-controlled, parallel-group, multicenter study. Parts 1 and 2 include a Screening Period, a 2-week Treatment Period, and a Safety Follow-up Visit. Part 2 also includes a 4- week Run-in Period before the Treatment Period and a 2- week Washout Period after the Treatment Period.

[00156] In previous studies, single doses of Compound I up to 2000 mg and multiple doses of Compound I up to 400 mg ql2h (ql2h means every twelve hours) were generally safe and well tolerated, except for the occurrence of treatment-emergent hemolysis in a subject who was found to have glucose-6-phosphate dehydrogenase (G6PD) deficiency, and possible occurrence of subclinical hemolysis in a second subject who was also found to have G6PD deficiency. Multiple doses of Compound I up to 300 mg ql2h in combination with Compound II (100 mg qd) (qd means once daily) and Compound III (150 mg ql2h) were generally safe and tolerated after 14 days of dosing.

Part i

[00157] In Part 1, three dose levels of Compound I (100, 200, and 300 mg ql2h) in triple combination with Compound II (100 mg qd) and Compound III (150 mg ql2h) is evaluated in subjects with the F508del/MF genotype.

[00158] Part 1 has three cohorts (Cohorts IA, IB, and IC). In Cohort 1A, the triple combination of Compound I at 100 mg ql2h, Compound Π at 100 mg qd, and Compound III at 150 mg ql2h is evaluated in subjects with the F508del/MF genotype. In Cohort IB, the triple combination of Compound I at 200 mg ql2h, Compound II at 100 mg qd, and Compound III at 150 mg ql2h is evaluated in subjects with the F508del/MF genotype. In Cohort 1C, the triple combination of Compound I at 300 mg ql2h, Compound II at 100 mg qd, and Compound III at 150 mg ql2h is evaluated in subjects with the F508del/MF genotype. Triple placebo is the comparator for all three cohorts

Part 2

[00159] In Part 2, two dose levels of Compound I (200 and 300 mg ql2h) in triple combination with Compound II (100 mg qd) and Compound III (150 mg ql2h) is evaluated in subjects with the F508del/F508del genotype. [00160] Part 2 has two cohorts (Cohorts 2A and 2B). In Cohort 2A, the triple combination of Compound I at 200 mg ql2h, Compound II at 100 mg qd, and Compound III at 150 mg ql2h is evaluated in subjects with the F508del/F508del genotype. In Cohort 2B, the triple combination of Compound I at 300 mg ql2h, Compound II at 100 mg qd, and Compound III at 150 mg ql2h is evaluated in subjects with the F508del/F508del genotype. The combination of placebo, Compound II, and Compound III is the comparator for both cohorts.

Table 8: Treatment Arms and Planned Doses for Parts 1 and 2

Cohort Treatment/ Compound I Compound II Compound III

Comparator Arms Dosage Dosage Dosage

1A Treatment 100 mg qd 100 mg qd 150 mg ql2h

Comparator Placebo Placebo Placebo

IB Treatment 200 mg qd 100 mg qd 150 mg ql2h

Comparator Placebo Placebo Placebo

1C Treatment 300 mg qd 100 mg qd 150 mg ql2h

Comparator Placebo Placebo Placebo

2A ^a Treatment 200 mg qd 100 mg qd 150 mg ql2h

Comparator Placebo 100 mg qd 150 mg ql2h

2B ^a Treatment 300 mg qd 100 mg qd 150 mg ql2h

Comparator Placebo 100 mg qd 150 mg ql2h

In Part 2, all subjects will also receive 100 mg qd of Compound II and Compound III 150 mg ql2h during (1) a 4 week Run-in Period prior to the 2 week Treatment Period and (2) a 4 week Washout Period following the 2 week Treatment Period.

[00161] Primary endpoints for the study include: safety and tolerability assessments based on adverse events (AEs), clinical laboratory values, standard 12-lead

electrocardiograms (ECGs), vital signs, and pulse oximetry. Secondary endpoints include: absolute change in sweat chloride concentrations from baseline at Day 15; absolute change in percent predicted forced expiratory volume in 1 second (ppFEVi) from baseline at Day 15; relative change in ppFEVi from baseline at Day 15; absolute change in Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain score from baseline at Day 15; and PK parameters of Compound I, Compound II, Compound III, and metabolites of

Compounds II and III.

Example 6: Phase 2 Study to Evaluate the Safety and Efficacy Study of

Compound I in Combination Therapy

[00162] In this Phase 2 randomized, double-blind study, Compound I (lOOmg, 200mg and 300mg ql2h) in combination with Compound II (lOOmg qd) and Compound III (150mg ql2h) in people with CF ages 18 and older who have one F508del mutation and one minimal function mutation and in people who have two copies of the F508del mutation was studied. Primary endpoints as described above in Example 6 were for safety and tolerability. Secondary endpoints included absolute change in ppFEVi and change in sweat chloride.

[00163] Safety Data: In Part 1 of the study, involving people who had one F508del mutation and one minimal function mutation (F/MF), the triple combination regimen was generally well tolerated. The majority of adverse events were mild or moderate. The most common adverse events (>10%), regardless of treatment group, were infective pulmonary exacerbation of cystic fibrosis, productive cough, diarrhea, cough, headache, sputum increased, and fatigue. There was one drug interruption due to an adverse event in the triple combination treatment group using 200mg of Compound I and one drug interruption due to an adverse event in the triple combination treatment group using 300mg of Compound I but none in the control group. An overview of treatment emergent adverse events (TEAEs) is provided in the following table:

Related TEAEs include related and possibly related

[00164] Safety Data: In Part 2 of the study, involving people who had two F508del mutations (F/F), the triple combination regimen was generally well tolerated. No serious or severe adverse events were reported. Two subjects discontinued treatment due to adverse events - one due to neumonia and one due to rash. One subject had a dose interruption due to increased blood bilirubin. An overview of treatment emergent adverse events (TEAEs) is provided in the following table:

" Pneumonia

b ash

c Increased bilirubin

[00165] 2-Week Efficacy Data in FSOSdel/ Minimal Function Patients (F/MF): In Part 1 of the study, the triple combination was evaluated for two weeks in 34 patients ages 18 and older who had one FSOSdel mutation and one minimal function mutation (8 in combined placebo, 6 in Compound I l OOmg, 10 in Corn pound 1 200mg, and 30 in Compound I 300mg). A summary of the within-group ppFEV (primary endpoint) and sweat chloride data (secondary endpoint) through Day 1 Sis provided below. 2 weeks of treatment with Compound I in triple combination with Compound II and Compound ΪΪΙ in subjects who had one FSOSdel mutation and one minimal function mutation resulted in statistically significant (1 -sided alpha = 5%) and clinically meaningful improvements in ppFEVi (5.7 - 9.7 percentage points), CFQ-R respiratory domain (18.6 - 21.8 points for 200 and 300 mg of Compound 1 arms), and sweat chloride (13.6 - 27.5 mmol/L), The treatment was safe and well tolerated with no safety findings of concern.

b 2 subjects had sweat chloride missing at Day 15

[00166] 2- Week Efficacy Data in F508del Homozygous Patients (F/F): In Part 2 of the study, the triple combination was evaluated for two weeks in 14 patients ages 18 and older who had two copies of the F508del mutation, who were already receiving the combination of Compound II and Compound III (4 weeks, 4 in placebo and 10 in Compound 1 200mg). A summary of the within-group lung function (ppFEV) (primary endpoint) and sweat chloride data (secondary endpoint) for the triple combination treatment period, from baseline (end of the 4-week Compound II/Compound III run-in period), through Day 15 is provided below.

[00167] 4- Week Efficacy Data in F508del Homozygous Patients (F/F): A summary of the within-group lung function (ppFEV) (primary endpoint) and sweat chloride data (secondary endpoint) from patients in Part 2 of the study who received the triple combination including 300 mg of Compound I for 4 weeks is provided below.

[00168] In summary, 2-4 weeks of Compound I in triple combination with

Compound II and Compound III in homozygous subjects for F508del in Part 2 study resulted in statistically significant and clinically meaningful improvements on top of Compound II and Compound III treatment in ppFEVi (6.5 - 7.3 percentage points) and sweat chloride (21.3 - 22.3 mmol/L). Treatment with Compound I in triple combination with Compound II and Compound III in homozygous subjects for F508del was generally safe and well tolerated; there were no serious AEs and all AEs were mild or moderate.

ssng ata om 2 su ects

Preclinical Toxicology Data

[00170] Preclinical reproductive toxicology studies of Compound I showed no adverse findings of note.

Other Embodiments

[00171] The foregoing discussion discloses and describes merely exemplary

embodiments of this disclosure. One skilled in the art will readily recognize from such discussion and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of this disclosure as defined in the following claims.