EA041854B1 - METHODS OF TREATMENT OF CYSIC FISSIDOSIS - Google Patents

Description

This application claims the priority of U.S. Provisional Application 62/517049 filed June 8, 2017, U.S. Provisional Application 62/533381 filed July 17, 2017, U.S. Provisional Application 62/562044 filed September 22, 2017, Provisional Application U.S. Patent Application 62/623,757, filed January 30, 2018, U.S. Provisional Application 62/633,021, filed February 20, 2018, U.S. Provisional Application 62/649,266, filed March 28, 2018, all of which are incorporated herein description by reference in full.

Disclosed herein is a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, pharmaceutical compositions containing the modulator, methods for treating cystic fibrosis, and a method for manufacturing the modulator.

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

In CF patients, mutations in CFTR endogenously expressed in the respiratory epithelium lead to a decrease in apical anion secretion, causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to increased mucus accumulation in the lungs and associated microbial infections, which ultimately cause death in CF patients. In addition to respiratory problems, patients with MB usually suffer from gastrointestinal problems and pancreatic insufficiency, which, if left untreated, lead to death. In addition, most men with cystic fibrosis are infertile and fertility is reduced among women with cystic fibrosis.

Sequence analysis of the CFTR gene has revealed various 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, more than 2,000 mutations in the CF gene have been identified; the CFTR2 database currently contains information on only 322 of these identified mutations and enough data to identify 281 mutations as causing disease. The most common 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 CF cases and is associated with severe disease.

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 move to the plasma membrane. As a result, the number of anion transport CFTR channels present in the membrane is much smaller than that observed in cells expressing wild-type CFTR, ie, CFTR that does not have mutations. In addition to reduced migration, the mutation results in defective membrane channel gating. Simultaneously, a reduced number of channels in the membrane and a defective gate mechanism lead to reduced transport of anions and fluids across the epithelium. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). The defective channels due to the F508del mutation are still functional, although less than in the case of wild-type CFTR channels. (Dalemans et al. (1991), Nature bond. 354: 526-528; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to F508del, another disease causing mutations in CFTR that results in impaired migration, synthesis, and/or channel gate mechanism could be regulated by activation or down-regulation to alter anionic secretion and modify disease progression and/or severity.

CFTR is a cAMP/ATP-mediated anion channel that is expressed in various cell types, including absorptive and secretory epithelial cells, where it regulates anionic flux across the membrane as well as the activity of other ion channels and proteins. In epithelial cells, normal CFTR function is critical to maintaining electrolyte transport throughout the body, including the tissue of the respiratory and digestive tracts. CFTR consists of approximately 1480 amino acids that code for a protein composed of a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. These two transmembrane domains are linked by a large polar regulatory (R) domain with multiple phosphorylation sites that regulate channel activity and cell migration.

Transport of chlorides is carried out due to the coordinated activity of ENaC and CFTR present in the apical membrane, and Na ⁺ -K ⁺ -ATPase pump and Cl-channels expressed on the basolateral surface of the cell. Secondary active transport of chlorides from the luminal region leads to the accumulation of intracellular chlorides, which can then be passively removed from the cell through Cl' channels, leading to vector transport. The location of Na ⁺ /2Cl'/K ⁺ -cotransporter, Na ⁺ -K ⁺ -ATPase pump and K ⁺ -channels on the basolateral surface and CFTR in the luminal region coordinates the secretion of chlorides due to CFTR in the luminal region. Because of water, it probably never actively transports itself, its flux through epithelial tissues depends on very small transepithelial osmotic gradients generated by a large sodium and chloride flux.

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Accordingly, there is a need for new methods of treating diseases mediated by CFTR.

This specification discloses Compound I and pharmaceutically acceptable salts thereof. Compound I can be depicted as having the following structure

The chemical name for compound I is K-(benzenesulfonyl)-6-[3-[2-[1(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2-[(4(S))-2,2 ,4-trimethylpirrolidin-1-yl]n-pyridine-3carboxamide.

This description also discloses pharmaceutical compositions containing compound I and/or at least one pharmaceutically acceptable salt thereof, and these compositions may further include at least one additional active pharmaceutical ingredient and/or at least one carrier. Also disclosed are methods for treating a CFTR-mediated disease of cystic fibrosis, comprising administering compound I and/or at least one pharmaceutically acceptable salt thereof, optionally as part of a pharmaceutical composition containing at least one additional component, to an object in need of such treatment. Also disclosed is a process for the preparation of compound I and/or pharmaceutically acceptable salts thereof.

Also disclosed are methods of treating a CFTR-mediated disease of cystic fibrosis, including the administration of H-(benzenesulfonyl)-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]nirazol-1-yl]-2-[(4(S ))2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (compound I), (R)-1-(2,2difluorobenzo[d] [1,3]]dioxol-5-yl)- X-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2yl)-H-indol-5-yl)cyclopropancarboxamide (compound II) and H-[2, 4-bis(1,1-dimethylethyl)-5hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide (compound III) or N-(2-(tert-butyl)-5hydroxy-4-(2- (methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (compound III-d), optionally as part of at least one pharmaceutical composition containing at least one additional component, to a patient in need of such treatment.

Brief description of the drawings

In FIG. 1 shows the XRPD of a spray dried dispersion of Compound I with HPMCAS-HG.

In FIG. 2 shows the modulated differential scanning calorimetry (MDSC) spectrum of a spray dried dispersion of Compound I with HPMCAS-HG.

In FIG. 3 is a typical list of CFTR genetic mutations.

Definitions.

As used herein, CFTR means a transmembrane conductance regulator in cystic fibrosis.

As used herein, mutations may refer to mutations in the CFTR gene or 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 change in nucleotides in a gene as a whole results in a mutation in the CFTR protein translated from that gene, or a frameshift(s).

The term F508del refers to a mutant CFTR protein that lacks the amino acid phenylalanine at position 508.

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

As used herein, a patient who is heterozygous for a particular gene mutation has that mutation in one allele and another mutation in the other allele.

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 CFTR activity. The increase in activity mediated by a CFTR modulator includes, but is not limited to, compounds that correct, potentiate, stabilize and/or enhance CFTR.

As used herein, the term CFTR corrector refers to a compound that facilitates the processing and migration of CFTR to increase the amount of CFTR on the cell surface. Compounds I and II disclosed herein are CFTR correctors.

As used herein, the term CFTR potentiator refers to a compound that increases the activity of CFTR protein channels located on the cell surface, resulting in enhanced ion transport. compounds III and III-d disclosed herein are CFTR potentiators.

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

Used in the present description, the term pharmaceutically acceptable salt refers to the salt form of the compounds of this disclosure, in which the salt is non-toxic. Pharmaceutically acceptable salts of the compounds of this disclosure include salts 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. describes in detail pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 1977, 66, 1-19.

Used in the present description, the term amorphous refers to a solid material that does not have long-range order in the position of its molecules. Amorphous solids are generally supercooled liquids in which the molecules are arranged randomly so that there is no well-defined arrangement, such as molecular packing, and no long-range order. Amorphous solids are usually isotropic, that is, they exhibit similar properties in all directions and do not have defined melting points. For example, an amorphous material is a solid material that does not have a distinct characteristic crystalline peak(s) in its diffractogram (XRPD) (ie, is not crystalline as determined by XRPD). Instead, one or more broad peaks (eg a halo) appear in its diffractogram (XRPD). Broad peaks are characteristic of an amorphous solid. See US 2004/0006237 for a comparison of XRPD of amorphous material and crystalline material. In some embodiments, the solid material may contain an amorphous compound, and the material may, for example, be characterized by the absence of a distinct characteristic crystalline peak(s) in its XRPD spectrum (i.e., the material is not crystalline, but is amorphous as determined by XRPD). Instead, one or more broad peaks (eg, a halo) may appear in the XRPD pattern of the material. See US 2004/0006237 for a comparison of XRPD of amorphous material and crystalline material. A solid material containing an amorphous compound may have, for example, a wider temperature range for melting the solid material than for melting a pure crystalline solid. Other methods such as, for example, Raman spectroscopy, infrared spectroscopy and solid state NMR can be used to characterize crystalline or amorphous forms.

In some embodiments, the implementation of the solid material may contain a mixture of crystalline solids and amorphous solids. The solid material prepared to contain an amorphous compound may also, for example, contain up to 30% crystalline solids. In some embodiments, the implementation of the solid material, prepared to contain an amorphous compound, may also, for example, contain up to 25, 20, 15, 10, 5 or 2% of crystalline solids. In embodiments where the solid material contains a mixture of crystalline solids and amorphous solids, characterization data, such as XRPD, may include both crystalline and amorphous solids. As used herein, the term substantially amorphous refers to a solid material having little or no long-range order in the position of its molecules. For example, substantially amorphous materials have less than 15% crystallinity (eg, less than 10% crystallinity or less than 5% crystallinity). It is also noted that the term substantially amorphous includes the descriptor amorphous, which refers to materials having no (0%) crystallinity.

As used herein, the term dispersion refers to a dispersed system in which one substance, the dispersed phase, is distributed in discrete units throughout a second substance (continuous phase or excipient). The size of the dispersed phase can vary significantly (for example, colloidal particles ranging in size from a nanometer to several microns). Typically, the dispersed phases may be solids, liquids or gases. In the case of a solid dispersion, the dispersed and continuous phases are both solids. In pharmaceutical applications, a solid dispersion may include a crystalline drug (dispersed phase) in an amorphous polymer (continuous phase); or alternatively, an amorphous drug (dispersed phase) in an amorphous polymer (continuous phase). In some embodiments, the implementation of the solid dispersion includes a polymer constituting the dispersed phase, and the drug is a continuous phase. Or, the solid dispersion includes a drug constituting the dispersed phase and a polymer constituting the continuous phase.

The terms patient and subject are used interchangeably and refer to an animal, including humans.

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 (eg, improvement in MB or symptom of MB, or reduction in severity of MB or symptom of MB). The exact amount of effective dose will depend on the goal of treatment and will be determined by a person skilled in the art using known methods (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

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As used herein, the terms treatment, treating, and the like generally mean an improvement in MB or its symptoms, or a decrease in the severity of MB or its symptoms in a subject. Treatments used herein include, but are not limited to: increasing subject height, increasing weight gain, reducing mucus in the lungs, improving pancreatic and/or liver function, reducing chest infections, and/or reducing cough or difficulty breathing. Improvement or reduction in the severity of any of these symptoms can be easily assessed in accordance with standard methods and techniques known in the art.

As used herein 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 a patient before, at the same time, or after each other.

The term approximately when used in connection with doses, amounts, or weight percent of the ingredients of a composition or dosage form, includes the meaning of a specific dose, amount, or weight percent, or a range of dose, amount, or weight percent that is recognized by a person skilled in the art to provide a pharmacological effect, equivalent to that obtained at the stated dose, amount, or percentage by weight.

Each of Compounds I, II, III and III-d and their pharmaceutically acceptable salts can independently be administered once a day, twice a day or three times a day. In some embodiments, at least one compound selected from compound I and pharmaceutically acceptable salts thereof is administered once a day. In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered twice daily. In some embodiments, at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered once a day. In some embodiments, at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered twice daily. In some embodiments, at least one compound selected from compound III or III-d and pharmaceutically acceptable salts thereof is administered once a day. In some embodiments, at least one compound selected from compound III or III-d and pharmaceutically acceptable salts thereof is administered twice daily.

One skilled in the art will understand 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 an amount equivalent to the free base concentration of the compound. It should be noted that the disclosed amounts of the compounds or their pharmaceutically acceptable salts herein are based on their free base form. For example, 100 mg of at least one compound selected from Compound I and its pharmaceutically acceptable salts includes 100 mg of Compound I, and the pharmaceutically acceptable salt concentration of Compound I is equivalent to 100 mg of Compound I.

As stated above, compound I is disclosed herein, which can be depicted as having the following structure:

The chemical name for compound I is N-(benzenesulfonyl)-6-[3-[2-[1(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2-[(4(S))-2,2 ,4-trimethylpyrrolidin-1-yl]pyridine-3carboxamide. Compound I may be in the form of a pharmaceutically acceptable salt thereof.

In some embodiments, Compound I (and/or at least one pharmaceutically acceptable salt thereof) may be administered in combination with at least one additional active pharmaceutical ingredient. In some embodiments, at least one additional active pharmaceutical ingredient is selected from:

(a) Compound II and pharmaceutically acceptable salts thereof.

The chemical name for compound II is (R)-1-(2,2-difluorobenzo[d][1,3]dioxole

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5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-H-indol-5yl)cyclopropanecarboxamide;

(b) compound III or compound III-d:

or

and its pharmaceutically acceptable salts.

The chemical name for compound III is K-(5-hydroxy-2,4-di-tert-butylphenyl)-4-oxo-H-quinoline-3-carboxamide and the chemical name for compound III-d is N-(2- (tert-butyl)-5-hydroxy-4-(2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)phenyl)-4-oxo-1,4dihydroquinoline -3-carboxamide; and (c) Compound IV

and its pharmaceutically acceptable salts.

The chemical name for compound IV is 3-(6-(1-(2,2difluorobenzo[b][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.

Suitable pharmaceutically acceptable salts are, for example, those disclosed in S. M. Berge, et al. J. Pharmaceutical Sciences, 1977, 66, 1-19. For example, tab. 1 of this article provides the following pharmaceutically acceptable salts.

Table 1

Acetate iodide benzathine Benzenesulfonate Isethionate Chlorprocaine Benzoate lactate Choline Bicarbonate Lactobionate Diethanolamine bitartrate Malat Ethylenediamine Bromide Maleate Meglumine Edetate calcium Mandelat Procaine Kamzilat Mesylate Aluminum Carbonate Methyl bromide Calcium Chloride Methyl nitrate Lithium Citrate Methyl sulfate Magnesium dihydrochloride Mukat Potassium Edetat Napsilate Sodium Edisilat Nitrate Zinc Estolat Pamoat (Embonat) Ezilat Pantothenate fumarate Phosphate/diphosphate Gluceptate Pol igal actur onat

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Gluconate salicylate Glutamate Stearate Glycolylarsanilate Subacetate Gexylresorcinate Succinate Hydrabamine Sulfate hydrobromide Tannat Hydrochloride Tartrate Hydroxysinaphthoate Theoclatus triethiodide

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 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, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, 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. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) _{4 salts} . This disclosure also provides for the quaternization of any basic nitrogen-containing groups of the compounds disclosed in the present description. Suitable non-limiting examples of alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium and magnesium. Additional 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 arylsulfonate. Other suitable non-limiting examples of pharmaceutically acceptable salts include besylate and glucosamine salts.

In some embodiments, at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered in combination with at least one compound selected from Compound II and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered in combination with at least one compound selected from Compound III and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered in combination with at least one compound selected from Compound IV and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered in combination with Compounds II or a pharmaceutically acceptable salt thereof and at least one compound selected from Compound III and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered in combination with at least one compound selected from Compound III and its pharmaceutically acceptable salts and at least one compound selected from Compound IV and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered in combination with at least one compound selected from Compound III-d and pharmaceutically acceptable salts thereof. In some embodiments, at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered in combination with at least one compound selected from Compound IV and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from compound I and its pharmaceutically acceptable salts is administered in combination with compounds II or a pharmaceutically acceptable salt thereof and at least one compound selected from compound III-d and its pharmaceutically acceptable salts. In some embodiments, at least one compound selected from compound I and its pharmaceutically acceptable salts is administered in combination with at least one compound selected from compound III-d and its pharmaceutically acceptable salts, and at least one compound, selected from compound IV and pharmaceutically acceptable salts thereof.

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Compounds I, II, III, III-d and IV and their pharmaceutically acceptable salts may each independently be administered once a day, twice a day, or three times a day. In some embodiments, at least one compound selected from compound I and pharmaceutically acceptable salts thereof is administered once a day. In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered twice daily. In some embodiments, Compound II, or pharmaceutically acceptable salts thereof, is administered once a day. In some embodiments, Compound II, or pharmaceutically acceptable salts thereof, is administered twice daily. In some embodiments, Compound III, or pharmaceutically acceptable salts thereof, is administered once a day. In some embodiments, Compound III, or pharmaceutically acceptable salts thereof, is administered twice daily. In some embodiments, Compound III-d, or pharmaceutically acceptable salts thereof, is administered once a day. In some embodiments, Compound III-d, or pharmaceutically acceptable salts thereof, is administered twice daily. In some embodiments, Compound IV, or pharmaceutically acceptable salts thereof, is administered once a day. In some embodiments, Compound IV, or pharmaceutically acceptable salts thereof, is administered twice daily.

In some embodiments, at least one compound selected from compound I and pharmaceutically acceptable salts thereof is administered in an amount of 10 to 900 mg, 20 to 800 mg, 80 to 800 mg, 30 to 720 mg, 40 to 600 mg, 60 to 100 mg, 60 to 500 mg, 80 to 400 mg, 120 to 240 mg, 120 to 360 mg, 160 to 320 mg, 240 to 400 mg, 320 to 480 mg or 360 to 640 mg per day. In some embodiments, 80, 120, 160, 240, 320, 400, 480, 560, 640 or 720 mg of Compound I or its pharmaceutically acceptable salts are administered once a day. In some embodiments, 80, 120, 160, 240, 320 or 400 mg of Compound I or its pharmaceutically acceptable salts are administered twice daily.

One skilled in the art will understand 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 an amount equivalent to the free base concentration of the compound. It should be noted that the disclosed amounts of the compounds or their pharmaceutically acceptable salts herein are based on their free base form. For example, 100 mg of Compound I or a pharmaceutically acceptable salt thereof includes 100 mg of Compound I and a pharmaceutically acceptable salt concentration of Compound I equivalent to 100 mg of Compound I.

Compounds I, II, III, III-d, IV and their pharmaceutically acceptable salts may be contained in one pharmaceutical composition or in separate pharmaceutical compositions. Such pharmaceutical compositions can be administered once a day or several times a day, for example twice a day. As used herein, the phrase that a given amount of API (e.g., compound I, II, III, III-d, IV, or a pharmaceutically acceptable salt thereof) is administered once or twice a day or per day means that said given amount is administered in dosage once or twice a day. For example, the phrase that 50 mg of compound II or a pharmaceutically acceptable salt thereof is administered twice a day or a day means that 50 mg of compound II or a pharmaceutically acceptable salt thereof is administered in a dosage twice a day (for example, 50 mg of compound II or its pharmaceutically acceptable salt is administered in the morning and 50 mg of compound II or its pharmaceutically acceptable salt is administered in the evening).

In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; at least one compound selected from compound II and pharmaceutically acceptable salts thereof is contained in the second pharmaceutical composition; and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts is contained in the third pharmaceutical composition.

In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; at least one compound selected from compound II and pharmaceutically acceptable salts thereof is contained in the second pharmaceutical composition; at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts is contained in the third pharmaceutical composition; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is contained in the third pharmaceutical composition.

In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; and at least one compound selected from compound II and its pharmaceutically acceptable salts and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts are contained in the second pharmaceutical composition. In some embodiments, the second pharmaceutical composition contains half of the daily dose of said at least one compound selected from compound III or III-d and pharmaceutically acceptable salts thereof, and the other half of said at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts are administered in the third pharmaceutical composition.

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In some embodiments, at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts is contained in the first pharmaceutical composition. In some embodiments, the implementation of the first pharmaceutical composition is administered to the patient twice a day.

In some embodiments, the disclosure relates to a pharmaceutical composition containing at least one compound selected from Compound I and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier.

In some embodiments, the disclosure relates to a pharmaceutical composition containing at least one compound selected from compound I and its pharmaceutically acceptable salts, at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable carrier .

In some embodiments, the disclosure relates to a pharmaceutical composition containing at least one compound selected from compound I and its pharmaceutically acceptable salts, at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable carrier.

In some embodiments, the disclosure relates to a pharmaceutical composition containing at least one compound selected from compound I and its pharmaceutically acceptable salts at least one compound selected from compound II and its pharmaceutically acceptable salts at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable carrier.

In some embodiments, the disclosure relates to a pharmaceutical composition containing at least one compound selected from compound I and its pharmaceutically acceptable salts at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts at least one compound selected from compound IV and its pharmaceutically acceptable salts, and at least one pharmaceutically acceptable carrier.

In some embodiments, the implementation of the pharmaceutical compositions disclosed in the present description, contain at least one additional active pharmaceutical ingredient. In some embodiments, at least one additional active pharmaceutical ingredient is a CFTR modulator. In some embodiments, at least one additional active pharmaceutical ingredient is a CFTR corrector. In some embodiments, 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 the other is a CFTR potentiator.

In some embodiments, at least one additional active pharmaceutical ingredient is selected from mucolytics, bronchodilators, antibiotics, anti-infectives, and anti-inflammatory agents.

The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. In some embodiments, at least one pharmaceutically acceptable carrier is selected from pharmaceutically acceptable excipients and pharmaceutically acceptable adjuvants. In some embodiments, at least one pharmaceutically acceptable carrier is selected from pharmaceutically acceptable excipients, disintegrants, surfactants, binders, lubricants.

It should also be understood that the pharmaceutical composition of this disclosure, including a pharmaceutical composition containing the combinations described above, can be used in combination therapy; that is, the compositions can be administered simultaneously, before or after at least one additional active pharmaceutical ingredient or medical procedures.

Pharmaceutical compositions containing these combinations are useful in the treatment of cystic fibrosis.

In some embodiments, the implementation of the pharmaceutical composition disclosed in the present description contains at least one compound selected from compound I and its pharmaceutically acceptable salts, 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-HG. In some embodiments, the implementation of the pharmaceutical composition contains a solid dispersion of compound I in HPMCAS-HG. In some embodiments, the solid dispersion contains Compound I in HPMCAS-HG in a 1:1 weight ratio. In some embodiments, the solid dispersion contains a substantially amorphous compound I.

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As described above, the pharmaceutical compositions disclosed in the present description may additionally contain at least one pharmaceutically acceptable carrier. At least one pharmaceutically acceptable carrier may be selected from adjuvants and excipients. At least one pharmaceutically acceptable carrier used herein includes any and all solvents, diluents, other liquid excipients, dispersion agents, suspension agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, and sliding agents in accordance with the desired specific dosage form. 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, disclose various carriers used in pharmaceutical compositions and known methods for their preparation. Unless any standard carrier is incompatible with the compounds of this disclosure, such as causing any undesirable biological effect or otherwise interacting in an undesirable manner with any other component(s) of the pharmaceutical composition, its use is intended 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), buffering agents (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 silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, block copolymers of polyethylene and polyoxypropylene, lanolin, sugars (such as lactose, glucose and sucrose), starches (such as cornstarch and potato starch), cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (such as cocoa butter and suppository waxes), oils (t 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, buffer substances (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, sweeteners flavorings, fragrances, preservatives and antioxidants.

It should also be understood that the pharmaceutical composition of this disclosure, including a pharmaceutical composition containing any of the combinations described above, can be used in combination therapy; that is, the compositions can be administered simultaneously, before or after at least one active pharmaceutical ingredient or medical procedures.

In some embodiments, methods of disclosure comprise administering to a patient in need of such treatment at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound II, compound III or III-d, compound IV; and pharmaceutically acceptable salts of any of the foregoing.

Any suitable pharmaceutical compositions known in the art may be used for Compound I, Compound II, Compound III or III-d, Compound IV, and pharmaceutically acceptable salts thereof. Some typical 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, all 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 and some exemplary pharmaceutical compositions for Compound III-d and its pharmaceutically acceptable salts can be found in US 8865902, US 9181192 and US 9512079, all of which are incorporated into the present description by reference. Some exemplary pharmaceutical compositions for Compound IV and its pharmaceutically acceptable salts can be found in WO 2010/037066, WO 2011/127241, WO 2013/112804 and WO 2014/071122, all of which are incorporated herein by reference.

In some embodiments, a pharmaceutical composition containing at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered with a pharmaceutical composition containing Compound II and Compound III or III-d. Pharmaceutical compositions containing Compound II and Compound III are disclosed in PCT Publication WO 2015/160787, incorporated herein by reference. An exemplary embodiment is shown in the following table.

- 9 041854

Table 2 Typical tablet containing 100 mg Compound II and 150 mg Compound III

Ingredient Quantity in tablet (mg) intragranular Connection II SDD (spray dried dispersion) (80 wt% Compound II; 20 wt% HPMC) 125 Compound III SDD (80 wt% Compound III; 19.5 wt.% HPMCAS-HG; 0.5 wt% sodium lauryl sulfate) 187.5 Microcrystalline cellulose 131.4 Croscarmellose sodium 29.6 Total 473.5 Extragranular Microcrystalline cellulose 112.5 magnesium stearate 5.9 Total 118.4 Total number of uncoated tablets 591.9 film sheath Opadry 17.7 Total number of coated tablets 609.6

In some embodiments, a pharmaceutical composition containing Compound I is administered with a pharmaceutical composition containing Compound III or III-d. Pharmaceutical compositions containing Compound III are disclosed in PCT Publication WO 2010/019239, incorporated herein by reference. An exemplary embodiment is shown in the following table.

Table 3. Ingredients for a typical Compound III tablet

Composition of the tablet The percentage in the dose %wt./mass. Dose (mg) The consignment (G) Compound III SDD (80 wt% Compound III; 19.5 wt% HPMCAS-HG; 0.5 wt% sodium lauryl sulfate) 34.09% 187.5 23.86 Microcrystalline cellulose 30.51% 167.8 21.36 Lactose 30.40% 167.2 21.28 Croscarmellose sodium 3.000% 16.50 2,100 linen 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

Additional pharmaceutical compositions containing Compound III are disclosed in PCT Publication WO 2013/130669, incorporated herein by reference. Typical mini-tablets (~2 mm diameter, ~2 mm thickness, each mini-tablet weighs approximately 6.9 mg) were formulated to have approximately 50 mg Compound III per 26 mini-tablets and approximately 75 mg Compound III per 39 mini-tablets. tablets, using the amounts of ingredients listed in the table. 4 below.

- 10 041854

Table 4. Ingredients for 50 and 75 mg mini-tablets

Composition of the tablet Percentage in dose Dose (mg) Dose (mg) The consignment %w/w 50 mg content 75 mg content (G) Compound III SDD (80 wt.% Compound III; 19.5 wt% HPMCAS-HG; 0.5 wt% sodium lauryl sulfate) 35 62.5 93.8 1753.4 Mannitol 13.5 24.1 36.2 675.2 Lactose 41 73.2 109.8 2050.2 Sucralose 2.0 3.6 5.4 100.06 Croscarmellose sodium 6.0 10.7 16.1 300.1 colloidal dioxide silicon 1.0 1.8 2.7 50.0 magnesium stearate 1.5 2.7 4.0 74.19 Total 100 178.6 268 5003.15

In some embodiments, the pharmaceutical compositions are in the form of a tablet. In some embodiments, tablets are suitable for oral administration.

These combinations are useful in the treatment of cystic fibrosis.

The compounds, their pharmaceutically acceptable salts, and deuterated analogs of any of the foregoing, and pharmaceutical compositions may be used in the treatment of cystic fibrosis.

A CFTR mutation can affect the amount of CFTR, that is, the number of CFTR channels on the cell surface, or it can affect the function of CFTR, that is, the functional ability of each channel to open and transport ions. Mutations that affect the amount of CFTR include mutations that cause defective synthesis (class I defect), mutations that cause defective processing and migration (class II defect), mutations that cause reduced CFTR synthesis (class V defect), and mutations which reduce the surface stability of CFTR (class VI defect). Mutations that affect CFTR function include mutations that cause a defective gate mechanism (class III defect) and mutations that cause defective conductance (class IV defect).

In some embodiments, methods of treating, ameliorating, or symptomatic treating cystic fibrosis in a patient disclosed herein comprise administering an effective amount of a compound, a pharmaceutically acceptable salt thereof, or a deuterated analog of any of the foregoing; or a pharmaceutical composition described in this disclosure, to a patient, such as a human, in which said patient suffers from cystic fibrosis. In some embodiments, the patient has P508del/minimal function (MF), F508del/F508del genotypes, P508del/gateway genotypes, or P508del/residual function (RF) genotypes.

As used herein, minimal function (MF) mutations refer to mutations in the CFTR gene associated with minimal CFTR function (with little to no function of the CFTR protein) and include, for example, mutations associated with severe defects in the ability of the CFTR channel to open and close, known as defective channel gate or gate mutations; mutations associated with severe defects in cellular processing of CFTR and its delivery to the cell surface; mutations associated with the absence (or minimal) synthesis of CFTR; and mutations associated with severe channel conduction defects. Tab. C below includes a non-exhaustive list of minimal function CFTR mutations that are detected by FDA-approved genotyping. In some embodiments, a mutation is considered an MF mutation if it meets at least 1 of the following 2 criteria:

(1) biological certainty of the untranslated protein (genetic sequence predicts total absence of CFTR protein) or (2) in vitro testing that confirms lack of sensitivity to Compound II, Compound III, or a combination of Compound II and Compound III, and population-based evidence of clinical severity ( as reported in large patient registries).

- 11 041854

In some embodiments, minimal function mutations are mutations that result in substantially no function of the CFTR protein and are not responsive in vitro to Compound II, Compound III, or a combination of Compound II and Compound III.

In some embodiments, minimal function mutations are mutations that do not respond in vitro to Compound II, Compound III, or a combination of Compound II and Compound III. In some embodiments, minimal function mutations are mutations based on an in vitro study that meet the following criteria in in vitro experiments:

initial chloride transport that was <10% wt CFTR and <10% increase in chloride transport from baseline after adding TEZ, IVA, or TEZ/IVA in the assay.

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

mean sweat chloride >86 mmol/L and prevalence of pancreatic insufficiency (PI) >50%.

Patients with the 508del/minimal function genotype are defined as patients who are heterozygous for F508del-CFTR with a second CFTR allele containing a minimal function mutation. In some embodiments, patients with the 508del/minimal function genotype are patients who are heterozygous for F508del-CFTR with a second CFTR allele containing a mutation that results in a CFTR protein with minimal CFTR function (substantially no CFTR protein function) and that does not respond in vitro for Compound II, Compound III, or a combination of Compound II and Compound III.

In some embodiments, minimal function mutations can be determined using 3 main sources:

biological certainty for a mutation that responds (i.e. mutation class) population-based evidence of clinical severity (from the CFTR2 patient registry; accessed February 15, 2016) mean sweat chloride >86 mmol/L and prevalence of pancreatic insufficiency (NPV) >50% in vitro study Mutations resulting in initial chloride transport <10% WT CFTR were considered minimal function Mutations resulting in chloride transport <10% WT CFTR after addition of Compound II and/or Compound III were considered non-reactive .

As used herein, residual function mutations refer to class II-V mutations that have some residual chloride transport and result in a less severe clinical phenotype. Residual function mutations are mutations in the CFTR gene that result in a reduction in the amount of a protein or function on the cell surface that can cause partial CFTR activity.

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, 3272-26AA, 711+3AA, E56K,

P67L, R74W, D11OE, D11OH, R117C, L206W, R347H, R352Q, A455E, D579G, E831X, S945L, S977F, F1052V, R1070W, F1074L, D1152H, D1270N, E193K and K1060T.

For example, CFTR mutations that cause defective mRNA splicing, such as 2789+507A, result in decreased protein synthesis but deliver some functional CFTR to the cell surface to provide residual function. Other CFTR mutations that reduce conductance and/or gating, such as Rl 17H, result in a normal number of CFTR channels on the cell surface, but the functional level is low, resulting in residual function. In some embodiments, the residual function CFTR mutation is selected from R117H, S1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, A1067T, E193K, and K1O6OT. In some embodiments, the residual function CFTR mutation is selected from R117H, S1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E56K, and A1067T.

Residual CFTR function can be characterized at the cellular level (in vitro) using cellular assays such as the FRT assay (Van Goar, F. et al. (2009) PNAS Vol. 106, No. 44, 1882518830; and Van Goor, F. et al (2011) PNAS Vol 108, No 46, 18843-18846) to measure the amount of chloride transport across mutated CFTR channels. Residual function mutations reduce, but not completely eliminate, CFTR-dependent ion transport. In some embodiments, residual function mutations result in a decrease in CFTR activity of at least about 10% in an FRT assay. In some embodiments, residual function mutations result in a decrease in CFTR activity of up to about 90% in an FRT assay.

P508del/residual function patients are defined as patients who are heterozygous for F508del-CFTR with a second CFTR allele that contains a mutation that, when

- 12 041854 leads to a decrease in the amount or function of the protein on the cell surface, which can cause partial CFTR activity.

Patients with the P508del/gateway mutation genotype are defined as patients who are heterozygous for F508del-CFTR with a second CFTR allele that contains a gate defect-associated mutation and has been clinically demonstrated to be responsive to Compound III. Examples of such mutations include: G178R, S549N, S549r, G551D, G551S, G1244E, S1251N, S1255P and G1349D.

In some embodiments, the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein each independently result in an increase in chloride transport above the patient's baseline chloride transport.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation. In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation, and the genotypes are expected to respond and/or respond to any of the novel compounds disclosed herein, such as compound 1, compound II, compound III and/or compound IV based on in vitro and/or clinical data. In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation, and the genotypes are expected to respond and/or respond to any combinations of (i) novel compounds disclosed herein, such as compound 1 and (ii) compound II and/or compound III and/or compound IV based on in vitro and/or clinical data.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has a CFTR genetic mutation selected from any of the mutations listed in Table 1. A.

- 13 041854

078delT______________

1078delT_____________

11234V_____________

1154insTC____________

HSldelC___________

1213delT______

1248+1G->A.......................

1249-1G+A_________

124del23bp__________

1259insA______

1288insTA____________

1341+1G->A__________

1342-2A->C___________

1461ins4~~~

1471delA____________

1497delGG___________

1507del_______________

1525-lG^A_________

1525-2A+G

1548^^

1577delTA___________

16O9delCA

1677delTA .....~

1716G/A____________

1717-1G-»A__________

1717-8G-+A...............................

1782delA

1811+1.6kbA->G

1811+1G->C__________

1811+1.6kbA-»G......................

1811+1G-K________

1812-1G->A

1898+1G->A_________

1812-1G-+A__________

1824delA____________

182delT 1119delA

185+1G->T__________

1898+1G->T__________

1898+1G->A....................................... .......................

1898+1G-»C_________

1898+3A->G__________

1898+5G->T__________

1924del7_____________

1949del84

Table A. MB Mutations

2043delG~

2055del9^A__________ 21052117dell3insAGAAA 2118dell4____________ 2143delT______________ 2183AA->G+ ___ _

2183AAAG ~ ......

2183AA+G ³ 2183delAA->G#________ 2183delAAAG ~ '

2184delA .......... 2184insA______________ 2307insA______________ 2347delG 2556insAT_____________ 2585delT 2594delGT .....

.

296+lG^A__________ 2991del32_____________ 3007delG_____________ 3Q28delA_____________ 3040G^C......... 306in$A_______________ 306insA 1138insG 3120G-»A____________ 3121-1G-»A___________ 3121-2A^G ~

3121-977_3499+248 de!2515___________

3132delTG_______

3141del9

3171delC.....................

3195del6__________

3199del6____

3272-26A->G______

35OO-2A^G

3600+ZinsT

365 366insT_____

3659delC_________

3667ins4__________

3737delA_________

3791delC_________

3821delT__________

3849+10kbC-+T

3849+lOkbC->T

3850-lG^A______

3850-3T->G________

3850-IG->A________

3876delA_________

3878delG_________

3905lnsT__________

3905insT____

394delTT J

4OO5+1G->A

4005*2T->C_______

4005+lG^A

4005+IG->A_______

4010del4__________

4015delA_________

4016insT......~

4Q21dupT__________________

4040delA_________

4054-1G-+A._________

405+3A-K_______

405+IG->A_________

406-1G+A_______

406-IG->A_________

4209TGTT>A_

4209TGTTAAA

4279insA__________

4326delTC

- 14 041854

4374+1GAT D192G G27R 4374+IG->T D443Y G27X 4382delA D513G G314E 4428insGA D579G G330X 442delA D614G G458V 457TAT^G D836Y G463V 541delC D924N G480C 574delA D979V G542X 5T E1104X G55OX 621+1G/T E116K G551D G21+3A->G E1371X G551S 663delT E193K G576A 663delT 1548delG E193X G622D 675del4 E403D G628R 711+1G->T 711+3A->G E474K G628R(G->A) G970D E56K 711+1GAT E585X G673X 711+3AAG E588V G85E 711+5GAA E60K G91R 712-1G->T E822K G970R 7T E822X G970R 852del22 E831X H1054D 935delA E92K H1O85P 991del5 E92X H1085R A1OO6E F1016S H1375P A120T F1O52V H139R A234D F1074L H199R A349V F1099L H199Y A455E F191V H6O9R A613T F311del H939R A46D F311L I1OO5R A46Db F5O8C I1027T A559T F5O8del 11234V A559Tb F57SY I1269N A561E G1061R I1366N C276X G1069R I148T C524R G1244E 1175V C524X G1249R I3336K CFTRde!2,3 G126D I502T CFTRdele22-23 G1349D I506S D11OE G149R I506T D11OH G178R l507del D1152H G194R l507del D1270N G194V I6O1F

- 15 041854

I618T P5L R1283S I807M P67L R170H I980K P7S0L R258G IVS14b+5G->A P99L R31C K710X Q1100P R31L K710X Q1291H R334L K710X Q1291R R334Q L1O2R Q1313X R334W L1O65P Q1382X R347H L1077P Q1411X R347L L1077Pb Q1412X R347P L1254X Q22OX R352Q L1324P Q237E R352W L1335P Q237H Q452P R516G L138ins R553Q L1480P Q290X R553X L15P Q359K/T360K R560K L165S Q39X R560S L2O6W Q414 R560T L218X Q414X R668C L227R E585X R709X L32OV Q.493X R74W L346P Q525X R751L L453S Q552X R75Q. L467P Q685X R75X L467Pb Q890X R764X L558S Q890X R792G L571S Q98R R792X L732X Q98X R851X L927P R1066C R1066H R933G L967S S1118F L997F R1066M S1159F M1101K R1070Q. S1159P M11O1R R1070W S1196X M152V R1102X S1235R MIT M1V R1158X S1251N R1162L S1255P M265R R1162X S1255X M470V R117C S13F M952I R117G S341P M952T R117H S434X N1303K R117L S466X P205S R117P S489X P574H R1283M S492F

- 16 041854

S4X_______

S549N

S549R__

S549R(A->Q

5549R(T->G)

S589N~

S737F

S912L

S912X

S945L

S977F

T1O36N

T1O53I

T1246I

T338I _ _

T6Q4I

V1153E

V1240G

V1293G

V201M

V232D

V456A

V456F

V52OF

V562I

V754M

W1089X

W1098C

W1098R

W1098X

W12O4X

W1282R'

W1282X__

W361R

W401X

W496X

W57G

W57R........

W57X

W846X

Y1014C

Y1O32C

Y1092X_

Y109N~

Y122X

Y161D

Y161S

Y563D

Y563N

Y569C

Y569D

Y569Db

Y849X

Y913C

Y913X

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has a CFTR genetic mutation selected from

- 17 041854

G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V, G1069R, R117C, DI ЮН, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L , D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 312O+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 4O5 +1G->A, 4O6-1G->A, 4OO5+1G->A, 1812-1G>A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G ->A, 3121-1G->A, 4374+lG->T, 38501G->A, 2789+5G->A, 3849+10kbC->T, 3272-26A->G, 711+5G->A , 3120G->A, 1811+E6kbA>G, 711+3A->G, 1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/A , 1811+1G>C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T, 4005+2T->C, 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, G628, G628 G970D, H1054D, H10 85P, H1O85R, 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, Q23,1 R1066H, R117G, R117L, R117P, R1283M, R1283S, R170H, R258G, R31L, R334L, R334Q, R347L, R352W, R516G, R553Q, R751L, R792G, R933G, S1118F, S1959F, S1159F( ), S549R(T->G), S589N, S737F, S912L, T1036N, T1053I, T1246I, T604I, V1153E, V1240G, V1293G, V201M, V232D, V456A, V456F, V562I, W1098C, W1098R, W1282R, W1282R W57R, Y1014C, Y1032C, Y109N, Y161D, Y161S, Y563D, Y563N, Y569C, and Y913C.

In some embodiments, the patient has at least one combination mutation selected from

G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, Е193К, F1052V, G1069R, R117C, D110H, R347H, R352Q, Е56К, P67L, L206W, А455Е, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 312O+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 4O5+ 1G->A, 4O6-1G->A, 4OO5+1G->A, 1812-1G->A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G ->A, 3121-1G->A, 4374+lG->T, 385O-1G->A, 2789+5G->A, 3849+10kbC->T, 327226A->G, 711+5G->A , 3120G->A, 1811+L6kbA->G, 711+3A->G, 1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G/ A, 1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G>A, 1898+1G->T, 4005+2T->C and 621+3A->G.

In some embodiments, the patient has at least one combination mutation selected 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 , 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, Q2237 , Q98R, R1066C, R1066H, R117G, R117L, R117P, R1283M, R1283S, R170H, R258G, R31L, R334L, R334Q, R347L, R352W, R516G, R553Q, R751L, R792G, R933G, S1118F, S1159F, S1159P, S13F, 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.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has the CFTR G551D genetic mutation. 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 MB-causing mutation on the other allele. In some embodiments, the patient is heterozygous for the G551D genetic mutation on one allele and the other MB-causing genetic mutation on the other allele is any of

- 18 041854

F508del, G542X, N13O3K, W1282X, R117H, R553X, 1717-1G->A, 621+1G->T, 2789+5G->A, 3849+10kbC->T, R1162X, G85E, 312O+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 R117H.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has the CFTR F508del genetic mutation. 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 MB-causing genetic mutation on the other allele. In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation, including but not limited to,

G551D, G542X, N13O3K, W1282X, R117H, R553X,

1717-1G->A, 621+1G->T, 2789+5G->A, 3849+10kbC->T, R1162X, G85E, 312O+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 R117H.

In some embodiments, the patient has at least one combination mutation selected from

D443Y; G576A; R668C,

F508C; S1251N,

G576A; R668C,

G970R; M470V,

R74W; D1270N,

R74W; V201M and

R74W; V201M; D1270N.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V and G1069R. In some embodiments, the patient has a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, and S1251N. In some embodiments, the patient has a CFTR genetic mutation selected from E193K, F1052V, and G1069R. In some embodiments, the implementation of the method provides an increase in the transport of chlorides in comparison with the original transport of chlorides in the patient.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has a CFTR genetic mutation selected from R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L , R1070W, F1074L, D110E, D1270N and DI 152H.

In some embodiments, the patient has a CFTR genetic mutation selected from 1717-1G->A, 621+1G->T, 312O+1G->A, 1898+1G->A,

711+1G->T, 2622+lG->A, 4O5+1G->A, 4O6-1G->A, 4OO5+1G->A, 1812-1G->A, 1525-1G->A, 712 -1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 385O-1G->A, 2789+5G>A, 3849+10kbC ->T, 3272-26A->G, 711+5G->A, 3120G->A, 1811+L6kbA->G, 711+3A->G, 1898+3A->G, 1717-8G->A , 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C, 1898+5G->T, 385O-3T->G, IVS14b+5G->A, 1898+1G ->T, 4005+2T->C and 621+3A->G.

In some embodiments, the patient has a CFTR genetic mutation selected from 1717-1G->A, 1811+1.6kbA->G, 2789+5G->A, 3272-26A->G, and 3849+1 OkbC->T.

In some embodiments, the patient has a CFTR genetic mutation selected from 2789+5G->A and 3272-26A->G.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has a CFTR genetic mutation selected from

- 19 041854

G178R G551S G970R G1244E S1255P G1349D S549N S549R S125 IN E193K F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 312O+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 4O5+1G->A, 4O6-1G->A, 4OO5+1G->A, 1812-1G>A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+ 1G->A, 3121-1G->A, 4374+lG->T, 38501G->A, 2789+5G->A, 3849+10kbC->T, 3272-26A->G, 711+5G-> A, 3120G->A, 1811+1.6kbA>G, 711+3A->G, 1898+3A->G, 1717-8G->A, 1342-2A->C, 405+3A->C, 1716G /A, 1811+1G>C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+1G->T, 4005+2T->C and 621+3A->G , and human CFTR mutations selected from F508del, R117H and G551D.

In some embodiments, in the methods of treating, ameliorating, or symptomatic treating cystic fibrosis disclosed herein, the patient has a CFTR genetic mutation selected from

G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, Е193К, F1052V, G1069R, R117C, D110H, R347H, R352Q, Е56К, P67L, L206W, А455Е, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, D1152H, 1717-1G->A, 621+1G->T, 312O+1G->A, 1898+1G->A, 711+1G->T, 2622+lG->A, 4O5+ 1G->A, 4O6-1G->A, 4OO5+1G->A, 1812-1G>A, 1525-1G->A, 712-1G->T, 1248+1G->A, 1341+1G- >A, 3121-1G->A, 4374+lG->T, 38501G->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, 385O-3T->G, IVS14b+5G->A, 1898+1G->T, 4005+2T->C, 621+3A->G,

And a CFTR mutation selected from F508del, R117H and G551D; and CFTR mutations selected from F508del, R117H and G551D.

In some embodiments, the patient has a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, S1251N, E193K, F1052V, and G1069R, and a human CFTR mutation selected from F508del, R117H, and G551D. In some embodiments, the patient has a CFTR genetic mutation selected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549R, and S1251N, and a human CFTR mutation selected from F508del, R117H, and G551D. In some embodiments, the patient has a CFTR genetic mutation selected from E193K, F1052V, and G1069R and a human CFTR mutation selected from F508del, R117H, and G551D.

In some embodiments, the patient has a CFTR genetic mutation selected from R117C, D110H, R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270N, and D1152H, and a human CFTR mutation selected from F508del, R117H and G551D.

In some embodiments, the patient has a CFTR genetic mutation selected from 1717-1G->A, 621+1G->T, 312O+1G->A, 1898+1G->A,

711+1G->T, 2622+lG->A, 4O5+1G->A, 4O6-1G->A, 4OO5+1G->A, 1812-1G->A, 1525-1G->A, 712 -1G->T, 1248+1G->A, 1341+1G->A, 3121-1G->A, 4374+lG->T, 385O-1G->A, 2789+5G>A, 3849+10kbC ->T, 3272-26A->G, 711+5G->A, 3120G->A, 1811+1.6kbA->G, 711+3A->G, 1898+3A->G, 1717-8G-> A, 1342-2A->C, 405+3A->C, 1716G/A, 1811+1G->C, 1898+5G->T, 3850-3T->G, IVS14b+5G->A, 1898+ 1G->T, 4005+2T->C and 621+3A->G, and a human CFTR mutation selected from F508del, R117H and G551D. In some embodiments, the patient has 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, R117H and G551D. In some embodiments, the patient has a CFTR genetic mutation selected from

2789+5G->A and 3272-26A->G, and a human CFTR mutation selected from F508del, R117H.

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

In some embodiments, the MB-causing mutation is selected from Table 1. A. In some embodiments, the MB-causing mutation is selected from Table B. In some embodiments, the MB-causing mutation is selected from Table 1. C. In some embodiments, the MB-causing mutation is selected from FIG. 3. In some embodiments, the patient is heterozygous, having a MB-causing mutation in one CFTR allele selected from mutations listed

- 20 041854 data in the table in FIG. 3 and the MB-causing mutation in another CFTR allele is selected from the CFTR mutations listed in Table B:

Table B. CFTR Mutations

Q39X 621+1G^T A559T W57X 1248+1G—+A R560T E60X 1341+lG—*A R560S R75X 1717-1G-A A561E E92X 1811+1.6kbA—>G Y569D Q98X 1811+lG^C L1065P Y122X 1812-1G—*A R1066C L218X 1898+10—Λ R1066.M Q220X 2622+1G^A L1077P S276X 3120+lG^A H1085R Q290X 3120G—»A M1101K G33OX 3850-1G—+A 4005+1G—»A N1303K W401X 3849+10kbC-*T Q414X 4374+lG—»T 3272-26A—*G S434X S466X S489X Q493X W496X 663delT 2183AA—>G CFTRdel2,3 711+3A—*G E56K P67L 3659delC 394delTT R74W D110E Q525X G542X Q552X R553X E585X G673X Ti ΊΛΛν 2184insA DHOH 3905insT R117C 2184delA L206W 1078delT R347H 1154insTC R352Q 2183delAA^G A455E R709X Κ710Χ L732X R764X 2143delT D579G 1677deITA E831X 3876delA S945L 2307ms A S977F R785X R792X Ε822Χ W846X R851X Q890X S912X W1089X Υ1092Χ Ε1104Χ R1158X R1162X S1196X W1204X S1255X W1282X Q1313X 4382delA F1052V 4016insT R1070W 2347delG F1074L 3007delG D1152H 574delA D1270N 2711delT G178R 3791delC CFTRdele22-23 S549N S549R 457TAT-*G G551D 2043delG G551S 2869insG G12441 3600+2insT 3737delA 4040delA 541dclC A46D T338I S1251N S1255P G13491) 621+lG^T 711 + 1G—+Τ R347P 7II+5G >A L927P 712-1G—*T G85E 405+1G—>A 405+3A—>C S341P L467P 406-1G—*A I507del V520F

- 21 041854

Table C. CFTR Mutations

Criterion Mutation Truncated Mutations Q2X L218X Q525X R792X E1104X %P1 >50% and/or SwCF S4X Q220X G542X E822X W1145X >86 mmol/l W19X Y275X G550X W882X R1158X Not a full length protein G27X S276X Q552X W846X R1162X Q39X Q290X R553X Y849X S1196X W57X G33OX E585X R851X W1204X E60X W401X G673X Q890X L1254X R75X Q414X Q685X S912X S1255X 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 С524Х R785X R1102X Q1411X

Splicing mutations 185+IGA 711+5G 1717-8GA 2622 3121- %P1 >50% and/or SwCT >86 mmol/l Absence or 296+1G A A 712- 1717-IGA +1G A 2790- 1GA 3500- small mature mRNA 296+CAT IGA 1248+1 1811+1GA KA C 3040 2AA 3600+2in 405+1GA GA 1249- 1811+1.6kbAA G^ C ST 3850- 405+ZAA 1GA 1341+1 1811+1643GA (G97 OR) 3120 1GA 4005+1G 406-1G A GA 1525- 1812-IGA G^ A 3120 A 4374+1G 621+1GA 2AA 1525- 1GA 1898+1GA +1G A 3121- 2A^ G A

- 22 041854

711+1G—>T 1898+lG^C

small nucleotides) (<z 182delT 1078del T 1677delTA 2711 delT 3737delA insertion/deletion (ins/del) mutations with 306insA 1119del A 1782delA 2732Ϊ nsA 3791delC frame shift %P1 >50% and/or SWCF 306delTAG A 1138ins G 1824delA 28691nsG 3821delT >86 mmol/l Distorted and/or 365-366insT 1154ins TC 1833delT 28961 nsAG 3876delA processed protein 394delTT 1161del C 2043delG 2942Ϊ nsT 3878delG 442delA 1213del T 2143delT 2957 delT 3905insT 444delA 1259ins A 2183AA^G ^a 3007 delG 4016insT 457TAT^ G 1288ins TA 2184delA 3028 delA 4021dup T 541delC 1343del G 2184insA 3171 delC 4022insT 574delA 147Idel A 2307insA 3171i nsC 4040delA 663delT 1497del GG 2347delG 3271 delG G 4279insA 849delG 1548del G 2585delT 33491 nsT 4326delT C 935delA 1609del CA 2594delGT 3659 delC

Large (>3 CFTRdelel 6- nucleotides) CFTRdelel 17b 1461ins4 insertion/deletion CFTRdelel7a th (ins/del) mutations co CFTRdele2 ,17b 1924del7 frame shift CFTRdelel7a %PI >50% and/or SwCT CFTRdele2,3 -18 2055del9^A >86 mmol/l CFTRdele2-4 CFTR delel 9 2105-

- 23 041854

Distorted and/or 2117dell3insAGAAAA processed protein CFTRdele3-10,14b- CFTR delel 9- 16 21 2372del8 CFTRdele4-7 CFTRdele21 2721dell CFTRdele22- CFTRdele4-ll 24 2991del32 CFTRdele22, 3121- CFTR50kbdel 23 977_3499+248del2515 CFTRdup6b-10 124del23bp 3667ins4 CFTRdelel 1 602dell4 4010del4 CFTR delel 3.14a 852del22 4209TGTT^AA CFTRdelel 4b-17b 991del5 Class II, III, IV mutations, A46D ^to V520F ^Y569Db N130 not susceptible to 3K Compound II, G85E ^A559Tb L1065P Compound III or R347P R560T R1066C connection L467P ^to R560S ^L1077Pb P/Connection III I507del A561E M1101K %P1>50% and/or SwCl'>86 mmol/l

And

Not susceptible in vitro to Compound II, Compound III, or Compound P/Compound III

CFTR: transmembrane conductance regulator in cystic fibrosis;

SwCl: chloride content in sweat;

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: http://www.cftr2.org/. Accessed February 15, 2016;

Notes: %PI: Percentage of F508del-CFTR heterozygous patients in the CFTR2 Patient Registry with pancreatic insufficiency; SwCl: mean sweat chloride in F508del-CFTR heterozygous patients in the CFTR2 patient registry.

^and also known as 2183delAA^G.

^b unpublished data.

In some embodiments, the patient has F508del/MF (F/MF) genotypes (heterozygous for F508del and an MF mutation that is not expected to respond to CFTR modulators such as Compound III); genotype F508del/F508del (F/F) (homozygous for F508del); and/or F508del/gateway (F/G) genotypes (heterozygous for F508del and gatekeeper mutations known to respond to a CFTR modulator (e.g., compound III). In some embodiments, a patient with F508del/ genotypes MF (F/MF) has an MF mutation that is not expected to respond to Compound II, Compound III, and both Compound II and Compound III In some embodiments, a patient with F508del/MF (F/MF) genotypes has either one of the MF mutations in Table C.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation, including truncated mutations, splicing mutations, small (<3 nucleotides) insertion or deletion (ins/del) frameshift mutations; large (>3 nucleotides) insertion or deletion (ins/del) frameshift mutations; and class II, III, IV mutations not susceptible to Compound III alone or in combination with Compound II or Compound IV.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is a truncated mutation. In some certain variations

- 24 041854 antah implementation truncated mutation is a truncated mutation listed in table. WITH.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is a splicing mutation. In certain specific embodiments, the implementation of the splicing mutation is a splicing mutation listed in table. WITH.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is a small (<3 nucleotides) insertion or deletion (ins/del) frameshift mutation. In certain specific embodiments, the small (<3 nucleotides) insertion or deletion (ins/del) frameshift mutation is the small (<3 nucleotides) insertion or deletion (ins/del) frameshift mutation listed in Table 1. WITH.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation expected to be and/or responsive based on in vitro and/or clinical data to any combination of (i) a novel compound, selected from those disclosed in the present description (for example, compounds of Formula (I), (II), (III), (IV) or (V), and their pharmaceutically acceptable salts, and their deuterated derivatives), and (ii) compounds II and/or compounds III and/or compounds IV.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any MB-causing mutation expected to be and/or responsive based on in vitro and/or clinical data to a triple combination of a novel compound selected from those , which are disclosed in the present description (for example, compounds of formula (I), (II), (III), (IV) or (V), and their pharmaceutically acceptable salts, and their deuterated derivatives), and compounds II, and compounds III .

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is a large (>3 nucleotides) insertion or deletion (ins/del) frameshift mutation. In certain specific embodiments, the large (>3 nucleotides) insertion or deletion (ins/del) frameshift mutation is the large (>3 nucleotides) insertion or deletion (ins/del) frameshift mutation listed in Table 1. 5V.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is a Class II, III, IV mutation not susceptible to Compound III alone or in combination with Compound II or Compound IV. In certain certain embodiments, class II, III, IV mutations not receptive to Compound III alone or in combination with Compound II or Compound IV are class II, III, IV mutations not receptive to Compound III alone or in combination with Compound II or compound IV listed in table. WITH.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any of the mutations listed in Table 1. WITH.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any mutation other than F508del listed in Table 1. A, B, C and Fig. 3.

In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any of the mutations listed in Table 1. A. In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any of the mutations listed in Table B. In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any of the mutations listed in Table 1. C. In some embodiments, the patient is heterozygous for F508del and the other CFTR genetic mutation is any of the mutations listed in FIG. 3.

In some embodiments, the patient is homozygous for F508del.

In some embodiments, the patient is heterozygous, having one MB-causing mutation in one CFTR allele selected from the mutations listed in the Table in FIG. 3 and another MB-causing mutation in another CFTR allele is selected from the CFTR mutations listed in Table 3. WITH.

In some embodiments, the composition disclosed herein is useful for the treatment, amelioration, or symptomatic treatment of cystic fibrosis in patients who exhibit residual CFTR activity in the apical membrane of the respiratory and non-respiratory epithelium. The presence of residual CFTR activity on the surface of the epithelium can be easily detected using methods known in the art, for example, standard electrophysiological, biochemical or histochemical methods. Such ways

- 25 041854 identify CFTR activity using in vivo or ex vivo electrophysiological methods, measurement of SG concentration in sweat or saliva, or ex vivo biochemical or histochemical methods to monitor cell surface density. Using such methods, residual CFTR activity can be easily detected in patients who 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 R117H mutation. In some embodiments, the compositions disclosed herein are useful in the treatment, amelioration, or symptomatic treatment of cystic fibrosis in patients who show little or no residual CFTR activity. In some embodiments, the compositions disclosed herein are useful in the treatment, amelioration, or symptomatic treatment of cystic fibrosis in patients who show little or no residual CFTR activity in the apical membrane of the respiratory epithelium.

In some embodiments, the compositions disclosed herein are useful for treating or ameliorating cystic fibrosis in patients who exhibit residual CFTR activity using pharmacological methods. Such methods increase the amount of CFTR present on the cell surface, thus inducing hitherto absent CFTR activity in the patient or increasing the existing level of residual CFTR activity in the patient.

In some embodiments, the compositions disclosed herein are useful for treating or reducing the severity of cystic fibrosis in patients with certain genotypes that exhibit residual CFTR activity.

In some embodiments, the compositions disclosed herein are useful in the treatment, amelioration, or symptomatic treatment of cystic fibrosis in patients with certain clinical phenotypes, e.g., a mild or moderate clinical phenotype that typically correlates with the amount of residual CFTR activity in the apical epithelial membrane. Such phenotypes include patients exhibiting pancreatic sufficiency.

In some embodiments, the compositions disclosed herein are useful in the treatment, amelioration, or symptomatic treatment of patients diagnosed with pancreatic sufficiency, idiopathic pancreatitis, and congenital bilateral vas deferens, or moderate lung disease in which the patient exhibits residual CFTR activity.

In some embodiments, the implementation of this disclosure relates to a method of increasing or inducing the activity of an anion channel in vitro or in vivo, including the interaction of the channel with the composition disclosed in the present description. In some embodiments, the anion channel is a chloride channel or a bicarbonate channel. In some embodiments, the anion channel is a chloride channel.

In some embodiments of the methods of treating cystic fibrosis disclosed herein, the absolute change in percentage predicted patient forced expiratory volume in 1 second (ppFEV1) after 15 days of administration of at least one compound selected from compound I and pharmaceutically acceptable salts of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, is in the range from 3 to 40% relative to pFOEF1 in the patient prior to said administration.

In some embodiments of the methods for treating cystic fibrosis disclosed herein, the absolute change in percent predicted patient forced expiratory volume in 1 second (ppFEV1) after 29 days of administration of at least one compound selected from Compound I and pharmaceutically acceptable salts of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III and its pharmaceutically acceptable salts, is in the range from 3 to 40% relative to pFOEF1 in a patient prior to said administration. In some embodiments, the absolute change in pFOEF1 at 29 days is in the range of 4 to 40% relative to the patient's pFOEF1 prior to administration.

In some embodiments, the absolute change in pFOEF1 at 29 days is in the range of 6% to 40% relative to the patient's pFOEF1 prior to administration. In some embodiments, the absolute change in pFOEF1 at 29 days is in the range of 7 to 40% relative to the patient's pre-administration pFOEF1.

In some embodiments of the methods for treating cystic fibrosis disclosed herein, the absolute change in chloride content in sweat in a patient after 15 days of administration of at least one compound selected from compound I and pharmaceutically acceptable salts thereof of at least one compound selected from compound II and its pharmaceutically acceptable

- 26 041854 my salts, and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, is in the range from -2 to -65 mmol / l from the initial level, that is, relative to the content of chloride in sweat in the patient prior to the indicated administration. In some embodiments, the absolute change in chloride content in the sweat of the specified patient is in the range from -3 to -65 mmol/l. In some embodiments, the absolute change in chloride content in the sweat of the specified patient is in the range from -5 to -65 mmol/l. In some embodiments, the absolute change in sweat chloride content of said patient is in the range of -10 to -65 mmol/L. In some embodiments, the absolute change in chloride content in sweat of said patient is in the range of -20 to -65 mmol/L. In some embodiments, the implementation of the absolute change in chloride content in the sweat of the specified patient is in the range from -30 to -65 mmol/l. In some embodiments, the absolute change in sweat chloride content of said patient is in the range of -38 to -65 mmol/L. In some embodiments of the methods for treating cystic fibrosis disclosed herein, the absolute change in chloride content in sweat in a patient after 29 days of administration of at least one compound selected from compound I and pharmaceutically acceptable salts thereof of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, is in the range from -2 to -65 mmol / l from the initial level, that is, relative to the content of chloride in sweat in the patient prior to the indicated administration. In some embodiments, the absolute change in chloride content in the sweat of the specified patient is in the range from -3 to -65 mmol/l. In some embodiments, the absolute change in chloride content in the sweat of the specified patient is in the range from -5 to -65 mmol/l. In some embodiments, the absolute change in sweat chloride content of said patient is in the range of -10 to -65 mmol/L. In some embodiments, the absolute change in chloride content in sweat of said patient is in the range of -20 to -65 mmol/L. In some embodiments, the implementation of the absolute change in chloride content in the sweat of the specified patient is in the range from -30 to -65 mmol/l. In some embodiments, the absolute change in sweat chloride content of said patient is in the range of -38 to -65 mmol/L.

In some embodiments of the methods for treating cystic fibrosis disclosed herein, the absolute change in chloride content in sweat in a patient after 29 days of administration of at least one compound selected from compound I and pharmaceutically acceptable salts thereof of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III and its pharmaceutically acceptable salts, is in the range from -2 to -65 mmol / l from the initial level, that is, relative to the chloride content in the patient's sweat up to the specified introductions. In some embodiments, the absolute change in chloride content in the sweat of the specified patient is in the range from 5 to -65 mmol/l. In some embodiments, the absolute change in sweat chloride content of said patient is in the range of -10 to -65 mmol/L. In some embodiments, the absolute change in sweat chloride content of said patient is in the range of -2 to -65 mmol/L.

In some embodiments, the triple combinations are administered to a patient who has one F508del mutation and one minimal function mutation and who is not taking either of said at least one compound selected from Compound I and pharmaceutically acceptable salts thereof of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts.

In some embodiments, triple combinations are administered to a patient who has two copies of the F508del mutation and in which the patient has taken at least one compound selected from compound II and its pharmaceutically acceptable salts and at least one compound selected from compound III or III -d and its pharmaceutically acceptable salts, but not any of said at least one compound selected from Compound I and its pharmaceutically acceptable salts.

In some embodiments, the absolute change in pFOEF1 in a patient after 15 days of administration of at least one compound selected from Compound I and its pharmaceutically acceptable salts of at least one compound selected from Compound II and its pharmaceutically acceptable salts and at least one compound , selected from compound III or III-d and its pharmaceutically acceptable salts, is in the range from 3 to 35% relative to pFOV1 in a patient before said administration.

In some embodiments, the absolute change in pFOEF1 in a patient after 29 days of administration of at least one compound selected from compound I and its pharmaceutically acceptable salts of at least one compound selected from compound II and its pharmaceutical

- 27 041854 highly acceptable salts, and at least one compound selected from compound III and its pharmaceutically acceptable salts, is in the range from 3 to 35% relative to pPOFEV1 in a patient before said administration.

In some embodiments, the patient's absolute change in pFOEF1 relative to the patient's pFOEF1 prior to such administration of the triple combinations may be calculated as (value after baseline - baseline). Baseline is defined as the most recent non-missing measurement taken prior to the first dose of study drug during the treatment period (Day 1).

The exact amount of pharmaceutical composition required will vary from subject to subject, depending on the type, age and general condition of the subject, the severity of the disease, the particular agent, its route of administration, and the like. The compounds of this disclosure may be formulated in unit dosage form for ease of administration and dosage uniformity. As used herein, unit dosage form refers to a physically discrete unit of agent suitable for the patient to be treated. However, it should be understood that the total daily use of the compounds and compositions of this disclosure will be determined by the attending physician within the framework of sound medical judgment. The specific effective dose level for any particular patient or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the particular composition used; age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or simultaneously with the specific compound used, and like factors well known in the medical field. As used herein, the term patient means an animal, such as a mammal, and even preferably, such as a human.

In some embodiments, the disclosure also relates to methods of treatment using isotopically labeled compounds of the aforementioned compounds, which in some embodiments are referred to as Compound I', Compound II', Compound III', Compound III-d, or Compound IV'. In some embodiments, Compound I', Compound II', Compound III', Compound III-d, Compound IV', 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 that is different from the atomic mass or mass number of an atom that normally occurs in nature (isotopically labeled). Examples of isotopes that are commercially available and suitable for disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ 0, ¹⁷ 0 , ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl, respectively.

Isotopically labeled compounds and salts can be used in a number of preferred ways. They may be suitable for drug formulations and/or various types of assays such as tissue distribution assays. For example, tritium ( ³ H) and/or carbon-14 ( ¹⁴ C) labeled compounds are most suitable for various types of assays such as tissue substrate distribution assays due to relatively simple preparation and excellent detection capability. For example, deuterium-labeled ( ² H) compounds are therapeutically useful with potential therapeutic advantages over non- ² H-labeled compounds. In general, deuterium ( ² H) labeled compounds and salts can have higher metabolic stability than non-isotopically labeled compounds due to the kinetic isotope effect described below. Higher metabolic stability leads directly to increased in vivo half-life or reduced dosages, which may be desirable. Isotopically labeled compounds and salts can generally be prepared by following the procedures disclosed in the synthetic schemes and the corresponding description, in the examples and in the preparation part of this text, replacing the non-isotopically labeled reagent with a readily available isotopically labeled reagent.

In some embodiments, the isotopically labeled compounds and salts are labeled with deuterium ( ² H). In certain specific embodiments, the isotopically labeled compounds and salts are deuterium ( ² H) labeled in which one or more of their hydrogen atoms have been replaced by deuterium. In chemical structures, deuterium is represented as D.

Deuterium-labeled ( ² H) compounds and salts can drive the oxidative metabolism of the compound through a primary kinetic isotope effect. The primary kinetic isotope effect is a change in the rate of a chemical reaction that occurs as a result of the exchange of isotopic nuclei, which, in turn, is caused by a change in the ground state energies required to form a covalent bond after this isotopic exchange. The exchange of a heavier isotope generally results in a decrease in the ground state energy for a chemical bond and thus causes a decrease in rate-limiting bond cleavage. If the connection is broken

- 28 041854 originates in the region of the saddle point along the reaction coordinate with several products, the distribution ratios of the products can be essentially changed. To explain: if the deuterium is bound to the carbon atom in an unchangeable position, the differences in the rate k _M /k _D =2-7 are typical. For more information, see SL Harbeson and RD Tung, Deuterium In Drug Discovery and Development, Ann. Rep. Med. Chem. 2011, 46, 403-417, incorporated herein in its entirety by reference.

The concentration of the isotope(s) (eg, deuterium) included in the isotopically labeled compounds and the opening salt can be determined by the isotopic enrichment factor. Used in the present description, the term isotopic enrichment factor means the ratio between the isotopic composition and the natural composition of a particular isotope. In some embodiments, if a substituent in a compound of the disclosure is designated deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% inclusion of deuterium in each designated deuterium atom) of at least 4000 (60% inclusion of deuterium deuterium), at least 4500 (67.5% deuterium inclusion), at least 5000 (75% deuterium inclusion), at least 5500 (82.5% deuterium inclusion), at least 6000 (90% deuterium inclusion) , at least 6333.3 (95% deuterium inclusion), at least 6466.7 (97% deuterium inclusion), at least 6600 (99% deuterium inclusion), or at least 6633.3 (99.5% deuterium inclusion deuterium).

When discovering and developing therapeutic agents, one skilled in the art seeks to optimize pharmacokinetic parameters while maintaining desirable in vitro properties. It may be reasonable to assume that many compounds with poor pharmacokinetic profiles are subject to oxidative metabolism.

In some embodiments, the implementation of the connection III' used in the present description includes the deuterated compound disclosed in US patent No. 8865902 (which is incorporated in this description by reference) and STR-656.

In some embodiments, Compound III' is:

Exemplary embodiments of the disclosure include.

1) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 10 to 900 mg of at least one compound selected from compound I

and its pharmaceutically acceptable salts daily; and (B) at least one compound selected from:

(i) compounds II

(ii) compounds III or compounds III-d:

- 29 041854 and (iii) compounds IV

and pharmaceutically acceptable salts of any of the foregoing.

2) The method of embodiment 1, comprising administering to said patient: at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from (i) compound III and its pharmaceutically acceptable salts or (ii) compound III-d and its pharmaceutically acceptable salts.

3) The method of embodiment 1 comprising administering to said patient: at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

4) The method of embodiment 1, comprising administering to said patient: at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; at least one compound selected from (i) compound III and its pharmaceutically acceptable salts or (ii) compound III-d and its pharmaceutically acceptable salts; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

5) The method of embodiment 1, comprising administering to said patient: (i) at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound II and pharmaceutically acceptable salts thereof; (ii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from (a) Compound III and its pharmaceutically acceptable salts or (b) Compound III-d and its pharmaceutically acceptable salts; or (w) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

6) The method according to any one of embodiments 1-5, wherein 20 to 800 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

7) The method according to any one of embodiments 1-5, wherein 30 to 720 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

8) The method according to any one of embodiments 1-5, wherein 40 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

9) The method according to any one of embodiments 1-5, wherein 60 to 100 mg or 60 mg to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

10) The method according to any one of embodiments 1-5, wherein 60 to 500 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

11) The method according to any one of embodiments 1-5, wherein 80 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

12) The method according to any one of embodiments 1-5, wherein 120 to 200 mg or from

- 30 041854

120 to 450 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

13) The method of any one of embodiments 1-5, wherein 120 to 360 mg or 120 mg to 500 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

14) The method of any one of embodiments 1-5, wherein 160 to 320 mg or 160 to 450 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

15) The method according to any one of embodiments 1-5, wherein 240 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

16) The method according to any one of embodiments 1-5, wherein 320 to 480 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

17) The method according to any one of embodiments 1-5, wherein 360 to 640 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

18) The method according to any one of embodiments 1-5, wherein 380 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

19) The method according to any one of embodiments 1-5, wherein 160 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

20) The method according to any one of embodiments 1-5, wherein 320 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

21) The method according to any one of embodiments 1-5, wherein 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

22) The method according to any one of embodiments 1-5, wherein 400 or 720 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

23) The method of any one of embodiments 1-22, wherein at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered as a single dose once a day.

24) The method of any one of embodiments 1-22, wherein at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered in two doses daily.

25) The method according to any one of embodiments 1-3 and 5, wherein 25 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

26) The method according to any one of embodiments 1-3 and 5, wherein 50 to 150 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

27) The method according to any one of embodiments 1-3 and 5, wherein 75 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof are administered daily.

28) The method according to any one of embodiments 1-3 and 5, wherein 50 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

29) The method according to any one of embodiments 1-3 and 5, wherein 100 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

30) The method of any one of embodiments 1-3, 5 and 25-29, wherein at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered as a single dose once a day.

31) The method according to any one of embodiments 1-3, 5 and 25-29, wherein at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered in two doses daily.

32) The method of any one of embodiments 1, 2, 4 and 5, wherein: (i) 50 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 50 to 600 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

- 31 041854

33) The method of any one of embodiments 1, 2, 4, and 5, wherein: (i) 50 to 450 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 50 to 450 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

34) The method of any one of embodiments 1, 2, 4 and 5, wherein: (i) 100 to 400 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 100 to 400 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

35) The method of any one of embodiments 1, 2, 4 and 5, wherein: (i) 125 to 300 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 125 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

36) The method according to any one of embodiments 1, 2, 4 and 5, wherein: (i) 150 to 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 150 mg to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

37) The method of any one of embodiments 1, 2, 4 and 5, wherein: (i) 200 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 100 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

38) The method according to any one of embodiments 1, 2, 4 and 5, wherein: (i) 300 or 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily or (ii) 150 , 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered daily.

39) The method according to any one of embodiments 1, 2, 4 and 5, wherein: (i) 150 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts are administered twice a day or (ii) 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered once a day.

40) The method of any one of embodiments 1, 2, 4, 5, and 32-38, wherein at least one compound selected from Compound III or Compound III-d and pharmaceutically acceptable salts thereof is administered as a single dose of one once a day.

41) The method of any one of embodiments 1, 2, 4, 5, and 32-38, wherein the dose of at least one compound selected from compound III or compound III-d and its pharmaceutically acceptable salts is administered in two doses daily .

42) The method of any one of embodiments 1, 3, 4 and 5, wherein 100 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

43) The method of any one of embodiments 1, 3, 4 and 5, wherein 400 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

44) The method according to any one of embodiments 1, 3, 4 and 5, wherein 800 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

45) The method of any one of embodiments 1, 3, 4, 5 and 41-44, wherein 400 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered twice a day.

46) The method of any one of embodiments 1, 3, 4, 5, and 41-44, wherein the dose of at least one compound selected from Compound IV and its pharmaceutically acceptable salts is administered as a single dose per day or as two doses per day.

47) The method of embodiment 1 wherein said patient having cystic fibrosis is selected from F508del/minimal function patients, F508del/F508del genotypes, F508del/gateway genotypes, F508del/residual function patients, and patients with F508del genotypes/another CFTR genetic mutation expected to be and/or responsive to the triple combination of Compound I, Compound II, and Compound III based on in vitro and/or clinical data.

48) The method of Embodiment 1 wherein 80 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

49) The method of embodiment 1 wherein: (i) 50 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof are administered daily and/or 150 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts is administered daily or (ii) 50 to 200 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts is administered daily

- 32 041854 and/or from 100 to 400 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered daily.

50) The method according to any one of embodiments 1, 48 and 49, wherein: (i) 150 to 600 mg of at least one compound selected from compound III and pharmaceutically acceptable salts thereof; and/or 400 to 1000 mg of at least one compound selected from compound IV and its pharmaceutically acceptable salts are administered daily or (ii) 100 to 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable acceptable salts; and/or 400 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof are administered daily.

51) A method for treating cystic fibrosis, comprising daily administration to a patient in need of such treatment, a pharmaceutical composition containing:

(A) 10 to 900 mg of at least one compound selected from compound I

and its pharmaceutically acceptable salts;

(B) at least one compound selected from: (i) compound II

(ii) compounds III or compounds III-d:

and (iii) compound IV and pharmaceutically acceptable salts of any of the above; and (C) a pharmaceutically acceptable carrier.

52) The method of embodiment 51, wherein: (i) the pharmaceutical composition contains: at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound III and pharmaceutically acceptable salts thereof; or (ii) the pharmaceutical composition contains: at least one compound selected from compound I and pharmaceutically acceptable salts thereof; at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof.

53) The method of embodiment 51, wherein the pharmaceutical composition contains: at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

54) The method of embodiment 51, wherein: (i) the pharmaceutical composition contains: at least one compound selected from Compound I and pharmaceutically acceptable compounds thereof

- 33 041854 lei; at least one compound selected from compound III and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof; or (ii) the pharmaceutical composition contains: at least one compound selected from compound I and pharmaceutically acceptable salts thereof; at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

55) The method of Embodiment 51, wherein the pharmaceutical composition comprises: (A) (i) at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound II and pharmaceutically acceptable salts thereof; (ii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound III and pharmaceutically acceptable salts thereof; or (iii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof; or (B) (i) at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound II and pharmaceutically acceptable salts thereof; (ii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof; or (iii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

56) The method of any one of embodiments 51-55, wherein 20 to 800 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

57) The method of any one of embodiments 51-55, wherein 30 to 720 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

58) The method of any one of embodiments 51-55, wherein 40 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

59) The method of any one of embodiments 51-55, wherein 60 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

60) The method of any one of embodiments 51-55, wherein 60 to 500 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

61) The method of any one of embodiments 51-55, wherein 80 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

62) The method of any one of embodiments 51-55, wherein 120 to 450 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

63) The method of any one of embodiments 51-55, wherein 120 to 500 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

64) The method of any one of embodiments 51-55, wherein 160 to 450 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

65) The method of any one of embodiments 51-55, wherein 240 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

66) The method of any one of embodiments 51-55, wherein 320 to 480 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

67) The method of any one of embodiments 51-55, wherein 360 to 640 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

68) The method of any one of embodiments 51-55, wherein 380 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

69) The method of any one of embodiments 51-55, wherein 160 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

- 34 041854

70) The method of any one of embodiments 51-55, wherein 320 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

71) The method of any one of embodiments 51-55, wherein 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

72) The method of any one of embodiments 51-55, wherein 480 or 720 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

73) The method of any one of embodiments 51, 53 and 55, wherein 25 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

74) The method of any one of embodiments 51, 53, and 55, wherein 50 to 150 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

75) The method of any one of embodiments 51, 53 and 55, wherein 75 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

76) The method of any one of embodiments 51, 53 and 55, wherein 50 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

77) The method of any one of embodiments 51, 53 and 55, wherein 100 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

78) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 50 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 50 to 600 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

79) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 50 to 450 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 50 to 450 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

80) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 100 to 400 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 100 to 400 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

81) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 125 to 300 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 125 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

82) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 150 to 300 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 150 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

83) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 200 mg to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii ) 100 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

84) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 300 mg or 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered daily.

85) The method of any one of embodiments 51, 52, 54, and 55, wherein: (i) 150 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered twice daily, or (ii) 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered once a day.

86) The method of any one of embodiments 51, 53, 54 and 55, wherein 100 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

87) The method of any one of embodiments 51, 53, 54 and 55, wherein 400 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

- 35 041854

88) The method of any one of embodiments 51, 53, 54, and 55, wherein 800 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof are administered daily, or 400 mg of at least one compound selected from Compound IV and its pharmaceutically acceptable salts are administered twice a day.

89) The method of Embodiment 51 wherein said patient having cystic fibrosis is selected from F508del/minimal function patients, F508del/F508del genotype patients, F508del/gateway genotype patients, and F508del/residual function genotype patients.

90) The method of Embodiment 51, wherein said pharmaceutical composition contains 80 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof and is administered daily.

91) The method of embodiment 90, further wherein: (i) 50 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof are administered daily and/or 150 to 600 mg of at least one a compound selected from Compound III and its pharmaceutically acceptable salts is administered daily or (ii) 50 to 200 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts is administered daily and/or 100 to 400 mg at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof is administered daily.

92) The method of any one of embodiments 51, 90, and 91, wherein: (i) 150 to 600 mg of at least one compound selected from Compound III and pharmaceutically acceptable salts thereof; and/or 400 to 1000 mg of at least one compound selected from compound IV and its pharmaceutically acceptable salts are administered daily or (ii) 100 to 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable acceptable salts; and/or 400 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof are administered daily.

93) A method for treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) a first pharmaceutical composition containing 10 to 900 mg of at least one compound selected from compound I

and its pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier in which the specified first pharmaceutical composition is administered daily; and (B) a second pharmaceutical composition containing at least one compound selected from (i) compound II:

and (ii) compounds III or compounds III-d and (iii) compounds IV:

- 36 041854

and pharmaceutically acceptable salts of any of the foregoing, and a pharmaceutically acceptable carrier.

94) The method of Embodiment 93, wherein the first pharmaceutical composition comprises: (i) at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains at least one compound selected from compound II and its pharmaceutically acceptable salts; and at least one compound selected from compound III and pharmaceutically acceptable salts thereof; or (ii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains at least one compound selected from compound II and its pharmaceutically acceptable salts; and at least one compound selected from compound IIId and pharmaceutically acceptable salts thereof.

95) The method of embodiment 93, wherein the first pharmaceutical composition contains: at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains at least one compound selected from compound II and its pharmaceutically acceptable salts; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

96) The method of Embodiment 93, wherein the first pharmaceutical composition comprises: (i) at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains: at least one compound selected from compound III and its pharmaceutically acceptable salts; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof; or (ii) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains: at least one compound selected from compound III-d and its pharmaceutically acceptable salts; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

97) The method of Embodiment 93, wherein the first pharmaceutical composition comprises: (a) at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains: (i) at least one compound selected from compound II and its pharmaceutically acceptable salts; and at least one compound selected from compound III and pharmaceutically acceptable salts thereof; or (ii) at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof; or (b) at least one compound selected from compound I and pharmaceutically acceptable salts thereof; and the second pharmaceutical composition contains: (i) at least one compound selected from compound II and its pharmaceutically acceptable salts; and at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof; or (ii) at least one compound selected from compound II and pharmaceutically acceptable salts thereof; and at least one compound selected from compound IV and pharmaceutically acceptable salts thereof.

98) The method of any one of embodiments 93-97 wherein 20 to 800 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

99) The method of any one of embodiments 93-97 wherein 30 to 720 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

100) The method of any one of embodiments 93-97 wherein 40 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

101) The method of any one of embodiments 93-97, wherein 60 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

102) The method of any one of embodiments 93-97 wherein 60 to 500 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

103) The method of any one of embodiments 93-97 wherein 80 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

- 37 041854

104) The method of any one of embodiments 93-97 wherein 120 to 450 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

105) The method of any one of embodiments 93-97 wherein 120 to 500 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

106) The method of any one of embodiments 93-97, wherein 160 to 450 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

107) The method of any one of embodiments 93-97 wherein 240 to 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

108) The method of any one of embodiments 93-97 wherein 320 to 480 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

109) The method of any one of embodiments 93-97, wherein 360 to 640 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered daily.

110) The method of any one of embodiments 93-97 wherein 380 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

111) The method of any one of embodiments 93-97 wherein 160 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

112) The method of any one of embodiments 93-97 wherein 320 to 420 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

113) The method of any one of embodiments 93-97 wherein 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

114) The method of any one of embodiments 93-97 wherein 400 or 720 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

115) The method of any one of embodiments 93-114 wherein at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered as a single dose once a day.

116) The method of any one of embodiments 93-114 wherein at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered in two doses daily.

117) The method of any one of embodiments 93-95 and 97, wherein 25 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

118) The method of any one of embodiments 93-95 and 97 wherein 50 to 150 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

119) The method of any one of embodiments 93-95 and 97, wherein 75 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

120) The method of any one of embodiments 93-95 and 97, wherein 50 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

121) The method of any one of embodiments 93-95 and 97 wherein 100 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered daily.

122) The method of any one of embodiments 93-95, 97, and 117-121 wherein at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered as a single dose once a day.

123) The method of any one of embodiments 93-95, 97 and 117-121 wherein at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered in two doses daily.

124) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 50 to 600 mg of at least one compound selected from compound III and is pharmaceutically

- 38 041854 acceptable salts are administered daily or (ii) 50 to 600 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered daily.

125) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 50 to 450 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 50 to 450 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

126) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 100 to 400 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 100 to 400 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

127) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 125 to 300 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 125 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

128) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 150 to 300 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 150 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

129) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 200 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 100 to 300 mg of at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof are administered daily.

130) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 300 or 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 150 , 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered daily.

131) The method of any one of embodiments 93, 94, 96, and 97, wherein: (i) 150 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered twice daily, or (ii) 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered once a day.

132) The method of any one of embodiments 93, 94, 96, 97, and 124-130, wherein at least one compound selected from compound III or III-d and pharmaceutically acceptable salts thereof is administered once a day.

133) The method of any one of embodiments 93, 94, 96, 97, and 124-130, wherein the dose of at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts is administered twice daily.

134) The method of any one of embodiments 93 and 95-97, wherein 100 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof are administered daily.

135) The method of any one of embodiments 93 and 95-97, wherein 400 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

136) The method of any one of embodiments 93 and 95-97, wherein 400 to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered daily.

137) The method of any one of embodiments 93, 95-97 and 133-136, wherein 800 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered once a day.

138) The method of any one of embodiments 93, 95-97 and 133-136, wherein 400 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof is administered twice a day.

139) The method of Embodiment 93, wherein said patient having cystic fibrosis is selected from F508del/minimal function patients, F508del/F508del genotypes, F508del/gateway genotypes, and F508del/residual function patients.

140) The method of Embodiment 93, wherein said first pharmaceutical composition contains 80 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof administered daily.

141) The method of Embodiment 93 wherein: (i) 50 to 200 mg of at least one compound selected from Compound II and pharmaceutically acceptable salts thereof are administered daily and/or 150 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered daily, or (ii) 50 to 200 mg of at least one

- 39 041854 compounds selected from compound II and its pharmaceutically acceptable salts are administered daily and / or from 100 to 400 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered daily.

142) The method according to any one of embodiments 93, 140 and 141, wherein: (i) 150 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts, and/or 400 to 1000 mg at least one compound selected from compound IV and its pharmaceutically acceptable salts is administered daily, or (ii) from 100 to 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts, and/or from 400 up to 1000 mg of at least one compound selected from compound IV and pharmaceutically acceptable salts thereof are administered daily.

143) The method of any one of embodiments 93-142, wherein said second pharmaceutical composition is administered before, after, or simultaneously with said first pharmaceutical composition.

144) The method of any one of embodiments 93-143, further comprising administering to said patient a third pharmaceutical composition, said composition comprising: (i) at least one compound selected from Compound II, Compound III, Compound IV, and pharmaceutically acceptable salts; or (ii) at least one compound selected from compound II, compound III-d, compound IV, and pharmaceutically acceptable salts thereof.

145) The method of embodiment 144 wherein said third pharmaceutical composition is administered once a day.

146) The method of any one of embodiments 1, 51, and 93, comprising administering Compound I to said patient.

147) The method of any one of embodiments 1, 51, and 93, comprising administering to said patient a pharmaceutically acceptable salt of Compound I.

148) The method of any one of embodiments 1, 51, and 93, comprising administering compound II to said patient.

149) The method of any one of embodiments 1, 51 and 93, comprising administering to said patient a pharmaceutically acceptable salt of Compound II.

150) The method of any one of embodiments 1, 51 and 93, comprising administering compound III or compound III-d to said patient.

151) The method of any one of embodiments 1, 51 and 93, comprising administering to said patient a pharmaceutically acceptable salt of Compound III or a pharmaceutically acceptable salt of Compound III-d.

152) The method of any one of embodiments 1, 51, and 93, comprising administering compound IV to said patient.

153) The method of any one of embodiments 1, 51 and 93, comprising administering to said patient a pharmaceutically acceptable salt of Compound IV.

154) The method of any one of embodiments 1, 51, and 93, comprising administering to said patient: (i) a pharmaceutically acceptable salt of Compound I; compound II and compound III-d or (ii) a pharmaceutically acceptable salt of compound I; compounds II and compounds III.

155) The method of any one of embodiments 1, 51, and 93, comprising administering to said patient: (i) Compound I; compounds II and compounds III or (ii) compounds I; compounds II and compounds III-d.

156) The method of any one of embodiments 1, 51, and 93, comprising administering to said patient: (i) Compound I and Compound III, or (ii) Compound I and Compound III-d.

157) The method of any one of embodiments 1, 51 and 93, comprising administering to said patient: (i) a pharmaceutically acceptable salt of Compound I and Compound III, or (ii) a pharmaceutically acceptable salt of Compound I and Compound III-d.

158) The method of any one of embodiments 47, 89, or 139, wherein the F508del/minimal function genotype patient has a minimal function mutation selected from

- 40 041854

Mx<acn

MX C/AU I'm xx ear: LX--to · -Lk. -X - — —— - — L·X.L.^ - -L -L - _L CHA L. L A χτ . Λ,Μ .$4.4 *. /D/x hah /pit i:: see CH V ΆΆ,,Χ TO? 5 5 U+A ΒύΧ kau UUAG A' g: LX-MX milestones 06U A and \ m s Ά ⁴ A * H k Y OU: KUH AND HIM SU U x+yx 2' - 5 AH.L V y yy U U C 5 AH I'm at w; Q2MA YAHU K, AAH ... ..... A?g to A...

T WOO A -' 2 A “E-IG-T XX-AY ?A \ AA-A A;- .V- XX AG-A, 3.4 "<Y< x l - Ax T to-U ^{4 w} J A ¹ - ⁴ ' ⁴ ' U ..A X YU U+Ah DUU AH HU A : * ί X-v ABOUT- THEM ·?Α»«ν HUHHUA OOOO _λ Y , V--- i / 5/+)0—A AU-K—< / A—U

,χΙ,Α&Α MDh&A ;g A, A G MAUX- 5th5 I U'UUK' 3?UvAT U & Π AAJ MaxssedT -AEU1 Ά- MAG i iX.Λ ::¾ a : sa a a 4'4' Ms? A ASSis. TA A S-U a A ZZU'A CHUYA .4xx®A j S^s.-J№ A. 4$ UMU ^: G 'el ' GxAXT »M&iMG MJ SPIRIT *? U/A Uch+x1 £ A 2 ^L UMU WV— U'chzh1L 2 A SAT . _: .V son-in-law y: ^g W.&;4 mx j Ah.? , SGTYAU+.X ? ^v y y 3 .) AfeJ v ⁴ UU'U-.d AMU) - + A A Ax A zhАААУ 43 42 GTGTT-A4.

159) The method of any one of embodiments 47, 89, or 139, wherein the F508del/gateway patient has a gatelet mutation selected from G178R, S549N, S549R, G551D, G551S, G1244E, S1251N, S1255P, and G1349D.

160) The method of any one of embodiments 47, 89, or 139, wherein the F508del/residual function genotype patient has a residual function mutation selected from

2789+5G^A, 3849+lOkbC^T, 3272-26A^G, 711+3A^G,

Е56К, P67L, R74W, D110E, D110H, R117C, L206W, R347H, R352Q, А455Е, D579G, Е831Х, S945L, S977F, F1052V, R1070W, F1074L, D1152H, D1270N, Е193К, К1060Т, R117H, S1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, RI 162L, E56K, A1067T, E193K and K1060T.

161) The method of embodiment 51 or 93 wherein the pharmaceutically acceptable carrier is HPMCAS-HG.

162) The method of any one of embodiments 1-161, wherein: (i) 100 to 600 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered once a day; 50 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered once a day and 150 to 600 mg of compound III is administered twice a day or (ii) 100 to 600 mg of at least at least one compound selected from compound I and its pharmaceutically acceptable salts is administered once a day; 50 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered once a day; and 150 to 600 mg of compound III-d is administered twice a day.

163) The method of any one of embodiments 1-161, wherein: (i) 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered once a day; 100 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts are administered once a day and 150 or 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts are administered twice a day.

- 41 041854 day or (ii) 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts are administered once a day; 100 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts are administered once a day and 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered alone once a day.

164) The method of any one of embodiments 1-161, wherein: (i) 50 to 300 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered twice a day; 50 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts are administered twice a day and 150 or 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts are administered twice a day or (ii) 50 to 300 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered twice a day; 50 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts are administered twice a day and 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered alone once a day.

165) The method of any one of embodiments 1-161, wherein: (i) 100 to 600 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof is administered once a day and 150 to 600 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts is administered twice daily or (ii) 100 to 600 mg of at least one compound selected from Compound I and its pharmaceutically acceptable salts is administered once per day and from 150 to 600 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered once a day.

166) The method of any one of embodiments 1-161, wherein: (i) 50 to 300 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof are administered twice a day and 150 mg or 300 mg of at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered twice a day or (ii) 50 to 300 mg of at least one compound selected from Compound I and its pharmaceutically acceptable salts are administered twice per day and 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered once a day.

167) The method of any one of embodiments 162-166 wherein 80, 160, 240, 320, or 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof is administered once a day.

168) The method of any one of embodiments 162-166, wherein 160 or 240 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered twice a day.

169) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, or 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered once a day ; and 100 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts is administered once a day; and 150 or 300 mg of at least one compound selected from compound III and pharmaceutically acceptable salts thereof are administered twice a day or (ii) 80, 160, 240, 320 or 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts are administered once a day; and 100 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts is administered once a day; and 150 or 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts is administered once a day.

170) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, or 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered once a day ; and 100 mg of compound II is administered once a day; and 150 or 300 mg of compound III is administered twice a day or (ii) 80, 160, 240, 320 or 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts are administered once a day; and 100 mg of compound II is administered once a day; and 150, 200 or 300 mg of compound III-d is administered once a day.

171) The method of any one of embodiments 1-161, wherein: (i) 160 or 240 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered twice a day; 100 mg of compound II is administered once a day; and 150 or 300 mg of compound III is administered twice a day; or (ii) 160 or 240 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts are administered twice a day; 100 mg of compound II is administered once a day; and 150, 200 or 300 mg of compound III-d is administered once a day.

172) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, or 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof are administered once a day and 150 mg or 300 mg of at least one compound selected from compound III and pharmaceutically acceptable salts thereof are administered twice a day, or (ii) 80,

- 42 041854

160, 240, 320 or 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts are administered once a day and 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts are administered once a day.

173) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, or 400 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered once a day and 200 or 300 mg of compound III-d is administered once a day or (ii) 80, 160, 240, 320 or 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts are administered once a day and 150, 200 or 300 mg of compound III-d is administered once a day.

174) The method of any one of embodiments 1-161, wherein: (i) 160 or 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof are administered twice a day and 150 or 300 mg of the compound III is administered twice a day or (ii) 160 or 240 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts is administered twice a day and 150, 200 or 300 mg of compound III-d is administered once a day day.

175) The method of any one of embodiments 1-161, wherein: (i) 160 or 240 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof are administered twice daily and 150 or 300 mg of the compound III is administered twice a day or (ii) 160 or 240 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts is administered twice a day and 150, 200 or 300 mg of compound III-d is administered once a day day.

176) The method of any one of embodiments 1-161, wherein 80 to 800 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is administered daily.

177) The method of any one of embodiments 1-161 wherein 80, 160, 240, 320, 400, 480, 560, 640, or 720 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof are administered daily.

178) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, 400, 480, 560, 640, or 720 mg of at least one compound selected from Compound I and its pharmaceutical acceptable salts administered daily; 50 to 200 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts is administered once a day and 150 to 600 mg of compound III is administered twice a day or (ii) 80, 160, 240, 320 , 400, 480, 560, 640 or 720 mg of at least one compound selected from Compound I and its pharmaceutically acceptable salts are administered daily; 50 to 200 mg of at least one compound selected from compound II and pharmaceutically acceptable salts thereof is administered once a day; and 150 to 600 mg of compound III-d is administered once a day.

179) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, 400, 480, 560, 640, or 720 mg of at least one compound selected from Compound I and its pharmaceutical acceptable salts administered daily; 100 mg of compound II is administered once a day and 150 or 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts is administered twice a day or (ii) 80, 160, 240, 320, 400, 480 , 560, 640 or 720 mg of at least one compound selected from Compound I and its pharmaceutically acceptable salts are administered daily; 100 mg of compound II is administered once a day and 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts is administered once a day.

180) The method of any one of embodiments 1-161, wherein: (i) 80, 160, 240, 320, 400, 480, 560, 640, or 720 mg of at least one compound selected from compound I and its pharmaceutical acceptable salts administered daily; 50 mg of compound II is administered twice a day and 150 or 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts is administered twice a day or (ii) 80, 160, 240, 320, 400, 480 , 560, 640 or 720 mg of at least one compound selected from Compound I and its pharmaceutically acceptable salts are administered daily; 50 mg of compound II is administered twice a day and 150, 200 or 300 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable salts is administered once a day.

181) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 120 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, twice a day

- 43 041854

(B) 100 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts once a day or 50 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts twice a day

and (C) 150 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts, twice a day

182) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

(B) 100 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts once a day or 50 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts twice a day

and (C) 150 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts, twice a day

183) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

(B) 100 mg of at least one compound selected from compound II and its pharmaceutical

- 44 041854 sky acceptable salts, once a day or 50 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts, twice a day

and (C) 150 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts, twice a day

184) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 120 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, twice a day

(B) 100 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts once a day or 50 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts twice a day

and (C) 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts, twice a day

185) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

(B) 100 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts once a day or 50 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts twice a day

- 45 041854

and (C) 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts, twice a day

186) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, twice a day

(B) 100 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts once a day or 50 mg of at least one compound selected from Compound II and its pharmaceutically acceptable salts twice a day

and (C) 300 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts, twice a day

187) The method of any one of embodiments 181-186 wherein said patient having cystic fibrosis is selected from patients with F508del/minimal function genotypes, patients with F508del/F508del genotypes, patients with F508del/gateway genotypes, and patients with F508del genotypes /residual function.

188) The method of embodiment 187 wherein the F508del/minimum function genotype patient has a minimal function mutation selected from

- 46 041854

Mutation

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189) The method of Embodiment 187, wherein the F508del/gateway patient has a gatelet mutation selected from G178R, S549N, S549R, G551D, G551S, G1244E, S1251N, S1255P, and G1349D.

190) The method of embodiment 187, wherein the F508del/residual function patient has a residual function mutation selected from

2789+5G^A, 3849+1OkbC^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, S1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, E106T, A.06K1 and E56K1

191) The method of any one of embodiments 181-190, wherein the absolute change in percentage predicted forced expiratory volume in said patient in one second (ppFEV1) following 15 days of administration of said at least one compound selected from Compound I and pharmaceutically acceptable compounds thereof salts of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, is in the range from 3 to 40% relative to pFOEF1 in the patient prior to the introduction.

192) The method of embodiment 191, wherein said patient has one F508del mutation and one minimal function mutation, and wherein said patient has not taken any of said at least one compound selected from compound I and at least one pharmaceutically acceptable salt thereof a compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts.

- 47 041854

193) The method of embodiment 191 wherein said patient has two copies of the F508del mutation and wherein the patient has taken at least one compound selected from compound II and pharmaceutically acceptable salts thereof and at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, but not any of said at least one compound selected from Compound I and its pharmaceutically acceptable salts.

194) The method of any one of embodiments 181-193, wherein said absolute change in ppFEV ₁ in said patient is in the range of 3 to 35%.

195) The method of any one of embodiments 181-194, wherein said at least one compound selected from compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; said at least one compound selected from compound II and its pharmaceutically acceptable salts is contained in the second pharmaceutical composition; and said at least one compound selected from compound III or IIId and its pharmaceutically acceptable salts is contained in the third pharmaceutical composition.

196) The method of any one of embodiments 181-194, wherein said at least one compound selected from compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition and said at least one compound selected from compound II and its pharmaceutically acceptable salts, and said at least one compound selected from compound III or III-d and its pharmaceutically acceptable salts, are contained in the second pharmaceutical composition.

197) The method of embodiment 196, wherein said second pharmaceutical composition comprises half of the daily dose of said at least one compound selected from Compound III and pharmaceutically acceptable salts thereof and the other half of the daily dose of said at least one compound selected from Compound III and its pharmaceutically acceptable salts are administered to said patient in a third pharmaceutical composition.

198) The method of any one of embodiments 181-194, wherein said at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; said at least one compound selected from compound II and its pharmaceutically acceptable salts is contained in the second pharmaceutical composition and said at least one compound selected from (i) compound III and its pharmaceutically acceptable salts or (ii) compound III-d and its pharmaceutically acceptable salts is contained in the first pharmaceutical composition.

199) The method of any one of embodiments 181-194, wherein said at least one compound is selected from Compound I and pharmaceutically acceptable salts thereof, said at least one compound is selected from Compound II and pharmaceutically acceptable salts thereof, and said at least one compound selected from i) compound III and its pharmaceutically acceptable salts or (ii) compound III-d and its pharmaceutically acceptable salts are contained in the first pharmaceutical composition.

200) The method of embodiment 199 wherein the first pharmaceutical composition is administered to the patient twice a day.

201) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 120 mg of at least one compound selected from Compound I and pharmaceutically acceptable salts thereof twice a day (B) 100 mg of Compound II once a day or 50 mg of Compound II twice a day

ΌH and (C) 150 mg Compound III twice a day

- 48 041854

202) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 150 mg Compound III twice a day

203) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 150 mg Compound III twice a day

204) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 120 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, twice a day

- 49 041854

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

and (C) 300 mg Compound III twice a day

205) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 300 mg Compound III twice a day

206) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, twice a day

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

- 50 041854

and (C) 300 mg Compound III twice a day

207) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 200 mg Compound III-d once a day

208) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 200 mg Compound III-d once a day

- 51 041854

209) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 400 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 150 mg Compound III-d once a day

210) A method of treating cystic fibrosis, comprising administering to a patient in need of such treatment:

(A) 240 mg of at least one compound selected from compound I and pharmaceutically acceptable salts thereof, once a day

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

and (C) 150 mg Compound III-d once a day

211) The method of any one of embodiments 201-210 wherein said patient having cystic fibrosis is selected from F508del/minimal function patients, F508del/F508del genotype patients, F508del/gateway genotype patients, F508del genotype patients /residual function.

212) The method of embodiment 211 wherein the F508del/minimal function genotype patient has a minimal function mutation selected from

- 52 041854

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213) The method of embodiment 211 wherein the F508del/gateway patient has a gatelet mutation selected from G178R, S549N, S549R, G551D, G551S, G1244E, S1251N, S1255P, and G1349D.

214) The method of embodiment 211 wherein the F508del/residual function patient has a residual function mutation selected from

2789+5Cu A, 3849+YUKCHIT, 3272-26A^G, 711+3A^G, E56K, P67L, R74W, D110E, D110H, R117C, L206W, R347H, R352Q, A455E, D579G, E831X, S945L, S952F, F1072F , R1070W, F1074L, D1152H, D1270N, Е193К, К1060Т, R117H, S1235R, I1027T, R668C, G576A, M470V, L997F, R75Q, R1070Q, R31C, D614G, G1069R, R1162L, Е56К, А1067Т, Е193К и К1060Т.

215) The method of any one of embodiments 201-214, wherein the absolute change in percent predicted forced expiratory volume in said patient in one second (ppFEV1) following 15 days of administration of said at least one compound selected from Compound I and pharmaceutically acceptable compounds thereof salts; Compound II and Compound III range from 3% to 40% relative to the patient's pFOEF1 prior to said administration.

216) The method of embodiment 211, wherein said patient has one F508del mutation and one minimal function mutation, and wherein said patient has not taken any of said at least one compound selected from Compound I and pharmaceutically acceptable salts thereof; compounds II and compounds III.

217) The method of embodiment 211 wherein said patient has two copies of the F508del mutation and wherein the patient has taken Compound II and Compound III but not any of said at least one compound selected from Compound I and pharmaceutically acceptable salts thereof.

- 53 041854

218) The method of any one of embodiments 201-210, wherein said absolute change in pFOEF1 in said patient is in the range of 3 to 35%.

219) The method of any one of embodiments 201-210, wherein said at least one compound selected from compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; said compound II is contained in the second pharmaceutical composition; and said compound III or compound III-d is contained in the third pharmaceutical composition.

220) The method of any one of embodiments 201-210, wherein said at least one compound selected from compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition and said compound II and said compound III or said compound III and said compound III-d is contained in the second pharmaceutical composition.

221) The method of embodiment 216 wherein said second pharmaceutical composition comprises half of the daily dose of said Compound III and the other half of the daily dose of Compound III is administered to said patient in a third pharmaceutical composition.

222) The method of any one of embodiments 201-210, wherein said at least one compound selected from Compound I and pharmaceutically acceptable salts thereof is contained in the first pharmaceutical composition; said compound II is contained in the second pharmaceutical composition; and said compound III or compound III-d is contained in the first pharmaceutical composition.

223) The method of any one of embodiments 201-210, wherein said at least one compound is selected from Compound I and pharmaceutically acceptable salts thereof; said compound II and said compound III or Compound III-d are contained in the first pharmaceutical composition.

224) The method of embodiment 219 wherein the first pharmaceutical composition is administered to the patient twice a day.

225) The method of any one of embodiments 181-191, wherein the absolute change in percentage predicted forced expiratory volume in said patient in one second (ppFEV1) following 15 days of administration of said at least one compound selected from Compound I and pharmaceutically acceptable compounds thereof salts; of at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III and its pharmaceutically acceptable salts, is in the range from 3% to 40% relative to pFOEF1 in the patient prior to said administration .

226) The method of any one of embodiments 181-193 and 225, wherein said absolute change in pFEV1 in said patient is in the range of 3 to 35%.

227) The method of any one of embodiments 201-210, wherein the absolute change in percent predicted forced expiratory volume in said patient in one second (ppFEV1) following 15 days of administration of said at least one compound selected from Compound I and pharmaceutically acceptable compounds thereof salts; at least one compound selected from compound II and its pharmaceutically acceptable salts, and at least one compound selected from compound III and its pharmaceutically acceptable salts, is in the range from 3 to 40% relative to the patient's pFOEF1 prior to said administration.

228) The method of any one of embodiments 201-210 and 227, wherein said absolute change in pFEV1 in said patient is in the range of 3 to 35%.

Methods for obtaining compounds.

General experimental techniques.

Reagents and starting materials were obtained from commercial sources, unless otherwise indicated, and were used without purification. Proton and carbon NMR spectra were obtained either on a Bruker Biospin DRX 400 MHz FTNMR spectrometer operating at 1H and ¹³ C resonance frequencies of 400 and 100 MHz, respectively, or on a 300 MHz NMR spectrometer. One-dimensional proton and carbon spectra were obtained using a broadband probe (BBFO) with sample rotation at 20 Hz at a digital resolution of 0.1834 and 0.9083 Hz/Pt, respectively. All proton and carbon spectra were temperature controlled at 30° C. using standard, previously published pulse sequences and standard processing parameters. The final purity of the compounds was determined by reverse phase UPLC using an Acquity UPLC BEN C ₁₈ column (50 x 2.1 mm, 1.7 µm particles) manufactured by Waters (pn: 186002350) and a dual gradient from 1-99% mobile phase B within 3.0 minutes. Mobile phase A=H ₂ O (0.05% CF ₃ CO ₂ H). Mobile phase B=CH ₃ CK (0.035% CF3CO2H). Flow rate=1.2 ml/min, injection volume=1.5 μl and column temperature=60°C. The 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 an accuracy of 0.1 Da and a minimum resolution of 1000 amu. in the detection range

- 54 041854 ionization using electrospray ionization (ESI) using hydrogen ion (H ⁺ ). The optical purity of methyl-(2S)-2,4-dimethyl-4-nitropentanoate was determined using a chiral gas chromatography (GC) analysis on an Agilent 7890A/MSD 5975C instrument using a Restek Rt-eDEXcst column (30mx0.25mmx0.25um_df ), with a flow rate of 2.0 ml/min (carrier gas H ₂ ), at an inlet temperature of 220°C and an oven temperature of 120°C, 15 minutes.

Compounds I, II, III and III-d can be prepared by any suitable method in the art, for example, using PCT Publication Nos. WO 2011/133751 and WO 2015/160787 and US Patent No. 8865902.

Example 1 Synthesis of compound I.

Part A. Synthesis of (4S)-2,2,4-trimethylpyrrolidine hydrochloride

Stage 1. Synthesis of methyl 2,4-dimethyl-4-nitropentanoate l _x A. ψψΑ O Base f II NO ₂ ¹

Tetrahydrofuran (THF, 4.5 L) was added to a 20 L glass reactor and stirred under N ₂ at room temperature. 2-Nitropropane (1.5 kg, 16.83 mol) and 1.8 diazabicyclo[5.4.0]undec-7-ene (DBU) (1.282 kg, 8.42 mol) were then charged to the reactor and the jacket temperature was raised to 50°C. As soon as the contents of the reactor approached 50° C., methyl methacrylate (1.854 kg, 18.52 mol) was slowly added over 100 minutes. The reaction temperature was maintained at or close to 50° C. for 21 hours. The reaction mixture was concentrated in vacuo, then transferred back to the reactor and diluted with methyl tert-butyl ether (MTBE) (14 L). Added 2 M HCl (7.5 l) and the mixture was stirred for 5 minutes, then left to stand. Two transparent layers were visible - the lower yellow aqueous phase and the upper green organic phase. The aqueous layer was removed and the organic layer was stirred again with 2 M HCl (3 L). After separation, the HCl washes were recombined and stirred with MTBE (3 L) for 5 minutes. The aqueous layer was removed and all organic layers were combined in the reactor and stirred with water (3 L) for 5 minutes. After separation, the organic layers were concentrated in vacuo to give a cloudy green oil. It was dried over MgSO ₄ and filtered to give methyl 2,4-dimethyl-4-nitropentanoate as a clear green oil (3.16 kg, 99% yield). 1H NMR (400 MHz, chloroform-d) δ 3.68 (s, 3H), 2.562.35 (m, 2H), 2.11-2.00 (m, 1H), 1.57 (s, 3H) , 1.55 (s, 3H), 1.19 (d, J=6.8 Hz, 3H).

Stage 2. Synthesis of methyl-(2S)-2,4-dimethyl-4-nitropentanoate

The reactor was loaded with purified water (2090 l; 10 vol.) and then monobasic potassium phosphate (27 kg, 198.4 mol; 13 g/l to load water). The pH of the reactor contents was adjusted to 6.5 (±0.2) with a 20% (w/v) potassium carbonate solution. Racemic methyl 2,4-dimethyl-4-nitropentanoate (209 kg; 1104.6 mol) and Palatase 20000L lipase (13 L, 15.8 kg; 0.06 vol) were charged to the reactor.

The reaction mixture was brought to 32±2° C. and stirred for 15-21 hours, and the pH was maintained at 6.5 using the pH value with automatic addition of 20% potassium carbonate solution. When the racemic starting material was converted to >98% e.i. S-enantiomer, as determined by chiral GC, external heating was turned off. The reactor was then charged with MTBE (35 L; 5 vol) and the aqueous layer was extracted with MTBE (3 times, 400-1000 L). The combined organic extracts were washed with aqueous Na ₂ CO ₃ (4 times, 522 L, 18% w/w 2.5 vol.), water (523 L; 2.5 vol.) and 10% aqueous NaCl (314 L, 1.5 vol.). The organic layer was concentrated in vacuo to give methyl (2S)-2,4-dimethyl-4-nitropentanoate as a mobile yellow oil (>98% ee, 94.4 kg; 45% yield).

Stage 3. Synthesis of (3S)-3,5,5-trimethylpyrrolidin-2-one

The 20 L reactor was purged with N ₂ . The vessel was charged successively with deionized water-washed wet Raney Ni® (2800 grade, 250 g), methyl-(2S)-2,4-dimethyl-4-nitropentanoate (1741 g, 9.2 mol) and ethanol (13.9 L , 8 vol.) The reaction mixture was stirred at 900 rpm and the reactor was purged with H ₂ and maintained at ~2.5 bar. The reaction mixture was then heated to 60°C for 5 hours. The reaction mixture was cooled and filtered to remove Raney nickel and the solid was washed

- 55 041854 ethanol (3.5 l, 2 vol.). The ethanol solution of the product was combined with a second portion of equal volume and concentrated under vacuum to reduce to the minimum volume of ethanol (~1.5 volume). Heptane (2.5 L) was added and the suspension was again concentrated to ~1.5 volume. This was repeated 3 times; the resulting suspension was cooled to 0-5°C, filtered under vacuum and washed with heptane (2.5 l). The product was dried under vacuum for 20 minutes, then transferred to drying trays and dried in a vacuum oven at 40°C overnight to give (3S)-3,5,5-trimethylpyrrolidin-2-one as a white crystalline solid ( 2.042 kg, 16.1 mol, 87%). 1H NMR (400 MHz, chloroform-d) δ 6.39 (s, 1H), 2.62 (ddc, J=9.9, 8.6, 7.1 Hz, 1H), 2.17 (dd, J=12.4, 8.6Hz, 1H), 1.56(dd, J=12.5, 9.9Hz, 1H), 1.31(s, 3H), 1.25(s, 3H ), 1.20(d, J=7.1 Hz, 3H).

Stage 4. Synthesis of (4S)-2,2,4-trimethylpyrrolidine hydrochloride

About z^

i) LiAlH ₄ ii) HC1

Lithium aluminum hydride pellets (2.5 kg, 66 mol) and dry THF (60 L) were charged to a 120 L glass lined reactor and heated to 30°C. The resulting suspension was charged with (S)-3,5,5trimethylpyrrolidin-2-one (7.0 kg, 54 mol) in THF (25 L) over 2 hours while maintaining the reaction temperature at 30 to 40°C. After completion of the addition, the reaction temperature was raised to 60-63°C and maintained overnight. The reaction mixture was cooled to 22°C, then carefully quenched by adding ethyl acetate (EtOAc) (1.0 L, 10 mol) followed by the addition of a mixture of THF (3.4 L) and water (2.5 kg, 2.0 eq. .) and then a mixture of water (1.75 kg) with 50% aqueous sodium hydroxide (750 g, 2 eq. water with 1.4 eq. sodium hydroxide relative to aluminum) followed by the addition of 7.5 liters of water. After the addition was complete, the reaction mixture was cooled to room temperature and the solid was removed by filtration and washed with THF (3x25 L). The filtrate and washings were combined and treated with 5.0 L (58 mol) of aq. 37% HCl (1.05 eq.) keeping the temperature below 30°C. The resulting solution was concentrated by vacuum distillation to form a suspension. Isopropanol (8 L) was added and the solution was concentrated to almost dryness by vacuum distillation. Isopropanol (4 L) was added and the product was suspended by heating to approximately 50°C. Added MTBE (6 l) and the suspension was cooled to 2-5°C. The product was collected by filtration and washed with 12 L MTBE and dried in a vacuum oven (55°C/300 Torr/N2 outlet) to give (4S)-2,2,4-trimethylpyrrolidine HCl as a white crystalline solid (6.21 kg, yield 75%). 1H NMR (400 MHz, DMSO-d6) δ 9.34 (br.d, 2H), 3.33 (dd, J=11.4, 8.4 Hz, 1H), 2.75 (dd, J= 11.4, 8.6 Hz, 1H), 2.50-2.39 (m, 1H), 1.97 (dd, J=12.7, 7.7 Hz, 1H), 1.42 (s , 3H), 1.38 (dd, J=12.8, 10.1 Hz, 1H), 1.31 (s, 3H), 1.05 (d, J=6.6 Hz, 3H).

Part B. Synthesis of N-(benzenesulfonyl)-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1yl]-2-[(4S)-2,2,4-trimethylpyrrolidine-1- yl]pyridine-3-carboxamide (compound I)

About η l

Cl

2) GDI, PhSO ₂ NH ₂

- 56 041854

Synthesis of starting materials.

Synthesis of tert-butyl-2,6-dichloropyridine-3-carboxylate

A solution of 2,6-dichloropyridine-3-carboxylic acid (10 g, 52.08 mmol) in THF (210 ml) was treated successively with di-tert-butyl dicarbonate (17 g, 77.89 mmol) and 4-(dimethylamino)pyridine ( 3.2 g, 26.19 mmol) and stirred overnight at room temperature. At this point, 1 N HCl (400 ml) was added and the mixture was vigorously stirred for about 10 minutes. The product was extracted with ethyl acetate (2x300 ml) and the combined organic layers were washed with water (300 ml) and brine (150 ml) and dried over sodium sulfate and concentrated under reduced pressure to give 12.94 g (96% yield) of t- butyl 2,6-dichloropyridine-3-carboxylate as a colorless oil. ESI-MS m/z calc. 247.02, found 248.1 (M+1)+; retention time: 2.27 minutes. Ή NMR (300 MHz, CDCl ₃ ) ppm 1.60 (s, 9H), 7.30 (d, J=7.9 Hz, 1H), 8.05 (d, J=8.2 Hz, 1H).

Synthesis of tert-butyl-3-oxo-2,3-dihydro-1H-pyrazole-1-carboxylate

A 50 L Syrris controlled reactor was started and the jacket was adjusted to 20° C. with stirring at 150 rpm, reflux (10° C.) and nitrogen purge. MeOH (2.860 L) and methyl-(E)-3-methoxyprop-2-enoate (2.663 kg, 22.76 mol) were added and the reactor was capped. The reaction mixture was heated to an internal temperature of 40°C and the system was set to maintain the jacket temperature of 40°C. Hydrazine hydrate (1300 g, 55% w/w, 22.31 mol) was added portionwise via addition funnel over 30 minutes. The reaction mixture was heated to 60°C for 1 hour. The reaction mixture was cooled to 20°C and triethiamin (2.483 kg, 3.420 L, 24.54 mol) was added in portions while maintaining the reaction temperature <30°C. A solution of Boc anhydride (4.967 kg, 5.228 L, 22.76 mol) in MeOH (2.860 L) was added in portions while maintaining the temperature <45°C. The reaction mixture was stirred at 20° C. for 16 hours. The reaction solution was partially concentrated to remove MeOH, which resulted in the formation of a clear light amber oil. The resulting oil was transferred to a 50 L reactor, stirred and water (7.150 L) and heptane (7.150 L) were added. The additions caused a small amount of product to fall out. The aqueous layer was poured into a clean container and the interface and the heptane layer were filtered to separate the solid (product). The aqueous layer was transferred back to the reactor and the collected solid was placed back into the reactor and mixed with the aqueous layer. An addition funnel was added to the reactor and charged with acetic acid (1.474 kg, 1.396 L, 24.54 mol) and added dropwise. The jacket was set to 0°C to absorb the exothermic effect. After the addition was complete (pH=5), the reaction mixture was stirred for 1 hour. The solid was collected by filtration and washed with water (7.150 L) and washed a second time with water (3.575 L). The crystalline solid was transferred to a 20 L rotary evaporator flask and heptane (7.150 L) was added. The mixture was suspended at 45° C. for 30 minutes and 1-2 volumes of solvent were distilled off. The slurry in a rotary evaporator flask was filtered and the solids were washed with heptane (3.575 L). The solid was further dried under vacuum (50° C., 15 mbar) to give tert-butyl 5-oxo-H-pyrazole-2-carboxylate (2921 g, 71%) as a coarse crystalline solid. 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 7.98 (d, J=2.9 Hz, 1H), 5.90 (d, J=2.9 Hz, 1H ), 1.54 (s, 9H).

Synthesis of 2-[1 -(trifluoromethyl)cyclopropyl]ethanol

To a solution of lithium aluminum hydride (293 mg, 7.732 mmol) in THF (10.00 ml) in an ice bath was added dropwise 2-[1-(trifluoromethyl)cyclopropyl]acetic acid (1.002 g, 5.948 mmol) in THF (3.0 ml ) for 30 minutes while maintaining the reaction temperature below 20°C. The mixture was allowed to gradually warm to ambient temperature and stirred for 18 hours. The mixture was cooled in an ice bath and quenched successively with water (294 mg, 295 μl, 16.36 mmol), NaOH (297 μl 6 M, 1.784 mmol) and then water (884.0 µl, 49.07 mmol) to give a granular solid in the mixture. The solid was filtered off using celite and the precipitate was washed with ether. The filtrate was further dried over MgSO ₄ , filtered and concentrated in vacuo to give the product with residual THF and ether. The mixture was taken directly to the next step without further purification.

- 57 041854

Stage 1. tert-butyl-3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazole-1-carboxylate

tert-Butyl 5-oxo-H-pyrazole-2-carboxylate (1.043 g, 5.660 mmol), 2-[1(trifluoromethyl)cyclopropyl]ethanol (916 mg, 5.943 mmol) and triphenylphosphine (1.637 g, 6.243 mmol) were combined in THF (10.48 ml) and the reaction mixture was cooled in an ice bath. Diisopropyl azodicarboxylate (1.288 g, 1.254 ml, 6.368 mmol) was added dropwise to the reaction mixture and the reaction mixture was allowed to warm to room temperature over 16 hours. The mixture was evaporated and the resulting material was partitioned between ethyl acetate (30 ml) and 1 N sodium hydroxide (30 ml). The organic layer was separated, washed with brine (30 ml), dried over sodium sulfate and concentrated. The crude material was purified by silica gel chromatography eluting with a gradient of ethyl acetate in hexane (0-30%) to give mpem-butyl-3-[2-[1(trifluoromethyl)cyclopropyl]ethoxy]pyrazole-1-carboxylate (1.03 g , 57%). ESI-MS m/z calc. 320.13, found 321.1 (M+1)+; retention time: 0.72 minutes.

Step 2. 3-[2-[1-(Trifluoromethyl)cyclopropyl]ethoxy]-1H-pyrazole

tert-Butyl 3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazole-1-carboxylate (1.03 g, 3.216 mmol) was dissolved in dichloromethane (10.30 ml) with trifluoroacetic acid (2.487 ml, 32 .16 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated and the resulting oil was partitioned between ethyl acetate (10 ml) and saturated sodium bicarbonate solution. The organic layer was separated, washed with brine, dried over sodium sulfate and evaporated to give 3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]-1H-pyrazole (612 mg, 86%). ESI-MS m/z calc. 220.08, found 221.0 (M+1) ⁺ ; retention time: 0.5 minutes. ¹ H NMR (400 MHz, DMSO-d6) δ 11.86 (s, 1H), 7.50 (t, J=2.1 Hz, 1H), 5.63 (t, J=2.3 Hz, 1H), 4.14 (t, J=7.1 Hz, 2H), 2.01 (t, J=7.1 Hz, 2H), 0.96-0.88 (m, 2H), 0. 88-0.81 (m, 2H).

Stage 3. tert-Butyl-2-chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]pyridine3-carboxylate

tert-Butyl 2,6-dichloropyridine-3-carboxylate (687 mg, 2.770 mmol), 3-[2-[1(trifluoromethyl)cyclopropyl]ethoxy]-1H-pyrazole (610 mg, 2.770 mmol) and freshly ground potassium carbonate (459 mg, 3.342 mmol) were combined in anhydrous DMSO (13.75 ml). 1,4 Diazabicyclo[2.2.2]octane (DABCO, 62 mg, 0.5540 mmol) was added and the mixture was stirred at room temperature under nitrogen for 16 hours. The reaction mixture was diluted with water (20 ml) and stirred for 15 minutes. The resulting solid was collected and washed with water. The solid was dissolved in dichloromethane and dried over magnesium sulfate. The mixture was filtered and concentrated to give tert-butyl-2-chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1yl]pyridine-3-carboxylate (1.01 g, 84%) . ESI-MS m/z calc. 431.12, found 432.1 (M+1) ⁺ ; retention time: 0.88 minutes.

Step 4. 2-Chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]pyridine-3carboxylic acid

tert-Butyl 2-chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]pyridine-3-carboxylate (1.01 g, 2.339 mmol) and trifluoroacetic acid (1, 8 ml, 23.39 mmol) were combined in dichloromethane (10 ml) and heated at 40° C. for 3 hours. The reaction mixture was concentrated. Added

- 58 041854 hexane and the mixture was again concentrated to obtain 2-chloro-6-[3-[2-[1(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]pyridine-3-carboxylic acid (873 mg, 99%) ESI-MS m/z calc. 375.06, found 376.1 (M+1)+; retention time: 0.69 minutes.

Step 5. N-(Benzenesulfonyl)-2-chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1yl]pyridine-3-carboxamide

A solution of 2-chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]pyridine-3-carboxylic acid (0.15 g, 0.3992 mmol) and carbonyldiimidazole (77 mg, 0.4790 mmol) in THF (2.0 ml) was stirred for one hour and benzenesulfonamide (81 mg, 0.5190 mmol) and DBU (72 μl, 0.4790 mmol) were added. The reaction mixture was stirred for 16 hours, acidified with 1 M aqueous citric acid and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate and evaporated. The residue was purified by silica gel chromatography eluting with a gradient of methanol in dichloromethane (0-5%) to give N-(benzenesulfonyl)-2-chloro-6-[3-[2-[1(trifluoromethyl)cyclopropyl]ethoxy]pyrazole -1-yl]pyridine-3-carboxamide (160 mg, 78%). ESI-MS m/z calc. 514.07, found 515.1 (M+1)+; retention time: 0.74 minutes.

Step 6 N-(benzenesulfonyl)-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2[(4S)-2,2,4-trimethylpyrrolidin-1- yl]pyridine-3-carboxamide

A mixture of N-(benzenesulfonyl)-2-chloro-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1yl]pyridine-3-carboxamide (160 mg, 0.3107 mmol), (4S )-2,2,4-trimethylpyrrolidine hydrochloride salt (139 mg, 0.9321 mmol) and potassium carbonate (258 mg, 1.864 mmol) in DMSO (1.5 ml) was stirred at 130° C. for 17 hours. The reaction mixture was acidified with 1M aqueous citric acid and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate and evaporated to give the crude product, which was purified by reverse phase HPLC using a gradient of 10-99% acetonitrile in 5 mM aqueous HCl to give N-(benzenesulfonyl)-6-[3-[2 -[1(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (87 mg, 47%). ESI-MS m/z calc. 591.21, found 592.3 (M+1)+; retention time: 2.21 minutes. 1H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 8.19 (d, J=2.8 Hz, 1H), 8.04-7.96 (m, 2H), 7 .81 (d, J=8.2 Hz, 1H), 7.77-7.70 (m, 1H), 7.70-7.62 (m, 2H), 6.92 (d, J=8 .2 Hz, 1H), 6.10 (d, J=2.8 Hz, 1H), 4.31 (t, J=7.0 Hz, 2H), 2.42 (t, J=10.5 Hz, 1H), 2.28 (dd, J=10.2, 7.0 Hz, 1H), 2.17-2.01 (m, 3H), 1.82 (dd, J=11.9, 5.5 Hz, 1H), 1.52 (d, J=9.4 Hz, 6H), 1.36 (t, J=12.1 Hz, 1H), 1.01-0.92 (m, 2H), 0.92-0.85 (m, 2H), 0.65 (d, J=6.3 Hz, 3H).

Preparation of a Spray-Dried Dispersion (SDD) of Compound I A spray-dried dispersion of Compound I was prepared using a Buchi Mini Spray Dryer B290. HPMCAS-HG (6.0 grams) was dissolved in 200 ml MeOH/DCM (1/1) and Compound I (6.0 g) was added and stirred for 30 minutes to form a clear solution. The resulting solution was spray dried under the following conditions, resulting in a spray-dried dispersion of 50% 1/50% HPMCAS-HG compound (yield: 80%, solids loading: 6%).

Conditions Inlet temperature (°C) 77 Outlet temperature (°C) 39 Nitrogen pressure (psi) 95 Aspirator (%) 100 Pump (%) thirty Rotameter (mm) 60

- 59 041854 tFilter pressure (mbar) -50

Condenser temperature (°C) -10

Powder x-ray diffraction.

Powder X-ray diffraction measurements were made using a PANalytical's X-pert Pro diffractometer at room temperature with copper radiation (1.54060 A). The incident beam collimation system consisted of a variable divergence slit to provide a constant illuminated length on the sample and on the reflected beam side; a fast linear solid state detector with an active length of 2.12 degrees 2 theta measured in scan mode was used. The powder sample was packed in a concave area with a low silicon holder, and rotation was carried out to achieve better statistics. Symmetric scanning was measured from 4 to 40 degrees 2 theta with a step size of 0.017 degrees and a scan step time of 15.5 s.

In FIG. 1 shows the XRPD spectrum of SDD 50% compound I in HPMCAS-HG and shows that compound I is amorphous in SDD.

Modulated differential scanning calorimetry (MDSC).

MDSC was used to determine the glass transition temperature of an amorphous material. MDSC was performed using a TA Discovery DSC differential scanning calorimeter (TA Instruments, New Castle, DE). The instrument was calibrated with indium. Approximately 1-3 mg samples were weighed into sealed vials which were closed using single hole lids. The MDSC sample was scanned at -20 to 210°C at a heating rate of 2°C/min modulating +/-1°C for 1 minute. Data were collected and analyzed using TA Instruments Trios software (TA Instruments, New Castle, DE).

In FIG. 2 shows the MDSC SDD spectrum of 50% compound I in HPMCAS-HG and shows that the SDD has an initial temperature of approximately 75.6°C, an average temperature of approximately 82.7°C and a temperature correction of approximately 89.7°C.

Example 2. Synthesis.

Compounds II. (R)-1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2(1-hydroxy- 2-methylpropan-2-yl)-1 H-indol-5 -yl)cyclopropanecarboxamide

Step 1. (R)-Benzyl-2-( 1 -((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-6-fluoro-5-nitro-1 H-indol-2yl) -2-methylpropanoate and ((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl-2-( 1 -(((R)-2,2-dimethyl-1,3dioxolan-4 -yl)methyl)-6-fluoro-5-nitro-1H-indol-2-yl)-2-methylpropanoate.

Cesium carbonate (8.23 g, 25.3 mmol) was added to a mixture of benzyl 2-(6-fluoro-5-nitro-1H-indol-2-yl)-2-methylpropanoate (3.0 g, 8.4 mmol) and (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl-4-methylbenzenesulfonate (7.23 g, 25.3 mmol) in DMF (17 mL). The reaction mixture was stirred at 80° C. for 46 hours under nitrogen. 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 MgSO ₄ , filtered and concentrated. The crude product, a viscous brown oil which contains both products shown above, was taken directly to the next step without further purification. (R)-Benzyl-2-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-6-fluoro-5nitro-1H-indol-2-yl)-2-methylpropanoate , ESI-MS m/z calc. 470.2, found 471.5 (M+1) ⁺ . Retention time 2.20 minutes. ((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)methyl-2-(1-(((R)-2,2-dimethyl-1,3-dioxolan4-yl)methyl) -6-fluoro-5-nitro-1H-indol-2-yl)-2-methylpropanoate, ESI-MS m/z calc. 494.5, found 495.7 (M+1) ⁺ . Retention time 2.01 minutes.

Step 2. (R)-2-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-6-fluoro-5-nitro-1H-indol-2-yl)- 2methylpropan-1-ol.

The crude reaction mixture obtained in step (A) was dissolved in THF (42 ml) and cooled in an ice bath. LiAIH ₄ (16.8 ml 1 M solution, 16.8 mmol) was added dropwise. After the addition was complete, the mixture was stirred for an additional 5 minutes. The reaction mixture was quenched by adding water (1 ml), 15% NaOH solution (1 ml) and then water (3 ml). The mixture was filtered through

- 60 041854 celite and solids were washed with THF and ethyl acetate. The filtrate was concentrated and purified by column chromatography (30-60% ethyl acetate-hexane) to give (R)-2-(1-((2,2-dimethyl-1,3dioxolan-4-yl)methyl)-6-fluoro -5-nitro-1H-indol-2-yl)-2-methylpropan-1-ol as a brown oil (2.68 g, 87% over 2 steps). ESI-MS m/z calc. 366.4, found 367.3 (M+1)+. Retention time 1.68 minutes. 1H NMR (400 MHz, DMSO-d6) 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 3: (R)-2-(5-amino-1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-6-fluoro-1H-indol-2-yl) -2methylpropan-1-ol.

(R)-2-(1-((2,2-dimethyl-1,3-dioxolαn-4-yl)methyl)-6-fluoro-5-nitro-H-indol-2-yl)-2-methylnponαn -1ol (2.5 g, 6.82 mmol) was dissolved in ethanol (70 ml) and the reaction mixture was purged with N2. Then Pd-C (250 mg, 5 wt%) was added. The reaction mixture was again purged with nitrogen and then stirred under H ₂ (atm). After 2.5 hours, only partial conversion to product was observed by LCMS. The reaction mixture was filtered through celite and concentrated. The residue was re-subjected to the conditions indicated above. After 2 hours, LCMS showed complete conversion to product. The reaction mixture was filtered through celite. The filtrate was concentrated to give the product as a black solid (1.82 g, 79%). ESI-MS m/z calc. 336.2, found 337.5 (M+1) ⁺ . Retention time 0.86 minutes. ¹ H NMR (400 MHz, DMSO-d6) δ 7.17 (d, J=12.6 Hz, 1H), 6.76 (d, J=9.0 Hz, 1H), 6.03 (s, 1H), 4.794.76 (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 4. (R)-1-(2,2-difluorobenzo[a][1,3]dioxol-5-yl)-N-(1-((2,2-dimethyl-1,3-dioxolan-4yl )methyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide DMF (3 drops) was added to the stirred mixture of 1-(2,2-difluorobenzo [d][1,3]dioxol-5yl)cyclopropanecarboxylic acid (1.87 g, 7.7 mmol) and thionyl chloride (1.30 ml, 17.9 mmol). After 1 hour a clear solution formed. The solution was concentrated in vacuo 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 under 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-1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-6-fluoro -1H-indol-2-yl)-2-methylpropan-1ol (1.8 g, 5.4 mmol) and triethylamine (2.24 ml, 16.1 mmol) in dichloromethane (45 ml). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was washed with 1 N HCl, saturated NaHCO ₃ and brine, dried over MgSO ₄ and concentrated to give the product as a black foamy solid (3 g, 100%). ESI-MS m/z calc. 560.6, found 561.7 (M+1) ⁺ . Retention time 2.05 minutes. 1H NMR (400 MHz, DMSO-d6) 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), 1.341.33 (m, 6H), 1.18 (s, 3H) and 1.14-1.12 (m, 2H) ppm.

Step 5. (R)-1-(2,2-difluorobenzo[a][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-( 1hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.

(R)-1 -(2,2-difluorobenzo[d] [ 1,3 ]dioxol-5-yl)-N-(1-((2,2-dimethyl-1,3-dioxolαn-4-yl) methyl)-6-fluoro2-(1-hydroxy-2-methylpropan-2-yl)-H-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^OH.H ₂ O (204 mg, 1.1 mmol). The reaction mixture was heated at 80°C for 45 minutes. The solution was concentrated and then partitioned between ethyl acetate and saturated NaHCO ₃ solution. The ethyl acetate layer was dried over MgSO ₄ and concentrated. The residue was purified by column chromatography (50-100% ethyl acetate-hexane) to give the product as a creamy foamy solid. (1.3 g, 47%, >98% ee with SFC). ESI-MS m/z calc. 520.5, found 521.7 (M+1) ⁺ . Retention time 1.69 minutes. 1H NMR (400 MHz, DMSO-d6) 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-1,4-dihydroquinoline-3-carboxamide.

Part A. Synthesis of 4-oxo-1,4-dihydroquinoline-3-carboxylic acid

Stage 1. Diethyl ester of 2-phenylaminomethylenmalonic acid.

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 hours. The mixture was cooled to room temperature and dried at reduced pressure to obtain diethyl ester of 2-phenylaminomethylenemalonic acid in the form

- 61 041854 solid, which was used in the next step without further purification. Ή NMR (DMSO-a ₆ ) 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).

Stage 2. Ethyl ester of 4-hydroxyquinoline-3-carboxylic acid.

A 1 L three-neck flask equipped with a mechanical stirrer was charged with 2-phenylaminomethylenemalonic 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 hours. The mixture was cooled to room temperature and filtered. The residue was treated with an aqueous solution of HarCO3, filtered off, 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 the next step without further purification.

Stage 3. 4-Oxo-1,4-dihydroquinoline-3-carboxylic acid.

4-Hydroxyquinoline-3-carboxylic acid ethyl ester (15 g, 69 mmol) was suspended in sodium hydroxide solution (2 N, 150 ml) and stirred for 2 h at reflux. After cooling, the mixture was filtered and the filtrate was acidified to pH 4 with 2 N HCl. The resulting precipitate was collected by filtration, washed with water and dried under vacuum to give 4-oxo-1,4-dihydroquinoline-3-carboxylic acid as a pale white solid (10.5 g, 92%). Ή NMR (DMSOCH) δ 15.34 (s, 1H), 13.42 (s, 1H), 8.89 (s, 1H), 8.28 (d, >8.0 Hz, 1H), 7, 88 (m, 1H), 7.81 (d, >8.4 Hz, 1H), 7.60 (m, 1H).

Part B. Synthesis of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide

Stage 1. Methyl ester of 2,4-di-tert-butylphenyl ester of carbonic acid.

Methyl chloroformate (58 ml, 750 mmol) was added dropwise to a solution of 2,4-di-tert-butylphenol (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 bath to 0°C. The mixture was allowed to warm to room temperature with stirring overnight, then filtered through silica gel (approx. 1 L) using 10% ethyl acetate-hexane (~4 L) as eluent. The combined filtrates were concentrated to give 2,4-di-tert-butylphenyl carbonic acid methyl ester as a yellow oil (132 g, quant.). *H NMR (400 MHz, DMSO-b ₆ ) δ 7.35 (d, >2.4 Hz, 1H), 7.29 (dd, >8.5, 2.4 Hz, 1H), 7.06 (d, >8.4 Hz, 1H), 3.85 (s, 3H), 1.30 (s, 9H), 1.29 (s, 9H).

Step 2: 2,4-di-tert-butyl-5-nitrophenyl carbonic acid methyl ester and 2,4-di-tert-butyl-6-nitrophenyl carbonic acid methyl ester.

To a stirred mixture of 2,4-di-tert-butylphenyl carbonic acid methyl ester (4.76 g, 180 mmol) in conc. sulfuric acid (2 ml) cooled in an ice bath was added a chilled mixture of sulfuric acid (2 ml) and nitric acid (2 ml). The addition was carried out slowly so that the reaction temperature did not exceed 50°C. The reaction mixture 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 (MgSOg), concentrated and purified by column chromatography (0-10% ethyl acetate-hexane) to give a mixture of 2,4-di-tert-butyl-5-nitrophenyl carbonic acid methyl ester and methyl ester 2,4 -di-tert-butyl-6nitrophenyl carbonic acid ester as a pale yellow solid (4.28 g) which was used directly in the next step.

Step 3 2,4-Di-tert-butyl-5-nitrophenol and 2,4-di-tert-butyl-6-nitrophenol.

A mixture of 2,4-di-tert-butyl-5-nitrophenyl carbonic acid methyl ester and methyl

-62041854 carbonic acid 2,4-di-tert-butyl-6-nitrophenyl ester (4.2 g, 14.0 mmol) was dissolved in MeOH (65 ml) before addition of KOH (2.0 g, 36 mmol) . The mixture was stirred at room temperature for 2 hours. The reaction mixture was then acidified (pH 2-3) by adding conc. HCl and partitioned between water and diethyl ether. The ether layer was dried (MgSO ₄ ), concentrated and purified by column chromatography (0-5% ethyl acetate-hexane) to give 2,4-di-tert-butyl-5-nitrophenol (1.31 g, 29% over 2 steps) and 2,4-di-tert-butyl-6-nitrophenol. 2,4-Di-tert-butyl-5nitrophenol: 1H NMR (400 MHz, DMSO^) 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-tert-butyl-6-nitrophenol: ¹ H NMR (400 MHz, CDCf) 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 4 5-Amino-2,4-di-tert-butylphenol.

Pd-5 % mass, on activated carbon (900 mg). The reaction mixture was stirred at reflux for 2 hours, cooled to room temperature and filtered through celite. Celite was washed with methanol and the combined filtrates were concentrated to give 5-amino-2,4-di-tert-butylphenol as a gray solid (1.66 g, quant.). 1H NMR (400 MHz, DMSO^) δ 8.64 (s, 1H, OH), 6.84 (s, 1H), 6.08 (s, 1H), 4.39 (s, 2H, NH2), 1.27 (m, 18H); HPLC hold time 2.72 min, 10-99% CH3CN, 5 min. experience; ESIMS 222.4 m/z [M+H]+.

Stage 5. N-(5-hydroxy-2,4-di-tert-butylphenyl)-4-oxo-1 H-quinoline-3-carboxamide

Et ₃ N (63.0 ml, 451 mmol) at ambient temperature. The mixture became homogeneous and was allowed to stir for 10 minutes, after which 5-amino-2,4-di-tert-butylphenol (50.0 g, 226 mmol) was added in small portions. The mixture was allowed to stir overnight at ambient temperature. During the reaction, the mixture became heterogeneous. After all the acid was consumed (LC-MS analysis, MH+ 190, 1.71 min), the solvent was removed under vacuum. EtOH was added to the orange solid to form a slurry. The mixture was stirred on a rotary evaporator (bath temperature 65°C) for 15 min without placing the system under vacuum. The mixture was filtered and the captured solid was washed with hexane to give a white solid which was crystalline EtOH. Et ₂ O was added to the solid above to form a slurry. The mixture was stirred in a rotary evaporator (bath temperature 25°C) for 15 min without placing the system under vacuum. The mixture was filtered and the solid was taken up. This procedure was carried out a total of five times. The solid obtained after the fifth precipitation was placed under vacuum overnight to give N-(5-hydroxy-2,4-di-tert-butylphenyl)-4-oxo-H-quinoline-3-carboxamide as a white powdery solid substances (38 g, 52%). HPLC hold time 3.45 min, 10-99% CH3CN, 5 min. experience; 1H NMR (400 MHz, DMSO^) 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. 392.21; found 393.3 [M+H] ⁺ .

Example 4 Synthesis of N-(2-(tert-butyl)-4-(tert-butyl-d ₉ )-5-hydroxyphenyl)-4-oxo-1,4dihydroquinoline-3-carboxamide (compound III-d).

Step 1 2-(tert-Butyl-d ₉ )-4-(tert-butyl)-6-d-phenol.

To a solution of 4-tert-butylphenol (3.43 g, 22.7 mmol) and tert-butyl alcohol-d10 (3.00 ml, 31.8 mmol, 98 atom% D, Cambridge Isotope Laboratories, Inc.) in dichloromethane (40.0 ml) was added D ₂ SO ₄ (1.50 ml, 99.5 atom% D, Sigma-Aldrich). The reaction mixture was stirred at room temperature for 15 hours, then diluted with water and extracted with dichloromethane (3x100 ml). The organic layers were combined, washed with saturated NaHCO ₃ , dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The resulting oil was purified by column chromatography (SO ₂ , 0-15% ethyl acetate/heptane) to give 2-(tert-butyl-d ₉ )-4-(tert-butyl)-6-d-phenol (4.04 g , 83% yield) as a clear oil. 1H NMR (d ₆ -DMSO, 400 MHz) δ 9.04 (s, 1H), 7.12 (d, J=2.4 Hz, 1H), 6.98 (dd, J=3.8, 2 .5 Hz, 1H), 6.67 (d, J=8.3 Hz, 0.3H), 1.22 (s, 10H).

Step 2. 2-(tert-Butyl-d ₉ )-4-(tert-butyl)-6-d-phenylmethyl carbonate To a solution of 2-(tert-butyl-d ₉ )-4(tert-butyl)-6-d -phenol (4.04 g, 18.8 mmol), triethylamine (5.24 ml, 37.6 mmol) and N, Ndimethylaminopyridine (115 mg, 0.940 mmol) in CH ₂ Cl ₂ (40.0 ml) at 0 °С methyl chloroformate was added

- 63 041854 (2.17 ml, 28.2 mmol). The reaction mixture was stirred at room temperature for 15 hours and additional trimethylamine (1.30 ml, 9.33 mmol) and methyl chloroformate (0.550 ml, 7.15 mmol) were added. After stirring for an additional 1 hour, the reaction mixture was diluted with 10% ethyl acetate/heptane and filtered through a plug of silica gel. The silica plug was then rinsed with additional 10% ethyl acetate/heptane. The filtrate was combined and concentrated in vacuo to give 2-(t-butyl-d9)-4-(t-butyl)-6-(1-phenylmethyl carbonate (4.69 g, 91% yield) as a light yellow oil which carried on without purification 1H NMR (d ₆ DMSO, 400 MHz) δ 7.33 (d, J=2.4 Hz, 1H), 7.30-7.20 (m, 1H), 7.06 (d , J=8.5 Hz, 0.3H), 3.84 (d, J=0.7 Hz, 3H), 1.28 (s, 9H).

Step 3 2-(tert-Butyl-d ₉ )-4-(tert-butyl)-6-d-5-nitrophenol.

To a solution of 2-(tert-butyl-d ₉ )-4-(tert-butyl)-6-d-phenylmethyl carbonate (4.69 g, 17.2 mmol) in sulfuric acid (2.00 ml) at 0°C a mixture of sulfuric acid and nitric acid 1:1 (4.00 ml) was added dropwise. The reaction mixture was then stirred at room temperature for two hours, then slowly added to ice water with vigorous stirring. The resulting suspension was extracted with ethyl acetate (3x100 ml) and the combined organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated to give an amber oil containing a mixture of regioisomers. This crude oil was then dissolved in MeOH (100 ml) and KOH (3.50 g) was added. The reaction mixture was stirred at room temperature for 2 hours, then acidified to pH=2 with concentrated HCl. The resulting solution was extracted with diethyl ether (3x100 ml), dried (MgSO ₄ ), filtered and concentrated. The residue was then purified by column chromatography (SiO ₂ , 0-5% ethyl acetate/heptane) to give 2-(tert-butyl-d ₉ )-4-(tert-butyl)-6-d-5-nitrophenol (1, 33 g, 30%) as a light yellow solid. MS (ESI) 260.2 [(M-H) ^- ].

Step 4. 5-Amino-2-(tert-butyl-d9)-4-(tert-butyl)-6-d-phenol Solution of 2-(tert-butyl-d ₉ )-4-(tert-butyl)-6- d-5-nitrophenol (1.33 g, 5.11 mmol) and ammonium formate (1.29 g, 20.4 mmol) in ethanol (60.0 ml) were heated to reflux. At this time, 10% Pd/C (650 mg, 50% moisture) was added in small portions and the reaction mixture continued to stir at reflux for two hours. The reaction mixture was then cooled to room temperature, diluted with THF, filtered through Celite® and concentrated in vacuo to give 5-amino-2(tert-butyl-d9)-4-(tert-butyl)-6-d-phenol (1, 19 g, 100%) as a pink solid. MS (ESI) 232.3 [(M+H)+].

Step 5 5-Amino-2-(tert-butyl-d ₉ )-4-(tert-butyl)phenol.

5-Amino-2-(tert-butyl-d ₉ )-4-(tert-butyl)-6-d-phenol (298 mg, 1.29 mmol) was dissolved in 5 M HCl in 2-propanol (20 ml) and the reaction the mixture was stirred at room temperature for 15 hours. Then the reaction mixture was concentrated under vacuum and collected back into 5 M HCl in 2-propanol (20 ml). After stirring for an additional 15 hours at room temperature, the reaction mixture was concentrated in vacuo and diluted with saturated aqueous sodium bicarbonate (100 ml). The resulting aqueous solution was extracted with dichloromethane (3x50 ml). The organic layers were combined, dried (Na ₂ SO ₄ , filtered and concentrated in vacuo to give 5amino-2-(tert-butyl-d ₉ )-4-(tert-butyl)phenol (240 mg, 81%) as a pink solid 1H NMR (d6-DMSO, 400 MHz) δ 8.62 (s, 1H), 6.83 (s, 1H), 6.08 (s, 1H), 1.27 (s, 9H).

Step 6 N-(2-(tert-Butyl)-4-(tert-butyl-d ₉ )-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (compound III-d).

To a solution of 5-amino-2-(tert-butyl-d ₉ )-4-(tert-butyl)phenol (240 mg, 1.04 mmol), 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (purchased from Matrix Scientific, 99 mg, 0.521 mmol) and N,Ndiisopropylethylamine (181 μl, 1.04 mmol) in DMF (6.00 ml) were added HATU (198 mg, 0.521 mmol). The reaction mixture was stirred at room temperature for three hours, then diluted with saturated NaHCO ₃ and extracted with ethyl acetate (3x50 ml). The combined organic extracts were washed with water (3x20 ml), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The resulting residue was purified by column chromatography (SiO2, 0-70% ethyl acetate/heptane) to give N-(2-(tert-butyl)-4-(tert-butyl-d ₉ )-5-hydroxyphenyl)-4-oxo -1,4-dihydroquinoline-3-carboxamide (compound III-d) (80 mg, 38% yield) as a white solid. 1H NMR (d ₆ -DMSO, 400 MHz) δ 12.88 (s, 1H), 11.81 (s, 1H), 9.19 (s, 1H), 8.86 (s, 1H), 8, 32 (dd, J=8.1, 1.4 Hz, 1H), 7.86-7.77 (m, 1H), 7.75 (d, J=8.2 Hz, 1H), 7.51 (s, 1H), 7.15 (s, 1H), 7.09 (s, 1H), 1.37 (s, 9H); MS (ESI) 402.3 [(M+H)+].

Example 5 Assays to detect and measure properties of F508del-CFTR modulator compounds.

Optical methods of membrane potential for the analysis of the properties of F508del-CFTR modulators.

Optical analysis was used to measure changes in membrane potential to determine the properties of compounds that modulate CFTR. The assay used fluorescent voltage sensitive dyes to measure changes in membrane potential using a fluorescent plate reader (eg, FLIPR III, Molecular Devices, Inc.) as an indication for increased functional F508del in NIH 3T3 cells. The driving force behind the reaction was the creation of a chloride ion gradient in combination with channel activation and simultaneous processing of the compound.

- 64 041854 one-step fluid addition after the cells have been pre-treated with a voltage-sensitive dye.

Method of analysis.

NIH3T3 mouse fibroblasts stably expressing F508del were used for optical measurements of membrane potential. Cells were maintained at 37°C in 5% CO2 and 90% humidity in Dulbecco's Modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, β-ME, 1 X penicillin/streptomycin, and 25 mM HEPES in culture flasks with a volume of 175 cm ² . For all optical assays, cells were seeded at 12,000 cells/well in 384-well Matrigel coated plates. For correction analysis, cells were cultured at 37° C. for 18-24 hours and treated with voltage sensitive dye. The cells were then activated and treated with Compound I. After 18-24 hours, the fluorescence of the voltage sensitive dye in the cells was measured to assess changes in membrane potential as an indication for increased functional F508del CFTR in NIH3T3 cells.

Using this method, Compound I had an EC ₅₀ of less than 3 μM and a % Efficacy >100% relative to Compound II.

Analysis using the Ussing chamber.

Ussing chamber experiments were performed on polarized airway epithelial cells expressing F508del to further characterize the F508del modulators identified in the optical analysis above. Airway epithelia without MB and with MB were isolated from bronchial tissue, cultured using methods well known in the art, and placed on Costar® Snapwell™ filters that were pre-coated with NIH3T3-conditioned medium. After four days, the apical medium was removed and the cells were grown at the air-liquid interface for >14 days before use. This resulted in the formation of a monolayer of fully differentiated cylindrical cells that were ciliated, a feature that is characteristic of the airway epithelium. Human bronchial epithelial cells (HBE) without MB were isolated from non-smokers who had no known lung disease. CF-HBE cells have been isolated from patients homozygous for F508del (F508del/F508del HBE) or heterozygous for F508del and a MB transmembrane conductance regulator (MF) minimal function-associated mutation (MF) that is not expected to respond to the compound II, compound III compound III-d and a combination of compound II and III or compound II and III-d (F508del/MF-HBE).

HBE cells grown on Costar® Snapwell™ cell culture inserts were placed in a Ussing chamber (Physiologic Instruments, Inc., San Diego, CA) and transepithelial resistance and short circuit current were measured in the presence of a basolateral-apical Cl'(I _Sc ) gradient using a potential fixation system (Department of Bioengineering, University of Iowa, IA). Briefly, HBE cells were examined under potential clamp recording conditions (V _ho i _d =0 mV) at 37°C. The basolateral solution contained (in mM) 145 NaCl, 0.83 K ₂ HPO ₄ , 3.3 KH ₂ PO ₄ , 1.2 MgCl ₂ , 1.2 CaCl ₂ , 10 Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and the apical solution contained (in mM) 145 N-Gluconate, 1.2 MgCl ₂ , 1.2 CaCl ₂ , 10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

Analysis technique using the Ussing chamber

The concentration gradient of Cl ^- on the basolateral-apical membrane was set as follows. Normal Ringer's solution was used on the basolateral membrane, while apical NaCl was replaced with equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to obtain a large concentration gradient of Cl ^- across the epithelium. Compound I was added either to the basolateral side 18-24 hours prior to analysis or to the apical side during analysis. Forskolin (10 μM) was added to the apical side during the assay to stimulate CFTR-mediated Cl ^- transport. Chloride current was measured to assess the increase in functional CFTR in the cell membrane.

Example 6 Chloride transport experiments.

In one experiment using a Ussing chamber with F508del/F508del-HBE cells, Compound I enhanced chloride transport. The effect of compound I on chloride transport was additive to that of compound II. In addition, F508del-CFTR delivered to the cell surface either with Compound I alone or in combination with Compound II was potentiated with Compound III. The ternary combination of Compound I/Compound II/Compound III provided an excellent (approximately 3-fold) increase in chloride transport compared to 3 dual regimens in most of the conditions studied.

Example 7 F508del-CFTR processing and migration experiments in vitro.

In vitro, Compound I improved the processing and migration of F508del CFTR, thereby increasing the amount of functional F508del-CFTR protein on the cell surface. The CFTR protein delivered to the cell surface with Compound I alone or in combination with Compound II (Compound I/Compound II) was potentiated with Compound III. In human bronchial epithelial cells (HBE),

- 65 041854 studied in vitro, the triple combination of Compound I, Compound II and Compound III (Compound I/Compound II/Compound III) increased CFTR chloride transport to a greater extent than any of the dual combinations (Compound I/Compound II, Compound I/ compound III and compound II/compound III) or single components (compound I, compound II and compound III) under most conditions tested.

Processing and migration of F508del-CFTR was directly monitored by the appearance of a band from 170 to 180 kDa. This monitoring established that Compound I is a CFTR corrector as it facilitates the processing and migration of F508del-CFTR to increase the amount of functional F508del-CFTR on the cell surface.

Incubation of F508del/F508del-HBE cells for 16-24 hours with 1 μM Compound I alone or in combination with 3 μM Compound II resulted in increased steady-state levels reaching 6.5- and 18.7-fold over untreated levels, respectively. .

Example 8 Safety and Efficacy Study 1 of Compound I.

In healthy subjects, Compound I monotherapy has been shown to be safe and well tolerated at multiple doses up to 400 mg per day for 10 days.

To evaluate the safety and efficacy of compound I in combination with compound III and in triple combination (TC) with compound II and compound III in subjects with cystic fibrosis, a randomized, double-blind, placebo- and compound II/III-controlled, part 3, multicenter study was performed. Part 1 includes objects with genotypes F508del/MF (F/MF), Part 2 with genotype F508del/F508del (F/F), Part 3 with genotypes F508del/gate mechanism (F/G), Part 4 with genotype F508del/residual function (RF) and Part 5 includes F508del/MF (F/MF) genotype subjects that are dosed twice a day. The F508del/gateway (F/G) genotype means that the patient is heterozygous for F508del with a second CFTR allele carrying a gate defect mutation clinically demonstrated to be susceptible to Compound III.

Parts 1-4 each include a treatment period and a 4-week follow-up period to evaluate safety after that treatment period. Parts 1, 2, 3 and 4 of the study include a treatment period consisting of 2 dosing periods - Compound I dosing period (period 1) and Compound I washout period (period 2). Parts 3 and 4 also include a 4-week run-in period prior to treatment period 1. Patients are administered Compound II (100 mg daily/Compound III 150 mg every 12 hours) during the run-in period.

For all parts, to be eligible to enter the treatment period, after the screening period, subjects must have stable MB disease and remain on a stable MB treatment regimen for 28 days prior to Day 1 of the visit and must not have acute non-MB disease for 14 days prior to day 1 visit.

The dosing period for compound I (period 1) is 4 weeks. The total duration of treatment (Period 1+Period 2) is approximately 5 weeks for Part 1, 8 weeks for Part 2, and 8 weeks for Parts 3 and 4.

The treatment groups and doses for the Study Part of Compound I, Compound II, and Compound III are shown below. The following definitions apply to the dosing regimens below: q12h means every 12 hours; qd means once a day.

- 66 041854

Period 1 Period 2 Compound I Compound II Compound III Dosage Dosage Dosage Connection Connection II Dosage III Dosage Part 1 TK- 4OOMrqd lOOMrqd 150Mrql2h high TK- 240 mg qd 100 mg qd 150 mg ql2h average TK-low 80 mg qd 100 mg qd 150 mg ql2h DK- 400Mrqd Placebo 150Mrql2h high Triple Placebo Placebo Placebo placebo 100 Ml qd 150 mi q!2h lOOMrqd 150Mrql2h lOOMrqd 150Mrql2h Placebo 150 mg ql2h placebo placebo Part 2 Comm. Placebo lOOMrqd 150Mrql2h p/w TK- 400 Mrqd lOOMrqd 150Mrql2h high DK- 400Mrqd Placebo 150Mrql2h high part 3 Sucking Placebo lOOMrqd 150Mrql2h F/W TK- 400 mg qd 100 mg qd 150 mg ql2h high lOOMrqd 150Mrql2h lOOMrqd 150Mrql2h lOOMrqd 150Mrql2h 100 mg qd 150 mi ql2h lOOMrqd 150Mrql2h Part 4 Comm. Placebo lOOMrqd 150Mrql2h ΙΙ/ΠΙ TK- 400 mg qd 100 mg qd 150 mg ql2h high lOOMrqd 150Mrql2h lOOMrqd 150Mrql2h Part? placebo placebo placebo placebo TK- 160 or 240 mg 50 mg ql2h 150 mg ql2h high ql2h

Compounds I, II and III are orally administered within 30 minutes of the start of a fat-containing meal or snack, such as a MB standard meal or snack or standard meal. A standard MB meal or snack includes foods containing fat such as eggs, butter, peanut butter, cheese pizza, whole dairy products (such as whole milk, cheese and yogurt), etc. Compound I is in the form of 80 mg tablet(s). Compound II is in the form of 50 mg tablet(s). Compound III is in the form of 150 mg tablet(s). the compound II and III may also be in the form of fixed dose tablet(s) containing 100 mg compound II/150 mg compound III.

Primary endpoints for the study include: safety and tolerability assessments based on adverse events (AEs), clinical laboratory values, standard 12-lead ECG, vital signs, and pulse oximetry; and an assessment of efficacy based on the absolute percent change in patient predicted forced expiratory volume in 1 second (ppFEV1) from baseline to visit day 29. Secondary endpoints include: absolute change in sweat chloride concentrations from baseline to visit day 29; relative change in pFOEF1 from baseline to day 29 of the visit; absolute change in the Respiratory Domain Score on the Revised Cystic Fibrosis Questionnaire (CFQ-R) from baseline to Day 29 of the visit; and PK parameters of compounds I, II and III.

Part 1 results.

In Part 1, as shown in the tables below, for 4 weeks Compound I in triple combination with

- 67 041854 Compound II and Compound III in subjects heterozygous for F508del and with a minimal function (F/MF) ¹ mutation, aged 18 years and older, resulted in statistically significant and clinically significant improvements in pFOEF1 (10.2-13.3 percent point) and chloride content in sweat (43.7-51.4 mmol/l).

Specifically, at day 29, the mean absolute improvement in pFOEF1 was +10.2, +11.6, and +13.3 percentage points from baseline in patients who respectively received compound I triple combination regimens (80 mg per day). ), compounds II (100 mg per day) and compounds III (150 mg every 12 hours); compound I (240 mg daily), compound II (100 mg daily) and compound III (150 mg every 12 hours) and compound I (400 mg daily), compound II (100 mg daily) and compound III (150 mg every 12 hours). For placebo patients, the mean absolute increase in pFOEF1 was +0.3.

Placebo N=10 Compound I (80 mg, qd)/ Compound II (100 mg, qd)/, Compound III Compound I (240 mg, qd)/ Compound II (100 mg, qd)/ Compound III Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=ll (150 mg, ql2h) N=20 (150 mg, ql2h) N=22 Initial value ππΟΦΒι; Mean value (SD) 53.9 (12.0) 57.9 (10.8) 58.0(16.8) 59.6(15.4) Mean absolute within-group change from baseline to day 29* (SD) 0.3 (2.8) 10.2 (2.7) 11.6 (2.1) 13.3 (1.9) p-value (within test options)* 0.9053 0.0004 <0.0001 <0.0001

all p-values are within-group p-values based on mixed effects models; values expressed as after 29 days are the average values for 15 days and 29 days.

At day 29, the mean reduction in sweat chloride was -45.8, -437, and -51.4 mmol/L from baseline in patients who respectively received compound I triple combination regimens (80 mg daily), Compound II (100 mg per day) and Compound III (150 mg every 12 hours); compound I (240 mg daily), compound II (100 mg daily) and compound III (150 mg every 12 hours) and compound I (400 mg daily), compound II (100 mg daily) and compound III (150 mg every 12 hours). For placebo patients, the mean absolute increase in sweat chloride was 2.9.

Placebo N=10 Compound I (80 mg, qd)/ Compound II (100 mg, qd)/, Compound III (150 mg, q!2h) N=ll Compound I (240 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, q!2h) N=20 Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, q!2h) N=22 Initial value of SwCl; Average value 98.2(13.3) 102.7 (7.0) 100.5 (9.0) 100.7(11.6)

- 68 041854

(SD) Mean absolute within-group change from baseline to day 29* (SD) 2.9 (4.6) -45.8 (4.4) -43.7 (3.5) -51.4 (3.2) p-value (within test options)* 0.5338 <0.0001 <0.0001 <0.0001

# all p-values are within-group p-values based on mixed effects models; values expressed as after 29 days are the average values for 15 days and 29 days.

The secondary endpoint in the Triple Combination Part 1 study measured the mean absolute change in the respiratory domain of a validated CFQ-R patient outcome measure at day 29. The mean absolute improvements in patients who received the triple combination were 24.6 points (80 mg Compound I), 19.8 points (240 mg Compound I) and 21.8 points (400 mg Compound I). The improvement in patients who received placebo was 4.7 points. The CFQ-R results presented are based on mixed-effects models, not adjusted for the original CFQR.

The following is an overview of adverse events that occurred during treatment (AELI) after 29 days.

placebo N=10 Compound I (80 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=ll Compound I (240 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=20 Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=22 Compound I Triple Combination Total N=53 Objects with any NJVL 9 (90.0) 9(81.8) 14 (70.0) 17 (77.3) 40 (75.5) Objects with heavy 0 0 1 1 2 NJVL Objects with serious NJVL 3 1 3 0 4 Subjects with AELV leading to discontinuation of treatment 0 0 0 0 0 Subjects with AELV leading to drug discontinuation 0 0 1 0 1

Thus, in part 1 of the study, the triple combination regimen was generally well tolerated. Most adverse events were mild or moderate. Serious adverse events were reported in seven patients: three patients in the placebo group (2 with infectious pulmonary exacerbations and 1 with decreased respiratory function) and four in the placebo groups.

- 69 041854 triple combination (3 with infectious pulmonary exacerbations and 1 with influenza). None of these serious adverse events were considered treatment related, and none of them led to treatment discontinuation. The most common adverse events (>10%) regardless of treatment group were cough, headache, oropharyngeal (throat) pain, and increased sputum. There were no discontinuations due to adverse events. One patient discontinued treatment due to an adverse event in the triple combination treatment groups (rash). The rash resolved upon discontinuation of treatment and this patient subsequently resumed and completed triple combination treatment without further rash.

Part 2 results.

In Part 2, as shown in the tables below, within 4 weeks, Compound I in triple combination with Compound II and Compound III in subjects homozygous for F508del aged 18 years and older resulted in statistically significant and clinically significant improvements in pFOEF1 (9, 7 percentage points) and chloride content in sweat (42.2 mmol/l).

Specifically, at day 29, the mean absolute improvement in pFOEF1 was +9.7 percentage points from baseline in patients who received Compound I (400 mg daily), Compound II (100 mg daily), and compound III (150 mg every 12 hours). For patients receiving placebo together with Compound II (100 mg daily) and Compound III (150 mg every 12 hours), the mean absolute increase in pFOEF1 was 0.0.

Placebo/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=ll Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=18 Initial value ππΟΦΒι; Mean value (SD) 60.0 (12.6) 58.6(13.3) Mean absolute within-group change from baseline to day 29* (SE) 0.0 (1.9) 9.7 (1.5) p-value (within test options)* 0.9926 <0.0001

# η all p-values are within-group p-values based on mixed effects models; values expressed as days are averages for 15 days and 29 days.

At day 29, the mean decrease in sweat chloride was -42.2 mmol/L from baseline in patients treated with the triple combination regimens of Compound I (400 mg daily), Compound II (100 mg daily), and Compound III (150 mg every 12 hours). For patients receiving placebo together with Compound II (100 mg daily) and Compound III (150 mg every 12 hours), the mean absolute increase in sweat chloride was +3.0.

Placebo/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=ll Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=18 Initial value of SwCl; Mean value (SD) 96.6(11.4) 91.9(11.6) Mean absolute within-group change from baseline to day 29* (SE) 3.0 (2.8) -42.2 (2.2) p-value (within test options)* 0.2977 <0.0001

# all p-values are within-group p-values based on mixed effects models; values expressed as after 29 days are the average values for 15 days and 29 days.

- 70 041854

The secondary endpoint in the Triple Combination Part 2 study measured the mean absolute change in the respiratory domain of a validated CFQ-R patient outcome measurement at day 29. The mean absolute improvement in patients who received the triple combination was 20.1 points. The improvement in patients who received placebo rather than Compound I was 2.0 points. The CFQ-R results presented are based on mixed-effects models, not adjusted for the original CFQR.

The following is an overview of adverse events that occurred during treatment (AELI) after 29 days.

Placebo/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=ll Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=18 Objects with any NJVL 8 (72.7) 14 (77.8) Objects with severe NJVL Objects with serious NELV 1 ^a 0 Objects with NJVL leading to termination treatment 0 0 Subjects with AELV leading to drug discontinuation 0 0

^a PEx, ^b abdominal pain, ^c elevated levels of AST/CK.

Thus, in Part 2 of the study, the triple combination regimen was generally well tolerated. There were no serious adverse events in the triple combination group, and all adverse events in the triple combination group were mild or moderate. There were no discontinuations or interruptions of treatment. The most common adverse events (>10%), regardless of treatment group, were cough, infectious pulmonary exacerbation, nasal congestion, nausea, increased sputum, vomiting, and headache.

Example 9 Safety and Efficacy Study 2 of Compound I.

To assess the safety and efficacy of Compound I in triple combination (TC) with Compound II and Compound III-d in subjects with cystic fibrosis, a randomized, double-blind, triple placebo-controlled trial was performed in subjects with F508del/MF (F/MF) genotypes (e.g., heterozygous F508del with a second CFTR allele carrying the MF mutation described in Table C).

The treatment group and doses of Compound I, Compound II, and Compound III-d are shown below. The following definitions apply to the dosage regimens below: q12h means every 12 hours: qd means once a day.

Treatment period Compound I Compound II Compound III-d TK2-high 400 mg qd 100 mg qd 200 mg qd Triple placebo placebo placebo placebo

Primary endpoints for the study include: safety and tolerability assessments based on adverse events (AEs), clinical laboratory values, standard 12-lead ECG, vital signs, and pulse oximetry; and an assessment of efficacy based on the absolute percent change in patient predicted forced expiratory volume in 1 second (ppFEV1) from baseline to visit day 29. Secondary endpoints include: absolute change in sweat chloride concentrations from baseline to visit day 29; relative change in pFOEF1 from baseline to day 29 of the visit; absolute change in the Respiratory Domain Score on the Revised Cystic Fibrosis Questionnaire (CFQ-R) from baseline to Day 29 of the visit; and PK parameters of compounds I, II and IH-d.

Results.

As shown in the table below, for 4 weeks Compound I in triple combination with Compound II and Compound III-d in subjects heterozygous for F508del and with a minimal function mutation

- 71 041854 (F/MF), aged 18 years and older, resulted in statistically significant and clinically significant improvements in pFOEF ₁ (12.2 percentage points) and sweat chloride (38.1 mmol/l).

Specifically, at day 29, the mean absolute improvement in pFOEF1 was +12.2 percentage points from baseline in patients who received Compound I (400 mg daily), Compound II (100 mg daily), and compounds III-d (200 mg per day). For patients who received the triple placebo, the mean absolute change in pFOEF1 was -5.0. For Part 1 patients in the TK-high group discussed above who received the triple combination regimens of Compound I (400 mg daily), Compound II (100 mg daily) and Compound III (150 mg every 12 hours), the mean absolute change in pFOEF1 was +13.3.

Triple placebo N=6 Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III-d (200 mg, qd) N=19 PART D, TC high Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=22 Initial value ππΟΦΒι; Mean value (SD) 53.0(12.3) 59.8 (12.6) 59.6(15.4) Mean absolute within-group change from baseline to day 29* (SE) -5.0 (3.4) 12.2(1.9) 13.3 (1.9) p-value (within test options)* 0.1561 <0.0001 <0.0001

# all p-values are within-group p-values based on mixed effects models; values expressed as after 29 days are the average values for 15 days and 29 days.

At day 29, the mean decrease in sweat chloride was -33.6 mmol/L from baseline in patients treated with Compound I (200 mg daily), Compound II (100 mg daily), and Compound III-d (150 mg per day). For patients who received the triple placebo, the mean absolute reduction in sweat chloride was -2.2. For patients in Part D, TC-high who received triple combination regimens of Compound I (200 mg daily), Compound II (100 mg daily), and Compound III (150 mg every 12 hours), mean absolute reduction in sweat chloride was -39.1.

Triple placebo N=6 Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III-d (200 mg, qd) N=19 PART D, TC high Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, q!2h) N=22 Initial value of SwCl; Mean value (SD) 96.6 (4.3) 101.2 (9.5) 100.7(11.6) Mean absolute within-group change from baseline to day 29* (SD) -1.3 (5.3) -38.1 (3.0) -51.4 (3.2) p-value (within test options)* 0.8077 <0.0001 <0.0001

# all p-values are within-group p-values based on mixed effects models; values expressed as after 29 days are the average values for 15 days and 29 days.

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The secondary endpoint in the triple combination measured the mean absolute change in the respiratory domain of the CFQ rat at day 29. The mean absolute improvement for patients who received the triple combination with Compound III-d was 14.7 points. The improvement in patients who received the triple placebo was -4.1 points. The improvement in patients in Part 1, TK high who received the triple combination with Compound III was 21.0 points. The CFQ-R results presented are based on mixed-effects models, not adjusted for the original CFQR.

The following is an overview of adverse events that occurred during treatment (AELI) after 29 days.

Triple placebo N=6 Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III-d (200 mg, qd) N=19 PART D, TC high Compound I (400 mg, qd)/ Compound II (100 mg, qd)/ Compound III (150 mg, ql2h) N=22 Objects with any NJVL 6 (100.0) 15 (78.9) 17 (77.3) Objects with severe NJVL 1 yes, b,c R Objects with serious NELV 3 ^{2 e'd} 0 Subjects with AELV leading to discontinuation of treatment 0 1 ^b 0 Subjects with AELV leading to drug discontinuation 0 2 ^a'b 0

^a gap; and the same subject had a rash leading to an interruption;

^b dizziness, vomiting; and the same subject had a rash leading to an abort, which was later terminated due to hypersensitivity to the drug (swelling of the eyes);

^with hyperthermia, dyspnea, and pleuritic chest pain;

^d PEx and pneumonia;

$ hyperthermia/pain in the oropharynx/foot fracture.

Thus, the triple combination scheme was generally well tolerated. Most adverse events were mild or moderate. There were 2 dose interruptions - both due to rash. There was 1 treatment discontinuation due to drug hypersensitivity in one subject who had a dose interruption due to rash. A total of 2 subjects treated with the triple combination, including Compound III-d, had serious AEs, all of which occurred after the triple combination treatment period.

Example 10 Safety and Efficacy Study 3 of Compound I

The safety and tolerability of single and multiple escalating doses of 10 mg-240 mg Compound I alone and in triple combination with Compound II and Compound III (Compound II 100 mg daily/Compound III 150 mg every 12 hours) in healthy volunteers in a randomized double blind placebo-controlled study.

The safety and tolerability of Compound I was also assessed as part of a triple combination for two weeks in people with MB aged 18 years or older who have one F508del mutation and one minimal function mutation (3 in placebo and 9 in Compound I 120 mg each 12 hours). In this part of the study, sweat chloride was assessed as an additional endpoint and absolute change in pFOEF1 was assessed as part of the safety analysis.

Screening 4 weeks Treatment period 2 weeks Safety follow-up 3-5 weeks N=9 Connection 1+Connection P+Compound III N=3 Triple placebo

compound 1120 mg q12h/coeguHeHue II 50 mg q12h/coeguHeHue III 150 mg q12h.

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At day 15, the mean absolute improvement in pFOEF ₁ was +9.6 percentage points from baseline in patients who received Compound I (120 mg every 12 hours), Compound II, and Compound III triple combination regimens and mean reduction in chloride in sweat was -41.6 mmol/l. For placebo patients, the mean absolute reduction in pFOEF1 was -0.4 and the mean decrease in sweat chloride was -11.0.

Placebo N=3 Compound I/ Compound II/ Compound III N=9 Initial value ππΟΦΒι; Mean value (SD) 44.9 (9.6) 48.0(12.7) Mean Change at Day 15 (SD) -0.4(1.0) 9.6 (10.0) p-value (within test options)* 0.5398 0.0304

# one-sample t-test to N=8 for the triple combination on day 15.

placebo N=3 Compound I/ Compound II/ Compound III N=9 The initial content of chloride in sweat; Mean value (SD) 104.3 (4.9) 107.7 (10.5) Mean Change at Day 15 (SD) -11.0(12.5) -41.6(10.3) p-value (within test options)* 0.2667 <0.0001

# one-sample t-test N=8 for the triple combination on day 15.

The following is an overview of adverse events that occurred during treatment (AELI) after 15 days.

Placebo N=3 and (%) Compound I/ Compound II/ Compound III N=9 p(%) Objects with any NJVL 0 8 (88.9) Objects with severe NJVL 0 0 Objects with serious NELV 0 2 (22.2)

Subjects with AELV leading to discontinuation of treatment 0 0 Subjects with AELV leading to drug discontinuation 0 0

Example 11 Safety and Efficacy Study 3 of Compound I

In study 3A, the following groups of MCs and DCs will be studied in 100 subjects 12 years of age or older with cystic fibrosis who are homozygous for the F508del (F/F) mutation. The total duration of the study is approximately 16 weeks (4 weeks for screening, then 4 weeks for Compound II/Compound III run-in period, then 4 weeks for treatment period, followed by 4 weeks for safety follow-up period). During the compound II/compound III run-in period, all subjects will receive compound II 100 mg once daily (qd)/compound III 150 mg every 12 hours (q12h). After completion of the Compound II/Compound III run-in period, subjects will be randomized to the TC or placebo/DC group (approximately 50 patients in each group) during the treatment period. The treatment groups and doses to be evaluated are shown in the table below.

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Claims (23)

Introductory period Compounds II/ Compounds III Treatment period Group treatment Compound I Dose Compound II Dose Compound III Dose Treatment group Compound I Dose Compound II Dose Compound III Dose Triple combination (TK) 0 mg 100 mg qd 150 mg ql2h Triple combination (TK) 240 mg qd 100 mg qd 150 mg ql2h Compound P/Compound III (DC) 0 mg 100 mg qd 150 mg ql2h Connection P/Connection III 0 mg 100 mg qd 150 mg ql2h The primary endpoint of the study is the mean absolute change in lung function (mFEF1) from baseline at week 4 of triple combination treatment compared with patients who received placebo, tezacaftor, and ivacaftor. Secondary endpoints will also be measured at week 4 and include changes in patient-reported outcomes as measured by the respiratory domain in the Cystic Fibrosis Revised Questionnaire (CFQ-R) and changes in sweat chloride, among others. In study 3B, the following groups of TKs and DCs will be studied in cystic fibrosis (MB) subjects who are heterozygous for the F508del mutation and the MF mutation (F/MF subjects). The total duration of the study is approximately 32 weeks (4 weeks for the screening period, then 24 weeks for the treatment period, followed by 4 weeks for the safety follow-up period). Unlike Study 3A, Study 3B does not have a lead-in period. Subjects will be randomized to either the TC group or the triple placebo group. The doses to be evaluated are shown in the table below. Treatment groups and doses Treatment group Compound I Dose Compound II Dose Compound III Dose TC 240 mg qd 100 mg qd 150 mg ql2h Triple placebo 0 mg 0 mg 0 mg q12h: every 12 hours, qd: once a day, TC: triple combination. Example 12 Compound I Safety and Efficacy Study 4 To assess the long-term safety and efficacy of Compound I, in Study 4, patients who completed the treatment period in Study 3A or 3B will receive TA at the same doses as evaluated in Study 3A or 3B. The total duration of the study is approximately 100 weeks (including a 96-week treatment period (not including 4 weeks for Study 3A or 3B) followed by a 4-week safety follow-up period). Example 13 Preclinical Toxicological Data Preclinical reproductive toxicology studies of Compound I did not reveal any adverse results. Other embodiments. The foregoing discussion discloses and describes only exemplary embodiments of this disclosure. One of ordinary skill in the art will readily appreciate from such discussion and the accompanying drawings and claims that various changes, modifications and variations may be made therein without departing from the spirit and scope of this disclosure as defined in the following claims. CLAIM 1. A method for treating cystic fibrosis, comprising daily administration to a patient in need of such treatment: (A) 80 to 400 mg of at least one compound selected from compound I - 75 041854 and its pharmaceutically acceptable salts; and (B) at least one additional CFTR modulator compound selected from compound II and its pharmaceutically acceptable salts. 2. A method of treating cystic fibrosis, including daily administration to a patient in need of such treatment: (A) 80 to 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts; and (B) at least one additional CFTR modulator compound selected from compound III and Compound III-d and pharmaceutically acceptable salts thereof. 3. A method for the treatment of cystic fibrosis, including daily administration to a patient in need of such treatment: (A) 80 to 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts; and (B) at least one additional CFTR modulator compound selected from compound IV and its pharmaceutically acceptable salts. - 76 041854 4. The method of claim 2 further comprising administering to said patient at least one compound selected from compound IV and pharmaceutically acceptable salts thereof. 5. The method of claim 1 further comprising administering to said patient at least one compound selected from compound III and pharmaceutically acceptable salts thereof. 6. The method of claim 1 further comprising administering to said patient at least one compound selected from compound III-d and pharmaceutically acceptable salts thereof. 7. The method according to any one of claims 1 to 6, wherein 120 to 240 mg of Compound I or a pharmaceutically acceptable salt thereof is administered daily. 8. The method according to any one of claims 1 to 6, wherein 120 to 360 mg of Compound I or a pharmaceutically acceptable salt thereof is administered daily. 9. The method according to any one of claims 1 to 6, wherein 120 mg of Compound I or a pharmaceutically acceptable salt thereof is administered daily. 10. The method according to any one of claims 1 to 6, wherein 240 mg of Compound I or a pharmaceutically acceptable salt thereof is administered daily. 11. The method according to any one of claims 1 to 6, wherein 120 mg of Compound I or a pharmaceutically acceptable salt per dosage is administered twice a day. 12. The method of claim 1 wherein 50 to 150 mg of compound II or a pharmaceutically acceptable salt thereof is administered daily. 13. The method of claim 1 wherein 100 mg of compound II or a pharmaceutically acceptable salt thereof is administered daily. 14. The method according to claim 2, in which: (A) 50 to 600 mg of Compound III, or a pharmaceutically acceptable salt thereof, is administered daily; or (B) 50 to 600 mg of Compound III-d, or a pharmaceutically acceptable salt thereof, is administered daily. 15. The method according to claim 2, in which: (A) 125 to 300 mg of Compound III, or a pharmaceutically acceptable salt thereof, is administered daily; or (B) 125 to 300 mg of Compound III-d, or a pharmaceutically acceptable salt thereof, is administered daily. 16. The method according to claim 1, in which: (A) 75 mg of Compound III or a pharmaceutically acceptable salt per dosage is administered twice a day; or (B) 100 mg of Compound III-d or a pharmaceutically acceptable salt per dosage is administered once a day. 17. The method of claim 3, wherein 400 to 1000 mg of compound IV or a pharmaceutically acceptable salt thereof is administered daily. 18. Method for the treatment of cystic fibrosis, comprising daily administration to a patient in need of such treatment, from 80 to 400 mg of at least one compound selected from compound I and its pharmaceutically acceptable salts; and (A) 50 to 200 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts are administered daily and/or 150 to 600 mg of at least one compound selected from compound III and its pharmaceutically acceptable salts , administered daily; or (B) 50 to 200 mg of at least one compound selected from compound II and its pharmaceutically acceptable salts are administered daily and/or 100 to 400 mg of at least one compound selected from compound III-d and its pharmaceutically acceptable acceptable salts, injected daily. 19. The method according to any one of claims 1 to 6, wherein at least one compound selected from compound I, or a pharmaceutically acceptable salt thereof, is compound I. 20. The method according to any one of claims 1 to 6, wherein at least one compound selected from Compound I or a pharmaceutically acceptable salt thereof is a pharmaceutically acceptable salt of Compound I. 21. A method for the treatment of cystic fibrosis, including daily administration to a patient in need of such treatment: (A) 80 to 400 mg of Compound I or a pharmaceutically acceptable salt thereof; 50 mg of Compound II or a pharmaceutically acceptable salt thereof; and 150 or 300 mg of Compound III or a pharmaceutically acceptable salt thereof. 22. A method for the treatment of cystic fibrosis, including daily administration to a patient in need of such treatment: (B) 80 to 400 mg of Compound I or a pharmaceutically acceptable salt thereof; 50 mg of compound II or a pharmaceutically acceptable salt thereof; and 150, 200 or 300 mg of compound III-d or a pharmaceutically acceptable salt thereof. 23. The method according to item 21, in which 120 mg of compound I or its pharmaceutically acceptable salt; 50 mg of Compound II or a pharmaceutically acceptable salt thereof and 150 mg of Compound III or a pharmaceutically acceptable salt thereof are administered daily. -