Amino acid compositions and methods for treating cystic fibrosis

Abstract

Provided herein are amino acid compositions useful for increasing the translocation of cystic fibrosis transmembrane conductance (CFTR) protein from the cytoplasm to the cell membrane, particularly in epithelial cells. Methods for increasing the concentration of CFTR in the cell membrane, increasing chloride ion transport, and increasing water transport are also provided. These compositions and methods are useful for treating cystic fibrosis in subjects carrying one or more mutations in the CFTR protein. Use of these compositions for treating cystic fibrosis and in preparing medicaments for treating cystic fibrosis is also encompassed herein.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 752,847, filed October 30, 2018, the entirety of which is incorporated by reference herein for all purposes.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on October 25, 2019, is named 174821-013301PCT_SL.txt and is 25,987 bytes in size.

[0003] FIELD OF THEINVENTION Described herein are amino acid compositions useful for increasing the translocation of cystic fibrosis transmembrane conductance (CFTR) protein from cytoplasm to cell membrane.Methods are also presented for increasing the concentration of CFTR in cell membrane, increasing chloride ion transport, and increasing water transport.The compositions and methods described herein are useful for treating cystic fibrosis in subjects who carry one or more mutations in CFTR protein.The use of these compositions for treating cystic fibrosis and preparing medicaments for treating cystic fibrosis are also encompassed herein. [Background technology]

[0004] 2. Background of the Invention Cystic fibrosis (CF) is an inherited recessive disease caused by reduced or absent CFTR synthesis, protein misfolding and / or channel dysfunction, resulting in reduced chloride secretion, increased sodium absorption (ENaC), and impaired fluid homeostasis in airway, intestinal and pancreatic epithelial cells. CF is the most common fatal genetic disorder that can affect the lungs, liver, pancreas, kidneys, and intestine. Cystic fibrosis is inherited in an autosomal recessive manner, with each parent carrying a mutation in at least one allele of the gene encoding the cystic fibrosis transmembrane conductance (CFTR) protein, and one in four offspring carrying two mutated copies (e.g., alleles) of the CFTR gene. Thus, more than 75% of patients are diagnosed with cystic fibrosis by the age of 2 years (1). Although the life expectancy of people living with cystic fibrosis has increased in recent years, the median age of survival is currently about 40 years. Current treatments often involve burdensome supportive care designed to manage the increased risk of pulmonary infections and poor nutritional status. In addition, although small molecule therapies have recently been approved to treat the underlying genetic cause of CF, these treatments are less effective against the most common CFTR mutation, Phe508del, and are cost-prohibitive, exceeding $300,000 per patient per year (2).

[0005] Although over 2,000 CFTR mutations have been identified in cystic fibrosis affected patients, the vast majority of cystic fibrosis diagnoses display only a handful of mutations (3). The most common mutation (Phe508del) is a three-nucleotide deletion resulting in the loss of one codon for the amino acid phenylalanine (three-letter code: Phe, single-letter code: F). This mutation results in defects in CFTR protein processing (e.g., folding and transport to the cell membrane), resulting in little to no membrane expression of the chloride ion transporter protein CFTR, which is involved in the transport of ions and water across the cell membrane. In addition, the small amount of Phe508del CFTR that successfully translocates to the cell membrane is often functionally defective, as characterized by impaired chloride ion transport. In general, CFTR mutations result in dysregulation of the ion gradient across the membrane, resulting in a decrease in the osmolality of water flowing out of the epithelial cells and the appearance of a thick mucus layer covering the cells. The thick, nutrient-rich mucus serves as an optimal environment for the trapping and proliferation of bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus, resulting in persistent infections that often do not respond to antibiotics. In addition, many CF patients also develop other lung diseases such as bronchopulmonary aspergillosis and bronchiectasis, resulting in increased morbidity and mortality.

[0006] Targeted therapies for cystic fibrosis caused by certain mutations in CFTR have been developed, but most are only effective in patients with certain CFTR mutations and have reported side effects (2). One example is ivacaftor (VX-770, KAYLDECO™), a small molecule potentiator of CFTR, as described in U.S. Patent Application Publication No. 2014 / 0221424 and International Patent Application PCT / US2015 / 036691 (4). However, ivacaftor is intended for the treatment of CF in patients with the missense mutation Gly551Asp (G551D) on at least one allele of the CFTR gene, which encompasses approximately 4-5% of patients affected by CF (5). There is a clear unmet need for therapies directed at the treatment of cystic fibrosis, especially in patients carrying the most common CFTR mutation, Phe508del CFTR. [Brief description of the drawings]

[0007] [Figure 1A] FIG. 1A shows a graph of C18-free benzamil-insensitive current (μA) for the control (basal Ringer's) solution and various individual amino acids (AA) (n=4). [Figure 1B] FIG. 1B shows a graph of benzamil-insensitive current (μA) with C18 for the control (basal Ringer) solution and various individual AAs (n=4). [Figure 2A] FIG. 2A shows a graph of C18-free benzamil-insensitive current (μA) for the control (basal Ringer) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4) (n=4). [Figure 2B] FIG. 2B shows a graph of benzamil-insensitive current (μA) with C18 for control (basal Ringer) solution, basal Ringer with DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3, and CF4AA-4) (n=4). [Figure 3A] FIG. 3A shows a graph of the benzamil-sensitive current (μA) without C18 for the control (basal Ringer's) solution and various individual amino acids (AA) (n=4). [Figure 3B] FIG. 3B shows a graph of benzamil-sensitive current (μA) with C18 for the control (basal Ringer) solution and various individual amino acids (AA) (n=4). [Figure 4A] FIG. 4A shows a graph of benzamil-sensitive current (μA) with C18 for control (basal Ringer) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4) (n=4). [Figure 4B]FIG. 4B shows a graph of benzamil-sensitive current (μA) with C18 for control (basic Ringer) solution, basal Ringer with DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3, and CF4AA-4) (n=4). [Diagram 5] Figure 5 shows a graph depicting basal chloride flux. Unstimulated CF cells exposed to vehicle do not secrete chloride. However, chloride secretion was significantly increased in CF cells exposed to CF-5AA-3 (P<0.05; n=7). [Figure 6] Figure 6 shows a graph depicting basal chloride flux. Unstimulated CF cells exposed to vehicle and CF-5AA-3 have significantly higher sodium absorption compared to unstimulated normal HBEC (P<(0.001; n=6). [Figure 7A-7F] Figures 7A-7F show graphs depicting anion currents and stimulated chloride flux. Figure 7A shows that benzamil-insensitive currents (anion currents) of CF cells bathed in vehicle were significantly lower compared to normal HBECs, but CF-5AA-3 increased anion currents by more than 10-fold (n=4). Figure 7B shows that bumetanide-sensitive currents (chloride currents) were significantly higher in CF cells bathed in CF-5AA-3 compared to vehicle (n=4), but did not reach the values ​​of normal HBECs. Figures 7C, 7D, 7E, and 7F show that CF cells bathed in CF-5AA-3 had significantly higher anion peak currents (Figure 7E) and total chloride secretion (Figure 7F), and stimulation with FSK and GLPG1837 did not contribute to the increased values ​​in the presence of AA (Figures (7C,D)). [Figure 8A-8B]Figures 8A and 8B show graphs showing ENaC activity and blocked sodium flux. Figure 8A shows that CF cells bathed in vehicle or CF-5AA-3 had significantly higher ENaC activity (benzamil-sensitive current) compared to normal HBECs, but ENaC activity was slightly lower in CF cells bathed in CF-5AA-3 compared to vehicle (n=4). Figure 8B similarly shows that sodium absorption was significantly increased in CF cells, and was slightly lower in CF cells bathed in CF-5AA-3 (n=7). [Figure 9] 9 shows a graph depicting the effect of the indicated AA formulations on ENaC activity in CF cells. CF cells exposed to CF-4AA-3 were able to further reduce ENaC activity compared to vehicle and CF-5AA-3 (n=4). [Figure 10A-10B] 10A and 10B show graphs showing the effect of C18 (corrector; similar to lumacaftor) and / or VX661 (corrector; tezacaftor) in the presence of forskolin and enhancers on CFTRΔF508 cells. Ivacaftor: GLPG1837 (reversible enhancer); Symdeko: VX661 (tezacaftor) / C18. CF5AA-3 increased the current to a greater extent than C18 or Symdeko. [Figure 11] FIG. 11 shows a graph depicting 36 chloride flux studies in primary HBEC harboring CFTRΔF508 that demonstrated increased chloride secretion in response to CF-5AA-3. [Figure 12] 12 shows a graph demonstrating that CF5AA-3 increased anion current through CFTR in primary HBECs carrying CFTRΔF508. Anion current with CF5AA-3 was significantly higher and sustained compared to the triple combination. [Figure 13] 13 shows a graph showing that CF5AA-3 increased anion currents through Ano1. Anol-mediated currents with CF5AA-3 are significantly higher and sustained compared to currents observed with the triple combination. [Figure 14]FIG. 14 depicts a graph showing that CF5AA-3 increases apical anion secretion to the same extent as the indicated correctors and enhancers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The targeted embodiments are defined by the claims, not this summary. This summary is a high-level overview of various aspects and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all of the drawings, and the claims.

[0009] The present disclosure recognizes the need to provide more cost-effective and effective treatment options for cystic fibrosis patients. The amino acid compositions described herein may be particularly useful for treating CF patients carrying at least one Phe508del mutation on an allele of the CFTR gene. When the CFTR gene is homozygous for the Phe508del mutation, little to no CFTR is transported to the cell membrane, rendering treatment with CFTR enhancers (e.g., ivacaftor) that enhance the function of CFTR present in the cell membrane substantially ineffective. The present disclosure provides compositions that have been shown to increase the translocation of both wild-type and Phe508del CFTR proteins from the cytoplasm to the cell membrane. In particular, the compositions described herein are particularly effective in increasing the number of mutant CFTR proteins on the cell membrane. Additionally, methods are provided herein for treating diseases in which CFTR dysfunction exists (e.g., cystic fibrosis).

[0010] Preparations - Amino acids In a first aspect, the present invention provides a formulation comprising as free amino acids cysteine ​​and proline and at least one additional free amino acid selected from the group consisting of glycine, tyrosine and lysine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0011] In one embodiment, the free amino acid is an L-amino acid.

[0012] In one embodiment, the formulation is free of free amino acids other than at least one additional amino acid selected from the group consisting of cysteine, proline and glycine, tyrosine and lysine.

[0013] In one embodiment, the formulation consists essentially of the free amino acids cysteine ​​and proline, and one or more additional free amino acids selected from the group consisting of glycine, tyrosine and lysine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0014] In one embodiment, the formulation consists of the free amino acids cysteine ​​and proline, and one or more additional free amino acids selected from the group consisting of glycine, tyrosine and lysine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0015] In one embodiment, the formulation comprises, consists of, or consists essentially of the free amino acids cysteine, proline and glycine, and optionally one or more additional free amino acids selected from the group consisting of tyrosine and lysine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0016] In one embodiment, the formulation comprises, consists of, or consists essentially of cysteine, proline and glycine as free amino acids, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0017] In one embodiment, the formulation comprises, consists of, or consists essentially of the free amino acids cysteine, proline and tyrosine, and optionally one or more additional free amino acids selected from the group consisting of glycine and lysine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0018] In one embodiment, the formulation comprises, consists of, or consists essentially of cysteine, proline and tyrosine as free amino acids, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0019] In one embodiment, the formulation comprises, consists of, or consists essentially of the free amino acids cysteine, proline and lysine, and optionally one or more additional free amino acids selected from the group consisting of glycine and tyrosine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0020] In one embodiment, the formulation comprises, consists of, or consists essentially of cysteine, proline and lysine as free amino acids, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0021] In one embodiment, the formulation comprises, consists of, or consists essentially of the free amino acids cysteine, proline, glycine and tyrosine, and optionally additionally comprises lysine as a free amino acid, provided that at least one of the free amino acids is an L-amino acid.

[0022] In one embodiment, the formulation comprises, consists of, or consists essentially of cysteine, proline, glycine and tyrosine as free amino acids, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0023] In one embodiment, the formulation comprises, consists of, or consists essentially of, as free amino acids, cysteine, proline, glycine and lysine, with the proviso that at least one of the free amino acids is an L-amino acid, and optionally additionally comprises, as a free amino acid, tyrosine.

[0024] In one embodiment, the formulation comprises, consists of, or consists essentially of cysteine, proline, glycine and lysine as free amino acids, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0025] In one embodiment, the formulation comprises, consists of, or consists essentially of, as free amino acids, cysteine, proline, lysine and tyrosine, with the proviso that at least one of the free amino acids is an L-amino acid, and optionally additionally comprises glycine as a free amino acid.

[0026] In one embodiment, the formulation comprises, consists of, or consists essentially of cysteine, proline, lysine and tyrosine as free amino acids, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0027] In one embodiment, the formulation comprises, consists of, or consists essentially of the free amino acids cysteine, proline, glycine, tyrosine and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0028] In one embodiment, the formulation is free of other free amino acids.

[0029] In a second aspect, the present invention provides a formulation comprising cysteine ​​and proline as free amino acids and at least one additional free amino acid selected from the group consisting of glycine, tyrosine, lysine and valine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0030] In one embodiment of the second aspect, the free amino acid is an L-amino acid.

[0031] In one embodiment of the second aspect, the formulation does not comprise any free amino acids other than cysteine, proline and at least one additional amino acid selected from the group consisting of glycine, tyrosine, lysine, and valine.

[0032] In one embodiment of the second aspect, the formulation comprises, consists of, or consists essentially of cysteine, proline, and valine as free amino acids, with the proviso that at least one of the free amino acids is an L-amino acid.

[0033] In one embodiment of the second aspect, the formulation comprises, consists of, or consists essentially of cysteine, proline, valine, and glycine, with the proviso that at least one of the free amino acids is an L-amino acid.

[0034] In one embodiment of the second aspect, the formulation comprises, consists of, or consists essentially of cysteine, proline, valine, and tyrosine as free amino acids, with the proviso that at least one of the free amino acids is an L-amino acid.

[0035] In one embodiment, any one of the formulations of the second aspect does not contain other free amino acids.

[0036] Formulations - Excipients etc. In one embodiment, the formulation further comprises water.

[0037] In one embodiment, the formulation is a pharmaceutical formulation.

[0038] In one embodiment, the formulation further comprises a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient.

[0039] In one embodiment, the formulation is sterile.

[0040] In one embodiment, the formulation is formulated for administration by enteral, pulmonary, inhalation, intranasal, or sublingual routes.

[0041] In one embodiment, each free amino acid is present in a therapeutically effective amount.

[0042] Treatment method In a further aspect, the present invention provides the above formulation for use as a medicament.

[0043] In one embodiment of the invention, the formulation is for use in the treatment of cystic fibrosis.

[0044] The invention also provides the use of the formulation described above in the manufacture of a medicament for the treatment of cystic fibrosis.

[0045] In a further aspect, the present invention provides a method for treating a subject suffering from cystic fibrosis, comprising administering to a subject suffering from cystic fibrosis a formulation as described herein above, wherein the administration reduces at least one symptom of cystic fibrosis.

[0046] In one embodiment, the subject with cystic fibrosis expresses wild-type CFTR. In one embodiment, the subject has a mutation in the CFTR gene. In one embodiment, the subject expresses wild-type and mutant CFTR. In one embodiment, the subject has cystic fibrosis in which there is a CFTR protein that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR, and the wild-type CFTR sequence comprises SEQ ID NO: 1. In one embodiment, the mutation in CFTR comprises Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or a combination thereof, where X is any amino acid. In one embodiment, the CFTR is a Phe508del CFTR mutant.

[0047] In one embodiment, the above formulations are used in combination with an additional therapeutic agent.

[0048] In one embodiment, the additional therapeutic agent comprises at least one of a small molecule drug, a protein drug, a nucleic acid drug, or a combination thereof. In one embodiment, the additional therapeutic agent is at least one of a CFTR enhancer, a CFTR corrector, a CFTR read-through agent, or a combination thereof. In one embodiment, the additional therapeutic agent is a CFTR enhancer. In one embodiment, the CFTR enhancer is ivacaftor.

[0049] Further aspects of the invention In a further aspect of the invention, there is provided a kit comprising the pharmaceutical formulation described above and instructions for administering to a subject or for contacting a biological sample with the formulation.

[0050] In a further aspect of the invention, there is provided a method for increasing the number of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins present on the plasma membrane of at least one cell, comprising contacting at least one cell with an effective amount of the pharmaceutical formulation described above, wherein the contacting promotes at least one of CFTR folding or trafficking of CFTR to the plasma membrane, increasing the number of CFTR proteins present on the plasma membrane of the cell.

[0051] In one embodiment, the number of wild-type CFTR proteins on the cell membrane is increased. In one embodiment, the number of mutant CFTR proteins on the cell membrane is increased. In one embodiment, the number of CFTR proteins on the cell membrane that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR is increased, where wild-type CFTR comprises SEQ ID NO: 1. In one embodiment, one or more of the CFTR proteins comprise Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or a combination thereof, where X is any amino acid. In one embodiment, one or more of the CFTR proteins comprise a Phe508del mutation. In one embodiment, increasing the number of CFTR proteins present on the cell membrane is detected by an increase in chloride ion transport out of the cell. In one embodiment, increasing the number of CFTR proteins present on the cell membrane is associated with an increase in water transport out of the cell. In one embodiment, the cell is an epithelial cell. In one embodiment, the epithelial cell is a lung epithelial cell. In one embodiment, the pulmonary epithelial cells are bronchial epithelial cells. In one embodiment, the bronchial epithelial cells are isolated from a subject suffering from cystic fibrosis.

[0052] In certain aspects, there is provided a pharmaceutical formulation comprising, as free amino acids, therapeutically effective amounts of cysteine ​​and proline, and a therapeutically effective amount of at least one additional free amino acid consisting of glycine, tyrosine or lysine, with the proviso that at least one of the free amino acids is an L-amino acid, wherein the pharmaceutical formulation consists essentially of cysteine, proline, and the at least one additional free amino acid.

[0053] In certain embodiments of the pharmaceutical formulation, the free amino acid is an L-amino acid. In another particular embodiment, the pharmaceutical formulation further comprises water. In another particular embodiment, the pharmaceutical formulation further comprises a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In another particular embodiment, the pharmaceutical formulation is sterile. In another particular embodiment, the pharmaceutical formulation is formulated for administration by enteral, pulmonary, inhalation, intranasal, or sublingual routes.

[0054] In certain embodiments of the pharmaceutical preparation, the pharmaceutical preparation comprises, consists essentially of, or consists of a therapeutically effective amount of cysteine ​​and proline as free amino acids and a therapeutically effective amount of at least one additional free amino acid consisting of glycine, tyrosine, or lysine, and does not contain other free amino acids. Recited amino acid combinations include, for example, cysteine, proline, and glycine; cysteine, proline, and tyrosine; cysteine, proline, and lysine; cysteine, proline, glycine, and tyrosine; cysteine, proline, glycine, and lysine; cysteine, proline, tyrosine, and lysine; and cysteine, proline, glycine, tyrosine, and lysine.

[0055] In another particular embodiment of the pharmaceutical preparation, the pharmaceutical preparation comprises, as free amino acids, a therapeutically effective amount of cysteine ​​and proline, and a therapeutically effective amount of at least one additional free amino acid consisting of glycine, tyrosine, lysine, or valine, and no other free amino acids. Recited amino acid combinations include, for example, cysteine, proline, and valine; cysteine, proline, valine, and glycine; and cysteine, proline, valine, and tyrosine.

[0056] In another particular embodiment of the pharmaceutical formulation, the pharmaceutical formulation comprises, as free amino acids, a therapeutically effective amount of each of cysteine, proline, glycine, tyrosine, and lysine, and no other free amino acids. In another particular embodiment of the pharmaceutical formulation, the pharmaceutical formulation consists essentially of, or consists of, as free amino acids, a therapeutically effective amount of each of cysteine, proline, glycine, tyrosine, and lysine.

[0057] In a particular embodiment, the pharmaceutical formulation is for use in therapy. In a more particular embodiment, the pharmaceutical formulation is for use in the treatment of cystic fibrosis. In another particular embodiment, the pharmaceutical formulation is used in the manufacture of a medicament for the treatment of cystic fibrosis.

[0058] In a further aspect, a method for treating a subject suffering from cystic fibrosis is provided, comprising administering to the subject suffering from cystic fibrosis a pharmaceutical formulation as described herein, wherein the administration reduces at least one symptom of cystic fibrosis. In a particular embodiment of the method, the subject expresses wild-type CFTR. In another particular embodiment, the subject has a mutation in the CFTR gene. In another particular embodiment, the subject expresses wild-type CFTR and mutant CFTR. In another particular embodiment, the subject suffers from cystic fibrosis in which there is a CFTR protein that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR, and the wild-type CFTR sequence comprises SEQ ID NO: 1. In another particular embodiment, the mutation in CFTR comprises Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or a combination thereof, where X is any amino acid. In another particular embodiment, the CFTR is a Phe508del CFTR mutant. In another particular embodiment of the method, the method further comprises administering an additional therapeutic agent. In another particular embodiment of the method, the additional therapeutic agent comprises at least one of a small molecule drug, a protein drug, a nucleic acid drug, or a combination thereof. In another particular embodiment of the method, the additional therapeutic agent is at least one of a CFTR enhancer, a CFTR corrector, a CFTR read-through agent, or a combination thereof. In another particular embodiment of the method, the additional therapeutic agent is a CFTR enhancer. In another particular embodiment of the method, the CFTR enhancer is ivacaftor.

[0059] In certain embodiments of the method, the pharmaceutical preparation comprises, consists essentially of, or consists of a therapeutically effective amount of cysteine ​​and proline as free amino acids, and a therapeutically effective amount of at least one additional free amino acid consisting of glycine, tyrosine, or lysine, and does not contain other free amino acids. Recited amino acid combinations include, for example, cysteine, proline, and glycine; cysteine, proline, and tyrosine; cysteine, proline, and lysine; cysteine, proline, glycine, and tyrosine; cysteine, proline, glycine, and lysine; cysteine, proline, tyrosine, and lysine; and cysteine, proline, glycine, tyrosine, and lysine.

[0060] In another particular embodiment of the method, the pharmaceutical formulation comprises, as free amino acids, a therapeutically effective amount of each of cysteine, proline, glycine, tyrosine, and lysine, and no other free amino acids. In another particular embodiment of the method, the pharmaceutical formulation consists essentially of, or consists of, as free amino acids, a therapeutically effective amount of each of cysteine, proline, glycine, tyrosine, and lysine.

[0061] In a further aspect, a method is provided for increasing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the cell membrane of at least one cell, comprising contacting at least one cell with an effective amount of the pharmaceutical formulation described herein, wherein the contacting promotes at least one of CFTR folding or CFTR transport to the cell membrane, thereby increasing the number of CFTR proteins present on the cell membrane of the cell. In a particular embodiment of the method, the number of wild-type CFTR proteins on the cell membrane is increased. In another particular embodiment of the method, the number of mutant CFTR proteins on the cell membrane is increased. In another particular embodiment of the method, the number of CFTR proteins on the cell membrane that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR on the cell membrane is increased, and the wild-type CFTR comprises SEQ ID NO:1. In another particular embodiment of the method, one or more of the CFTR proteins comprises Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or a combination thereof, where X is any amino acid. In another particular embodiment of the method, one or more of the CFTR proteins comprises a Phe508del mutation. In another particular embodiment of the method, the increase in the number of CFTR proteins present on the cell membrane is detected by an increase in chloride ion transport from the cell. In another particular embodiment of the method, the increase in the number of CFTR proteins present on the cell membrane is associated with an increase in water transport from the cell. In another particular embodiment of the method, the cell is an epithelial cell. In a more particular embodiment of the method, the epithelial cell is a lung epithelial cell. In a further particular embodiment of the method, the lung epithelial cell is a bronchial epithelial cell. In another particular embodiment of this method, bronchial epithelial cells are isolated from a subject suffering from cystic fibrosis, and the results of in vitro assays performed on such cells provide an indication of which therapeutic agent and / or combination thereof will provide therapeutic benefit to the subject from whom the cells were isolated. In another particular embodiment of this method, the method further comprises administering an additional therapeutic agent.In another particular embodiment of the method, the additional therapeutic agent comprises at least one of a small molecule drug, a protein drug, a nucleic acid drug, or a combination thereof.In another particular embodiment of the method, the additional therapeutic agent is at least one of a CFTR enhancer, a CFTR corrector, a CFTR read-through agent, or a combination thereof.In another particular embodiment of the method, the additional therapeutic agent is a CFTR enhancer.In another particular embodiment of the method, the CFTR enhancer is ivacaftor.

[0062] In certain embodiments of the method, the pharmaceutical preparation comprises, consists essentially of, or consists of a therapeutically effective amount of cysteine ​​and proline as free amino acids, and a therapeutically effective amount of at least one additional free amino acid consisting of glycine, tyrosine, or lysine, and does not contain other free amino acids. Recited amino acid combinations include, for example, cysteine, proline, and glycine; cysteine, proline, and tyrosine; cysteine, proline, and lysine; cysteine, proline, glycine, and tyrosine; cysteine, proline, glycine, and lysine; cysteine, proline, tyrosine, and lysine; and cysteine, proline, glycine, tyrosine, and lysine.

[0063] In another particular embodiment of the method, the pharmaceutical formulation comprises, as free amino acids, a therapeutically effective amount of each of cysteine, proline, glycine, tyrosine, and lysine, and no other free amino acids. In another particular embodiment of the method, the pharmaceutical formulation consists essentially of, or consists of, as free amino acids, a therapeutically effective amount of each of cysteine, proline, glycine, tyrosine, and lysine.

[0064] In another particular embodiment, a kit is provided that comprises a pharmaceutical formulation comprising, consisting essentially of, or consisting of the free amino acids cysteine, proline, glycine, tyrosine, and lysine and instructions for administering to a subject or instructions for contacting a biological sample with the composition.

[0065] In another aspect, the disclosure provides a composition comprising, consisting essentially of, or consisting of cysteine, proline, glycine, tyrosine, and lysine for use in treating cystic fibrosis. In a further aspect, the present invention provides the use of a composition of amino acids for treating cystic fibrosis in a subject in need thereof. In a further aspect, the present invention provides the use of a composition of amino acids in the preparation of a medicament for the treatment of cystic fibrosis in a subject in need thereof.

[0066] In one aspect, the disclosure provides a composition comprising, consisting essentially of, or consisting of cysteine, proline, glycine, tyrosine, and lysine. In an embodiment, the amino acid is an L-amino acid. In an embodiment, the composition further comprises water. In another aspect, the composition further comprises a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In an embodiment, the composition is sterile. In another aspect, the composition is formulated for administration by enteral, pulmonary, inhalation, intranasal, or sublingual routes.

[0067] In an embodiment, the subject suffers from cystic fibrosis in which wild-type CFTR is present. In one aspect, the subject has a mutation in the CFTR gene. In a further aspect, the subject suffers from cystic fibrosis in which both wild-type and mutant CFTR are present. In yet another aspect, the subject suffers from cystic fibrosis in which a CFTR protein is present that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR provided by SEQ ID NO:1. In yet another aspect, the CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del CFTR mutant, where X is any amino acid. In an embodiment, the CFTR is a Phe508del CFTR mutant.

[0068] Also provided herein is a method for increasing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the cell membrane, comprising contacting the cell with an effective amount of a composition comprising, consisting essentially of, or consisting of cysteine, proline, glycine, tyrosine, and lysine. In some aspects, the effective amount increases the number of CFTR proteins present on the cell membrane. In further aspects, the number of wild-type CFTR proteins on the cell membrane is increased. In further aspects, the number of mutant CFTR proteins on the cell membrane is increased. In some aspects, the number of CFTR proteins on the cell membrane that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR provided by SEQ ID NO:1 is increased. In some embodiments, one or more of the CFTR proteins are Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del mutants, where X is any amino acid. In further embodiments, one or more of the CFTR proteins is a Phe508del CFTR mutant. In some embodiments, chloride ion transport out of the cell is increased. In further embodiments, water transport out of the cell is increased. In further embodiments, the cell is an epithelial cell. In some embodiments, the epithelial cell is a lung epithelial cell. In some embodiments, the lung epithelial cell is a bronchial epithelial cell. In further embodiments, the bronchial epithelial cell is obtained from a subject suffering from cystic fibrosis. The cell may be in vitro, in vivo, or ex vivo.

[0069] In some aspects, the methods provided herein further comprise administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a small molecule drug, a protein drug, or a nucleic acid drug. In some embodiments, the additional therapeutic agent is a small molecule drug. In further embodiments, the additional therapeutic agent is a CFTR enhancer, corrector, or read-through agent. In further embodiments, the additional therapeutic agent is a CFTR enhancer. In some aspects, the CFTR enhancer is ivacaftor. In some embodiments, the additional therapeutic agent is a CFTR corrector. In some embodiments, the CFTR corrector is lumcaftor. The additional therapeutic agent may be included in any of the compositions described herein (e.g., the composition further comprises an additional therapeutic agent). The additional therapeutic agent may be administered simultaneously with, prior to, or following administration of any of the compositions described herein (e.g., combination therapy).

[0070] In a further aspect, the disclosure provides a method for treating cystic fibrosis, comprising administering to a subject in need thereof a composition described herein, wherein the composition is formulated for administration by enteral, pulmonary, inhalation, intranasal, or sublingual routes. In some embodiments, the composition is formulated for pulmonary administration to the subject. In some embodiments, the composition is formulated for enteral administration to the subject. In some embodiments, the composition is formulated for oral administration to the subject.

[0071] In a further aspect, the present disclosure provides a method for treating cystic fibrosis, comprising administering a composition described herein to a subject in need thereof. In an embodiment, the subject suffers from cystic fibrosis, in which the subject has a mutation in the CFTR gene. In an embodiment, the subject suffers from cystic fibrosis, in which the subject is heterozygous for both wild-type and mutant CFTR. In an embodiment, the subject suffers from cystic fibrosis, in which the subject is heterozygous for a first mutant CFTR and a second mutant CFTR (e.g., the mutations are different). In an embodiment, the subject suffers from cystic fibrosis, in which the subject is homozygous for mutant CFTR (e.g., both alleles have the same mutation). In an embodiment, the subject suffers from cystic fibrosis, in which the subject is homozygous for wild-type CFTR (e.g., both alleles are not mutated). In some embodiments, the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del CFTR mutant, where X is any amino acid. In some embodiments, the mutant CFTR is a Phe508del CFTR mutant. In some embodiments, the CFTR protein present in the subject is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR as provided by SEQ ID NO: 1. In some embodiments, the CFTR protein present in the subject is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of Phe508del CFTR as provided by SEQ ID NO: 2.

[0072] Information regarding the diagnosis and treatment of various diseases, including cystic fibrosis and related pulmonary diseases, can be found in Longo, D. et al. (eds.), Harrison's Principles of Internal Medicine, 18th Edition; McGraw-Hill Professional, 2011. Information regarding various therapeutic agents and human diseases, including pulmonary diseases, can be found in Brunton, L. et al. (eds.) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th Edition, McGraw Hill, 2010 and / or Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill / Appleton & Lange; 11th Edition (June 2009). All patents, patent applications, books, articles, documents, databases, websites, publications, references, etc. mentioned herein are incorporated by reference in their entirety. In the event of any discrepancy between this specification and any of the incorporated references, this specification (including any amendments thereto) shall prevail. The applicant reserves the right, for example, to amend this specification based on any of the incorporated materials and / or to correct obvious errors. The content of any of the incorporated materials does not limit the present invention. Standard art-accepted meanings of terms are used herein unless otherwise indicated. Standard abbreviations for various terms are used herein.

[0073] In certain aspects, the compositions provided herein comprise, consist essentially of, or consist of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine, and are free of other free amino acids.

[0074] Also provided herein is a use of a composition comprising cysteine, proline, glycine, tyrosine and lysine for treating cystic fibrosis in a subject in need thereof.

[0075] Also provided herein is a kit comprising a composition comprising, consisting essentially of, or consisting of cysteine, proline, glycine, tyrosine, and lysine; and instructions for administering to a subject or for contacting a biological sample with the composition.

[0076] All combinations of the embodiments described separately are envisaged.

[0077] The details of certain embodiments of the invention are set forth in the Detailed Description of Specific Embodiments, as set forth below. Other features, objects, and advantages of the invention will become apparent from the definition, examples, figures, and claims.

[0078] definition Explanations and specific information relating to various terms used in the present disclosure are collected herein for convenience.

[0079] The term "agent" is used herein to refer to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound can be any agent that can be represented by a chemical formula, chemical structure, or sequence. Examples of agents include, for example, small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotides, aptamers), lipids, polysaccharides, and the like. In general, an agent can be obtained using any suitable method known in the art. A skilled artisan will select an appropriate method based, for example, on the nature of the agent. An agent can be at least partially purified. In some embodiments, an agent can be provided as part of a composition, which can include, in addition to the agent in various embodiments, for example, counterions, aqueous or non-aqueous diluents or carriers, buffers, preservatives, or other components. In some embodiments, an agent can be provided as a salt, ester, hydrate, or solvate. In some embodiments, an agent is cell-permeable, e.g., within the scope of typical agents that are taken up by cells and act to produce a biological effect within a cell, e.g., a mammalian cell. Certain compounds may exist in specific geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)-isomers, racemic mixtures thereof, and other mixtures thereof, are encompassed by the present disclosure in various embodiments, unless otherwise indicated. Certain compounds may exist in various states or protonation states, may have various configurations, may exist as solvates (e.g., water (i.e., hydrates) or common solvents), and / or may have different crystal forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable. The term "agent" may also encompass "therapeutic agent." The terms "compound" and "agent" may be used interchangeably.

[0080] An "effective amount" or "effective dose" of an agent (or a composition containing such an agent) refers to an amount sufficient to achieve a desired biological and / or pharmacological effect when delivered to a cell or organism, for example, according to a selected administration form, route, and / or schedule. The phrases "effective amount" and "therapeutically effective amount" are used interchangeably. One of ordinary skill in the art will understand that the absolute amount of a particular agent or composition that is effective may vary depending on factors such as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, and the like. One of ordinary skill in the art will further understand that an "effective amount" may be contacted with a cell or administered to a subject in a single dose or through the use of multiple doses in various embodiments. In an embodiment, an effective amount is an amount that increases the transport of CFTR to the cell membrane of a cell. In an embodiment, an effective amount is an amount that increases the translocation of CFTR from the cytoplasm of a cell to the cell membrane. In an embodiment, an effective amount is an amount that increases chloride ion transport out of a cell. In an embodiment, an effective amount is an amount that increases water transport out of a cell. In an embodiment, an effective amount is an amount that reduces and / or treats a symptom of a pulmonary disease. In one embodiment, an effective amount is an amount that reduces and / or treats a symptom of cystic fibrosis.

[0081] The term "consisting essentially of" as used herein limits the scope of ingredients and steps to those that do not substantially affect the specific materials or steps and the basic and novel feature(s) of the present invention, e.g., the composition and its use for the treatment of cystic fibrosis, and the method of treating cystic fibrosis. For example, by using "consisting essentially of," the therapeutic composition does not include any unspecified ingredients, including, but not limited to, free amino acids, dipeptides, oligopeptides, or polypeptides or proteins; and monosaccharides, disaccharides, oligosaccharides, polysaccharides, and carbohydrates that have a direct beneficial or detrimental therapeutic effect on the treatment of cystic fibrosis. By using the term "consisting essentially of," the composition may include substances that have no therapeutic effect on the treatment of cystic fibrosis. Such ingredients include carriers, excipients, adjuvants, flavorings, and the like that do not affect the health or function of the pulmonary epithelium.

[0082] Description of any aspect or embodiment herein using terms such as "comprising," "having," "including," or "containing," with reference to an element or elements, is intended to provide support for similar aspects or embodiments that "consist," "consist essentially of," or "substantially comprise" that particular element or elements, unless otherwise indicated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood to also describe a composition consisting of that element, unless otherwise indicated or clearly contradicted by context).

[0083] The term "gene" refers to a locus (e.g., region) of DNA composed of nucleotides. In general, a gene contains multiple regions, including an open reading frame, including one or more upstream or downstream regulatory sequences (e.g., enhancers / silencers, promoters, 5' non-coding sequences, 3' non-coding sequences) normally required to initiate transcription, one or more exons, and one or more introns. An "exon" is any part of a gene that codes for a portion of the final mature RNA that is translated into a protein sequence. An "intron" is any part of a gene that is removed by RNA splicing during maturation of the final mature RNA. A "cryptic exon" is an exon that can introduce a premature translation stop codon into the mature RNA or result in an atypical splicing pattern. The term "gene" may refer to a nucleic acid fragment that expresses a protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. A "native gene" refers to a gene as found in nature with its own regulatory sequences. Thus, a chimeric gene or chimeric construct may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. "Foreign" gene refers to a gene not normally found in the host organism but that is introduced into the host organism by gene transfer. Foreign genes may include native genes inserted into a non-native organism or chimeric genes.

[0084] The term "gene product" (also referred to herein as "gene expression product" or "expression product") encompasses products resulting from expression of a gene, such as RNA transcribed from a gene, and polypeptides resulting from translation of such RNA. It is understood that some gene products may be processed or modified, for example, in a cell. For example, RNA transcripts may be spliced, polyadenylated prior to mRNA translation, and / or polypeptides may be subjected to co-translational or post-translational processing, such as removal of secretory signal sequences, removal of organelle targeting sequences, or modifications such as phosphorylation, fatty acylation, and the like. The term "gene product" encompasses such processed or modified forms. Genomic, mRNA, and polypeptide sequences from various species, including human, are known in the art and are available in publicly accessible databases, such as those available at the National Center for Biotechnology Information (www.ncbi.nih.gov) or the Universal Protein Resource (www.uniprot.org). Databases include, for example, GenBank, RefSeq, Gene, UniProtKB / SwissProt, UniProtKB / Trembl, etc. In general, sequences in the NCBI reference sequence database, such as mRNA and polypeptide sequences, can be used as gene product sequences of the gene of interest. It should be understood that multiple alleles of a gene can exist between individuals of the same species. For example, differences in one or more nucleotides (e.g., about 1%, 2%, up to 3-5% of nucleotides) of a nucleic acid encoding a particular protein can exist between individuals of a given species. Due to the degeneracy of the genetic code, such variations often do not change the encoded amino acid sequence, although DNA polymorphisms that result in changes in the sequence of the encoded protein can exist.Examples of polymorphic variants can be found, for example, in the Single Nucleotide Polymorphism Database (dbSNP) available on the NCBI website at www.ncbi.nlm.nih.gov / projects / SNP / [Sherry, ST et al. (2001) dbSNP: The NCBI database of genetic variation Nucl Acids Res, 29: 308-311; Kitts, A. and Sherry, S. (2009) The single nucleotide polymorphism database (dbSNP) of nucleotide sequence variation in the NCBI Handbook (Internet); McEntyre, J., Ostell, J. (eds.). Bethesda (MD): National Center for Biotechnology Information (US); 2002 (www.ncbi.nlm.nih.gov / bookshelf / br.fcgi?book=handbook&part=ch5)]. Multiple isoforms of a protein may exist, for example, as a result of alternative RNA splicing or editing. In general, where aspects of the present disclosure relate to genes or gene products, unless otherwise indicated, embodiments relating to allelic variants or isoforms are encompassed, where applicable. Certain embodiments may be directed to a particular sequence(s), for example, a particular allele(s) or isoform(s).

[0085] The term "amino acid" includes all known amino acids that contain an amine (-NH2) functional group, a carboxyl (-COOH) functional group, and a side chain ("R") group specific to each amino acid. "Amino acid" includes the 21 amino acids encoded by the human genome (i.e., proteinogenic amino acids), amino acids encoded or produced by bacteria or single-cell organisms, and naturally occurring amino acids. For purposes of this disclosure, the conjugate acid forms of amino acids with basic side chains (arginine, lysine, and histidine) or the conjugate base forms of amino acids with acidic side chains (aspartic acid and glutamic acid) are essentially the same, unless otherwise specified. "Amino acid" also includes derivatives thereof that retain substantially the same activity or better activity in enhancing the effect of the compositions of the invention (e.g., increasing the number of CFTR proteins in cell membranes, increasing chloride ion transport out of cells, treating cystic fibrosis). These derivatives may be, for example, enantiomers and may include both D and L forms of the amino acids. These derivatives may be "natural" or "unnatural" amino acid derivatives (e.g., β-amino acids, homoamino acids, proline derivatives, pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted tyrosine derivatives, ring-substituted phenylalanine derivatives, linear core amino acids, and N-methyl amino acids), such as selenocysteine, pyrrolidine, iodotyrosine, norleucine, or norvaline. Other amino acid derivatives include, but are not limited to, those synthesized, for example, by acylation, methylation, glycosylation, and / or halogenation of amino acids. These include, for example, β-methyl amino acids, C methyl amino acids, and N-methyl amino acids. The amino acids described herein may exist as free amino acids. The term "free amino acid" refers to an amino acid that is not part of a peptide or polypeptide (e.g., not linked to another amino acid via a peptide bond). A free amino acid is free in solution, but may be associated with salts or other components in the solution.

[0086] The terms "protein", "peptide" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified by addition of a chemical entity, such as, for example, a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide may be a single molecule or a multi-molecular complex. In some embodiments, a protein comprises a homodimer or a heterodimer. A protein, peptide, or polypeptide may simply be a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be natural, recombinant, or synthetic, or any combination thereof. A protein may comprise distinct domains, e.g., a nucleic acid binding domain (e.g., a gRNA binding domain of Cas9 that directs the protein to bind to a target site) and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous portion, e.g., an amino acid sequence that constitutes a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is complexed with or bound to a nucleic acid, e.g., RNA. In some embodiments, a protein comprises a ligand binding domain. In some embodiments, a protein comprises an active site (e.g., a site of biological or enzymatic activity). In some embodiments, a protein comprises an allosteric site (e.g., a site of a protein that can bind to a ligand that may be distant from the active site). Any of the proteins provided herein may be produced by any method known in the art.For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods of recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)], the entire contents of which are incorporated herein by reference.

[0087] "Identity" or "percent identity" is a measure of the degree to which two or more nucleic acid or polypeptide sequences are identical. The percent identity between a sequence of interest A and a second sequence B can be calculated by aligning the sequences, allowing for the introduction of gaps to maximize identity, determining the number of residues (nucleotides or amino acids) opposite to identical residues, dividing by the minimum of TGA and TGB (where TGA and TGB are the sum of the number of residues and internal gap positions in sequences A and B in the alignment), and multiplying by 100. When calculating the number of identical residues required to achieve a particular percent identity, the fraction should be rounded to the nearest integer. Sequences can be aligned by using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, gapped BLAST, etc. can be used to generate alignments and / or obtain percent identities. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc Natl Acad Sci USA, 87:22264-2268, 1990), modified as in Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873-5877, 1993, has been incorporated into the NBLAST and XBLAST programs of Altschul et al. [Altschul et al. (1990) J Mol Biol, 215:403-410]. In some embodiments, to obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. [Altschul et al. (1997) Nucleic Acids Res, 25:3389-3402]. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. See the website having the URL www.ncbi.nlm.nih.gov and / or McGinnis, S. and Madden, TL, W20-W25 Nucleic Acids Research, 2004, Vol. 32, Web Server Publication.Other suitable programs include CLUSTALW [Thompson, JD, Higgins, DG, and Gibson, TJ (1994) Nuc Acid Res, 22: 4673-4680], CLUSTAL Omega [Sievers, F., Wilm, A., Dineen, D. et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega Mol Sys Biol, 7: doi: 10.1038 / msb.2011.75], and GAP (GCG version 9.1; which implements the Needleman & Wunsch, 1970 algorithm [Needleman, SB, and Wunsch, CD (1970) J. Mol Biol, 48: 443-453]). Percent identity can be evaluated over a window of evaluation. In some embodiments, the window of evaluation may have a length of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100%, of the minimum length of the sequences to be compared. In some embodiments, the window of evaluation is at least 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000; 1,200; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500; or 5,000 amino acids. In some embodiments, no more than 20%, 10%, 5%, or 1% of the positions in either or both sequences over the evaluation window are occupied by gaps. In some embodiments, no more than 20%, 10%, 5%, or 1% of the positions in either or both sequences are occupied by gaps.

[0088] A "variant" of a particular polypeptide or polynucleotide may have one or more additions, substitutions, and / or deletions relative to the polypeptide or polynucleotide, which may be referred to as the "original polypeptide" or "original polynucleotide," respectively. The additions may be insertions or may be at either end. A variant may be shorter or longer than the original polypeptide or polynucleotide. The term "variant" encompasses "fragments." A "fragment" is a contiguous portion of a polypeptide or polynucleotide that is shorter than the original polypeptide. In some embodiments, a variant comprises or consists of a fragment. In some embodiments, a fragment or variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the length of the original polypeptide or polynucleotide, or longer. A fragment may be an N-terminal fragment, a C-terminal fragment, or an internal fragment. In some embodiments, a variant polypeptide comprises or consists of at least one domain of the original polypeptide. In some embodiments, a variant polypeptide or polynucleotide comprises or consists of a polypeptide or polynucleotide that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in sequence to the original polypeptide or polynucleotide. In some embodiments, a variant polypeptide or polynucleotide comprises or consists of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more than 100% of the polypeptide or polynucleotide of the original polypeptide or polynucleotide. In some embodiments, the sequence of the variant polypeptide comprises or consists of a sequence having N amino acid differences with respect to the original sequence, where N is any integer up to 1%, 2%, 5%, or 10% of the number of amino acids in the original polypeptide, and "amino acid differences" refer to amino acid substitutions, insertions, or deletions. In some embodiments, the substitutions are conservative substitutions. Conservative substitutions may be made, for example, on the basis of similarity in side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.In some embodiments, conservative substitutions are made according to Table A, where amino acids in the same block in the second column and amino acids in the same line in the third column may be substituted for each other with conservative substitutions. A conservative substitution replaces an amino acid in one line in the third column that corresponds to a block in the second column with an amino acid from another line in the third column in the same block in the second column.

[0089] Table A [Table 1]

[0090] In some embodiments, proline (P), cysteine ​​(C), or both are each considered within an individual group. Within a particular group, certain substitutions may be of particular interest in certain embodiments, such as the substitution of isoleucine for leucine (or vice versa), threonine for serine (or vice versa), or glycine for alanine (or vice versa).

[0091] In some embodiments, the variants are biologically active variants, i.e., the variants at least partially retain at least one activity of the original polypeptide or polynucleotide. In some embodiments, the variants at least partially retain two or more or substantially all of the known biologically significant activities of the original polypeptide or polynucleotide. The activity can be, for example, catalytic activity, binding activity, ability to function or participate in a biological structure or process, etc. In some embodiments, the activity of the variants can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the original polypeptide or polynucleotide, and in various embodiments can be up to about 100%, about 125%, or about 150% of the activity of the original polypeptide or polynucleotide. In some embodiments, a variant, e.g., a biologically active variant, comprises or consists of a polypeptide that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the original polypeptide, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% or 100% identical to the original polypeptide. In some embodiments, for example, the changes, e.g., substitutions or deletions, in the functional variant do not alter or remove amino acids or nucleotides known or predicted to be important for activity, e.g., known or predicted catalytic residues or residues involved in binding to substrates or cofactors. Variants can be tested in one or more suitable assays to assess activity.

[0092] As used herein, the term "small molecule" refers to an organic molecule having a mass of less than about 2 kilodaltons (kDa). In some embodiments, a small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, a small molecule is less than about 800 Daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a sugar. In some embodiments, a small molecule contains multiple carbon-carbon bonds and may contain one or more heteroatoms and / or one or more functional groups important for structural interactions with proteins (e.g., hydrogen bonding), such as amines, carbonyls, hydroxyls, or carboxyls, in some embodiments at least two functional groups. Small molecules often contain one or more cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups. In some embodiments, the small molecule is a therapeutically active agent, such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)). The small molecule may also form a complex with one or more metal atoms and / or metal ions. In this example, the small molecule is also referred to as a "small organometallic molecule." Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, and more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents. In some embodiments, the small molecule is a drug. Preferably, but not necessarily, a drug is one that has already been deemed safe and effective for use in humans or animals by an appropriate government agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.FR §§ 330.5, 331 to 361, and 440 to 460, which are incorporated herein by reference. All drugs listed are considered acceptable for use in accordance with the present invention.

[0093] The terms "composition" and "formulation" are used interchangeably.

[0094] The terms "administer," "administering," or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, into or onto a subject.

[0095] In various embodiments, a "subject" may be any vertebrate organism. A subject may be, for example, an individual to whom an agent is administered, or to whom a sample is obtained, or to whom a treatment is performed, for experimental, diagnostic, and / or therapeutic purposes. In some embodiments, the subject is a mammal, for example, a human, a non-human primate, or a rodent (e.g., mouse, rat, rabbit). Mammalian species that may benefit from the disclosed therapeutic methods include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domestic animals such as dogs, cats; farm animals such as horses, cows, pigs, sheep, goats, chickens; and animals such as mice, rats, guinea pigs, and hamsters. In an embodiment, the subject is a human. The human may be of either gender and at any stage of development. In an embodiment, the subject has been diagnosed with cystic fibrosis. In an embodiment, the subject has been diagnosed with cystic fibrosis caused by a CFTR mutation. In an embodiment, the subject has been diagnosed with cystic fibrosis caused by a Phe508del CFTR mutation.

[0096] Human CF animal models are known in the art and described herein. See, e.g., Example 4. See also Grubb et al. (Am J Physiol Lung Cell Mol Physiol 290:L270-L277, 2006); McCarron et al. (Respiratory Research 19:54, 2018); and Lavelle et al. (BioMed Research International Volume 2016, Article ID 5258727, page 14), the entire contents of each of which are incorporated herein by reference. Lavelle et al., for example, describe mouse and pig models of CF.

[0097] As used herein, in the context of treating a subject, "treat", "treatment", "treating" and similar terms refer to providing medical and / or surgical management of the subject. Treatment can include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to the subject. As used herein, the term "treatment" or any grammatical variants thereof (e.g., treat, treating, treatment, etc.) includes, but is not limited to, alleviating the symptoms of a disease or condition; and / or reducing, suppressing, inhibiting, ameliorating, or affecting the progression, severity, and / or extent of a disease or condition. The effect of treatment can also include reducing the likelihood of development or recurrence of a disease, or one or more symptoms or manifestations of a disease. A therapeutic agent may be administered to a subject who has a disease or who is at increased risk of developing a disease compared to members of the general population. In some embodiments, a therapeutic agent may be administered to a subject who has a disease but no longer shows evidence of the disease. An agent may be administered, for example, to reduce the likelihood of recurrence of a disease. A therapeutic agent may be administered prophylactically, i.e., prior to the onset of any symptoms or signs of a disease.

[0098] "Prophylactic treatment" refers to providing medical and / or surgical management to a subject who does not develop or show evidence of a disease, e.g., to reduce the likelihood that a disease will occur or to reduce its severity if the disease does develop. The subject may be identified as being at risk of developing a disease (e.g., having an increased risk compared to the general population or having risk factors that increase the likelihood of developing a disease).

[0099] As used herein, the term "amelioration" or grammatical variations thereof (e.g., ameliorate, ameliorating, and amelioration, etc.) includes, but is not limited to, delaying the onset of or reducing the severity of a disease or condition (e.g., cystic fibrosis or complications thereof). Amelioration, as used herein, does not require the complete absence of symptoms.

[0100] The terms "condition," "disease," and "disorder" are used interchangeably.

[0101] An "effective amount" of a compound described herein refers to an amount sufficient to elicit a desired biological response. In some embodiments, an effective amount is an amount sufficient to increase chloride ion transport. In some embodiments, an effective amount is an amount sufficient to modulate (e.g., increase) the function of a cystic fibrosis transmembrane conductance regulator (CFTR) protein (e.g., wild-type CFTR or mutant CFTR). In some embodiments, an effective amount is an amount sufficient to modulate (e.g., increase) the function of a Phe508del CFTR. In some embodiments, an effective amount is an amount sufficient to increase the translocation of CFTR from the cytoplasm to the cell membrane. An effective amount of a compound described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is a prophylactic treatment. In some embodiments, an effective amount is the amount of a compound described herein in a single dose. In some embodiments, an effective amount is the combined amount of a compound described herein in multiple doses.

[0102] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of a condition, and / or enhances the therapeutic effect of another therapeutic agent. In some embodiments, a therapeutically effective amount is an amount sufficient to increase chloride ion transport (e.g., increase chloride transport from epithelial cells). In some embodiments, a therapeutically effective amount is an amount sufficient to increase water transport (e.g., increase water transport from epithelial cells). In some embodiments, a therapeutically effective amount is an amount sufficient to modulate (e.g., enhance) the function of a cystic fibrosis transmembrane conductance regulator (CFTR) protein (e.g., wild-type CFTR or mutant CFTR). In some embodiments, the therapeutically effective amount is sufficient to modulate (e.g., enhance) the function of Phe508del CFTR. In some embodiments, the therapeutically effective amount is sufficient to increase the translocation of CFTR from the cytoplasm to the cell membrane. In some embodiments, the therapeutically effective amount is sufficient to treat a pulmonary disease. In some embodiments, the therapeutically effective amount is sufficient to treat cystic fibrosis.

[0103] As used herein, the term "salt" refers to any and all salts, including pharma- ceutically acceptable salts.

[0104] The term "carrier" may refer to any diluent, adjuvant, excipient, or vehicle with which the composition of the present disclosure is administered. Examples of suitable pharmaceutical carriers are described in Remington's Essentials of Pharmaceuticals, 21st Edition, Ed. Felton, 2012, which is incorporated herein by reference.

[0105] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0106] Pharmaceutically acceptable excipients used in the manufacture of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents, and fragrances may also be present in the composition. The exact amount of the composition containing an amino acid required to achieve an effective amount will vary from subject to subject, depending, for example, on the species, age, and general condition of the subject, the severity of side effects or disorders, the identity of the particular compound, the mode of administration, and the like. An effective amount may include a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of the compounds described herein. In some embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is 3 doses per day, 2 doses per day, 1 dose per day, 1 dose every other day, 1 dose every 3 days, 1 dose per week, 1 dose per 2 weeks, 1 dose per 3 weeks, or 1 dose per 4 weeks. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is 1 dose per day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is 2 doses per day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is 3 doses per day. In some embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the time between the first and last dose of the multiple doses is 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years, 20 years, or the lifespan of the subject, tissue, or cell. In some embodiments, the time between the first and last dose of the multiple doses is 3 months, 6 months, or 1 year. In some embodiments, the time between the first and last dose of the multiple doses is the lifespan of the subject, tissue, or cell.In some embodiments, the doses described herein (e.g., any of a single dose or multiple doses) independently comprise 0.1 μg to 1 μg, 0.001 mg to 0.01 mg, 0.01 mg to 0.1 mg, 0.1 mg to 1 mg, 1 mg to 3 mg, 3 mg to 10 mg, 10 mg to 30 mg, 30 mg to 100 mg, 100 mg to 300 mg, 300 mg to 1,000 mg, or 1 g to 10 g of a composition comprising an amino acid described herein. In some embodiments, the doses described herein independently comprise 1 mg to 3 mg of an amino acid described herein. In some embodiments, the doses described herein independently comprise 3 mg to 10 mg of an amino acid described herein. In some embodiments, the doses described herein independently comprise 10 mg to 30 mg of an amino acid described herein. In some embodiments, the doses described herein independently comprise 30 mg to 100 mg of an amino acid described herein.

[0107] The dosage ranges described herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent can be determined by a medical professional or one of skill in the art and can be lower than or the same as the amount administered to an adult.

[0108] The composition can be administered, for example, simultaneously with, prior to, or after one or more additional pharmaceutical or therapeutic agents that may be useful as a combination therapy. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for use in humans or animals by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules bound to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In some embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing a disease (e.g., a proliferative disease, a pulmonary disease, a gastrointestinal disease, a blood disease, a neurological disease, a pain condition, a psychiatric disorder, or a metabolic disorder). In some embodiments, the additional therapeutic agent is an agent useful for treating a pulmonary disease.

[0109] In some embodiments, the additional therapeutic agent is an agent useful for treating cystic fibrosis. In some embodiments, the agent useful for treating cystic fibrosis can be ivacaftor (KALYDECO®), lumacaftor (ORKAMBI®), ataluren, or tezacaftor. In some embodiments, the additional therapeutic agent is ivacaftor. In some embodiments, the additional therapeutic agent is lumacaftor. In some embodiments, the additional therapeutic agent is an agent useful for treating cystic fibrosis, managing symptoms associated with cystic fibrosis, or preventing a disease or infection (e.g., bacterial infection, viral infection, bronchitis, asthma, etc.) that occurs concomitantly with cystic fibrosis. Additional therapeutic agents useful for the purposes of this disclosure include dornase alfa systemic (Pulmozyme), azithromycin (e.g., Zithromax, Zmax), aztreonam (Kayston, Azactam), tobramycin (e.g., TOBI®, Nevcin, Beskis, KitavisPak, TOBI Podhaler), Amikin, pancrelipase (Creon, Zenpep, Pancrease, Biocase, Pertzye, Ultresa, Pangestyme), and the like. Examples of additional pharmaceutical agents include, but are not limited to, EC, Panocaps), gentamicin (Garamycin), and pancreatin. Each additional pharmaceutical agent may be administered at a dose and / or time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered in a single dose, together with each other and / or the compounds or compositions described herein, or separately in different doses. The particular combination to use in the dosing regimen will take into consideration the compatibility of the compounds described herein with the additional agent(s) and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, the additional pharmaceutical agent(s) in the combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in the combination will be lower than the levels utilized individually.In certain embodiments, a compound or pharmaceutical composition described herein can be administered in combination with an anti-cancer therapy, including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplant, bone marrow transplant), immunotherapy, and chemotherapy.

[0110] The term "lung disease" or "pulmonary disease" refers to a disease of the lungs. Examples of lung diseases include, but are not limited to, cystic fibrosis and conditions associated therewith, including bronchiectasis, bronchitis, asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, and pulmonary fibrosis.

[0111] Also included in the present disclosure are kits (e.g., pharmaceutical packs). The kits provided may include a pharmaceutical composition or compound described herein and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the kits provided may further include a second container, optionally including a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound provided in the first container and the second container described herein are combined to form one unit dosage form. In some embodiments, the kits described herein further include instructions for using the kit.

[0112] Detailed Description of Specific Embodiments CF is the most common fatal recessive genetic disorder affecting 1 in 2,500 to 1 in 3,500 newborns annually among people of European descent. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in reduced or absent CFTR synthesis, protein misfolding and / or channel dysfunction, leading to reduced chloride secretion, increased sodium absorption (ENaC), and impaired fluid homeostasis in airway, small intestine, and pancreatic epithelial cells. The CFTR gene is expressed in multiple tissues. CF is characterized by multisystem pathology involving the respiratory, gastrointestinal, pancreatic, and hepatobiliary systems. The main morbidity and mortality are associated with chronic pulmonary infection and inflammation.

[0113] CF mutations are classified as: Class I (22%) = no CFTR expression; Class II (88%) = misfolded CFTR protein; Class III (6%) = CFTR channel does not open; Class IV (6%) = defective CFTR channel; Class V (5%) = too little CFTR. Mutations may span more than one category. Drugs such as lumacaftor are used to address class II mutations (folding); drugs such as tezacaftor or ivacaftor are "potentiators" used to address class III-V mutations.

[0114] Recent advances in CF therapy using small molecules that selectively activate CFTR activity or correct protein misfolding have shown limited success in treating subjects with CFTRΔF508 mutations. The inventors have identified a combination of selected amino acids (AA) that can stimulate chloride secretion and reduce ENaC activity, thereby increasing apical chloride channel activity and decreasing sodium absorption on the apical membrane. Thus, the combination of selected AA and its composition are presented as a therapeutic agent for treating subjects suffering from CF, alone and in combination with other CF therapeutic agents, such as, for example, lumacaftor, tezacaftor, and / or ivacaftor. Also included herein is the use of the selected AA combination for CF treatment or in the preparation of a medicament for treating CF, alone and / or in combination with other CF therapeutic agents, such as, for example, lumacaftor, tezacaftor, and / or ivacaftor.

[0115] As described herein, we experimentally compared fully differentiated primary normal human bronchial epithelial cells with homozygous CFTRΔF508 (class II mutant) human bronchial epithelial cells. Briefly, the transepithelial short circuit current (I sc ), resistance (R), and unidirectional (J ms &J sm ) and 22 Na and 36 Net flux of Cl (J net ) was measured in an Ussing chamber. When CF cells were stimulated with forskolin and GLPG1837 (an alternative potentiator to ivacaftor) in the absence (control) or presence of CF-5AA-3 (an amino acid formulation for treating CF), peak current (FIG. 7E) and chloride secretion (FIG. 7F) were significantly improved in the presence of CF-5AA-3 (also called CF5AA-3). For further study details, see Example 2 below.

[0116] For example, the results shown in Figures 5-9 demonstrate that selected amino acid combinations (e.g., CF-5AA-3) ameliorate dysfunctional chloride and sodium channel activity in fully differentiated primary normal human bronchial epithelial cells (HBECs) homozygous for CFTRΔF508 by correcting and / or altering cell membrane channel function. Thus, selected amino acid formulations (e.g., CF-5AA-3) are proposed to complement existing standards of care in patients with CFTRΔF508 mutations.

[0117] In the presence of a corrector (C18; VX661, tezacaftor) and a potentiator (GLPG1837, an alternative to ivacaftor), the peak current more than doubled from baseline but remained lower than the basal peak current of CF-5AA-3 when used alone. Addition of the same triple cocktail to CF-5AA-3 further increased the current by 26% over CF-5AA-3 alone (Figures 10A and 10B).

[0118] In addition, it was further demonstrated that chloride flux was dramatically increased by CF-5AA-3 compared to triple cocktail (Figure 11) by direct action on both CFTR and ANO1 channels.CF-5AA-3 showed only slight effect on ENaC.For further study details, please refer to Example 2 below.

[0119] In summary, in vitro electrophysiological studies using primary human bronchial epithelial cells harboring the CFTRΔF508 mutation (Class II) show that CF-5AA-3 alone quantitatively increases chloride secretion (negative flux) in F508del human bronchial cells similar to common correctors and enhancers [Column AA DMSO BASAL vs. Column C18+GLPG], and that CF-5AA-3 in combination with a commonly used corrector (C18) and enhancer (GLPG) doubles chloride secretion (negative flux) when compared to the correctors and enhancers alone.

[0120] The results presented herein show that additional chloride channels may also be active in HBEC (e.g., SLC26A9). Activation of chloride channels other than CFTR suggests that CF-5AA-3 may provide relief to all five classes of CF mutations by inducing chloride secretion via CFTR-independent means. This is particularly noteworthy for subjects with CFTR class I mutations, for which no drug currently exists.

[0121] Compositions containing amino acids that increase translocation of CFTR to the cell membrane The compositions provided herein increase the translocation of both wild-type and mutant CFTR proteins from the cytoplasm to the cell membrane. In particular, the compositions described herein are particularly effective in increasing the number of Phe508del CFTR proteins on the cell membrane, resulting in the secretion of more chloride ions and water from epithelial cells, overcoming the formation of thick mucus that is a hallmark of cystic fibrosis.

[0122] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine, and optionally a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, glycine, tyrosine, and lysine. In certain embodiments, the composition consists essentially of or consists of a particular free amino acid, with no other free amino acids or negligible amounts of other free amino acids. These compositions include derivatives of amino acids, which in certain embodiments are derivatives of "natural" or "unnatural" amino acids. The composition includes, in certain embodiments, salts and / or prodrugs of amino acids. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, glycine, tyrosine, and lysine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, or all five free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine; and optionally, a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In yet another aspect, the composition comprises, consists essentially of, or consists of the free amino acids cysteine ​​and proline and at least one additional free amino acid selected from glycine, tyrosine, and / or lysine; and optionally, a pharma-ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0123] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from threonine and lysine; and optionally pharma- ceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and / or additional therapeutic agents. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids threonine and lysine. In certain embodiments, the composition consists essentially of, or consists of a particular free amino acid, with no other free amino acids or negligible amounts of other free amino acids. The composition comprises, in certain embodiments, a derivative of an amino acid, which is a derivative of a "natural" or "unnatural" amino acid. The composition comprises, in certain embodiments, a salt and / or prodrug of an amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids threonine and lysine. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from threonine and lysine.

[0124] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, and valine; and optionally, a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In one embodiment, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, and valine. In one embodiment, the composition consists essentially of, or consists of a particular free amino acid, with no other free amino acids or negligible amounts of other free amino acids. The composition comprises, in one embodiment, a derivative of an amino acid, which is a derivative of a "natural" or "unnatural" amino acid. The composition comprises, in one embodiment, a salt and / or prodrug of an amino acid. In one embodiment, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, and valine. In one embodiment, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, and valine. In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, or all three, free amino acids selected from cysteine, proline, and valine; and optionally a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0125] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine; and optionally, pharma- ceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and / or additional therapeutic agents. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, glycine, and valine. In certain embodiments, the composition consists essentially of, or consists of a particular free amino acid, with no other free amino acids or negligible amounts of other free amino acids. The composition comprises, in certain embodiments, a derivative of an amino acid, which is a derivative of a "natural" or "unnatural" amino acid. The composition comprises, in certain embodiments, a salt and / or prodrug of an amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, glycine, and valine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine. In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, three or more, or all four free amino acids selected from cysteine, proline, glycine, and valine; and optionally a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0126] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine; and optionally, pharma- ceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and / or additional therapeutic agents. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, valine, and tyrosine. In certain embodiments, the composition consists essentially of, or consists of a particular free amino acid, with no other free amino acids or with negligible amounts of other free amino acids. The composition comprises, in certain embodiments, a derivative of an amino acid, which is a derivative of a "natural" or "unnatural" amino acid. The composition comprises, in certain embodiments, a salt and / or prodrug of an amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids cysteine, proline, valine, and tyrosine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, three or more, or all four free amino acids selected from cysteine, proline, valine, and tyrosine; and optionally a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0127] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, and lysine; and optionally, a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In one embodiment, the composition comprises, consists essentially of, or consists of the free amino acids glycine, leucine, and lysine. In one embodiment, the composition consists essentially of, or consists of a particular free amino acid, with no other free amino acids or negligible amounts of other free amino acids. The composition comprises, in one embodiment, a derivative of an amino acid, which is a derivative of a "natural" or "unnatural" amino acid. The composition comprises, in one embodiment, a salt and / or prodrug of an amino acid. In one embodiment, the composition comprises, consists essentially of, or consists of the free amino acids glycine, leucine, and lysine. In one embodiment, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, and lysine. In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, or all three, free amino acids selected from glycine, leucine, and lysine; and optionally a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0128] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine; and optionally, pharma- ceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and / or additional therapeutic agents. In one embodiment, the composition comprises, consists essentially of, or consists of the free amino acids glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In one embodiment, the composition consists essentially of, or consists of a particular free amino acid, with no other free amino acids or with negligible amounts of other free amino acids. The composition comprises, in one embodiment, a derivative of an amino acid, which is a derivative of a "natural" or "unnatural" amino acid. The composition comprises, in one embodiment, a salt and / or prodrug of an amino acid. In one embodiment, the composition comprises, consists essentially of, or consists of the free amino acids glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, or all six free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine; and optionally, a pharma- ceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0129] Non-limiting examples of compositions containing amino acids that increase translocation of CFTR to the cell membrane are provided in Table B below.

[0130] Table B: Compositions containing amino acids [Table 2]

[0131] Each of the free amino acids, when present in the composition, may be present, for example, at the following concentrations: about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams / liter, or about 1.5 to about 1.7 grams / liter of proline; about 0.7 to about 1.7, about 0.9 to about 1.5, about 1.1 to about 1.3 grams / liter, or about 1.5 to about 1.7 grams / liter of glutamic acid; about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 grams / liter, or about 1.5 to about 1.7 grams / liter of glycine; 0.5 to about 1.7 grams per liter of glutamine; about 0.05 to about 0.4, about 0.1 to about 0.3 grams per liter, or about 1.5 to about 1.7 grams per liter of leucine; about 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter of alanine; about 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter of aspartate. phenylalanine at 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter; histidine at 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter; threonine at 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams per liter, or about 1.5 to about 1.7 grams per liter; isoleucine from about 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter; asparagine from about 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter; tryptophan from about 0.4 to about 1.5, about 0.7 to about 1.3, about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams per liter.

[0132] In some embodiments, the amino acids of the composition are free amino acids. In some embodiments, the amino acids of the composition are L-amino acids. In some embodiments, the amino acids of the composition are D-amino acids. In some embodiments, the amino acids of the composition are a combination of D- and L-amino acids.

[0133] In certain embodiments, the amino acids of the compositions described herein can be prodrugs of the free amino acids. The term "prodrug" refers to compounds that have cleavable groups and that, by solvation or under physiological conditions, yield compounds described herein that are pharma- ceutically active in vivo.

[0134] In some embodiments, the amino acid of the composition described herein can be an amino acid salt (i.e., an amino acid salt). The amino acid can be in the form of a salt with a cation (e.g., the salt of an amino acid with a negatively charged side chain in solution (e.g., glutamate and aspartate)), in the form of a salt with an anion (e.g., the salt of an amino acid with a positively charged side chain in solution (e.g., lysine, arginine, histidine)), and an inorganic compound. Exemplary amino acid salts are listed in Fleck M and Petrosyan AM, Salts of Amino Acids, 1st Edition; Springer International Publishing, 2014, which is incorporated herein by reference.

[0135] In some embodiments, the composition further comprises water.

[0136] In some embodiments, the composition further comprises a buffer. Exemplary buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0137] In one embodiment, H 2 PO 4 - ,HPO 4 2- , and P.O. 4 3- Phosphate ions such as HCO are used to buffer the compositions of the invention. In certain embodiments, the therapeutic composition contains HCO as a buffer. 3 - or CO 3 2- In another embodiment, the therapeutic composition uses HCO as a buffer. 3 - or CO 3 2- Do not use.

[0138] In certain embodiments, the composition comprises, for example, Na + ;K + ;HCO 3 - ;CO 3 2- ;Ca 2+ ;Mg 2+ ;Fe 2+;Cl - ;H 2 PO 4 - , H.P.O. 4 2- , and PO4 3- In another alternative embodiment, the composition contains one or more electrolytes selected from the group consisting of phosphate ions, zinc, iodine, copper, iron, selenium, chromium, and molybdenum. ... 3 - or CO 3 2- In some embodiments, the composition does not contain electrolytes. In some embodiments, the composition does not contain carbohydrates (e.g., disaccharides, oligosaccharides, or polysaccharides). In some alternative embodiments, the composition comprises Na + ;K + ;HCO 3 - ;CO 3 2- ;Ca 2+ ;Mg 2+ ;Fe 2+ ;Cl - ;H 2 PO 4 - , H.P.O. 4 2- , and PO4 3- and the like; zinc; iodine; copper; iron; selenium; chromium; and molybdenum.

[0139] In some embodiments, the composition further comprises a sugar, a vitamin, an electrolyte, a mineral, a protein, or a lipid. In some embodiments, the composition further comprises a sugar. In some embodiments, the composition further comprises a vitamin. In some embodiments, the composition further comprises an electrolyte. In some embodiments, the composition further comprises a mineral. In some embodiments, the composition further comprises a protein. In some embodiments, the composition further comprises a lipid.

[0140] In certain embodiments, the composition does not contain one or more components selected from oligosaccharides, polysaccharides, and carbohydrates; oligopeptides, or polypeptides or proteins; lipids; short-, medium-, and / or long-chain fatty acids; and / or foods containing one or more of the above-mentioned nutrients.

[0141] The composition may have a pH in the range of about 2.5 to about 8.5. In some embodiments, the pH of the composition is in the range of about 2.5 to about 6.5, about 3.0 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH of the composition is in the range of about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.2 to about 7.8. In some embodiments, the composition has a pH of, for example, about 2.5 to about 8.5. In some embodiments, the composition has a pH of about 2.5 to about 6.5, about 2.5 to about 6.0, about 3.0 to about 6.0, about 3.5 to about 6.0, about 3.9 to about 6.0, about 4.2 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH is about 6.5 to about 8.5, about 7.0 to about 8.5, about 7.0 to about 8.0, about 7.2 to about 8.0, or about 7.2 to about 7.8. In some embodiments, the pH is about 7.3 to about 7.5. In some embodiments, the pH is about 7.3 to about 7.4. In some embodiments, the pH is about 7.4 to about 7.5. In some embodiments, the pH is about 7.4.

[0142] In some embodiments, the composition has a total osmolality of about 100 mosm to 280 mosm, or any value therebetween. In some embodiments, the total osmolality is about 150 mosm to 260 mosm. In other embodiments, the composition has a total osmolality of any value below 280 mosm.

[0143] In certain embodiments, the composition is sterile.

[0144] The compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing into association the compound of the compositions described herein (i.e., free amino acid(s)) with the carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single or multi-dose unit.

[0145] The relative amounts of the active ingredient(s), pharma- ceutically acceptable excipient, and / or any additional ingredients in the pharmaceutical compositions described herein will vary depending on the identity, size, and / or condition of the subject being treated, and will further depend on the route by which the composition is to be administered. The compositions may contain from 0.1% to 100% (w / w) active ingredient.

[0146] The compounds or compositions described herein can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and / or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., efficacy and / or effectiveness) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, and / or in enhancing the activity of CFTR in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and / or alter metabolism, inhibit excretion, and / or alter distribution in a subject or cell. It is also understood that the therapeutic methods used may achieve desired effects for the same disorder and / or achieve different effects. In certain embodiments, the pharmaceutical compositions described herein that include the compounds described herein and the additional pharmaceutical agents exhibit a synergistic effect that is not present in pharmaceutical compositions that include one of the compounds and the additional pharmaceutical agents but not both.

[0147] The compositions described herein can be administered simultaneously with, prior to, or after one or more additional pharmaceutical or therapeutic agents that are different from the compound or composition and may be useful, for example, as a combination therapy. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucosal proteins, lipoproteins, synthetic polypeptides or proteins, small molecules bound to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In some embodiments, the additional agent is a pharmaceutical agent useful for treating and / or preventing a disease (e.g., a proliferative disease, a blood disease, a neurological disease, a pain condition, a psychiatric disorder, a pulmonary disease, or a metabolic disorder). In some embodiments, the additional pharmaceutical agent is useful for treating a pulmonary disease. In certain embodiments, the additional pharmaceutical agents are useful for treating cystic fibrosis. Each additional agent may be administered at a dose and / or time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered in a single dose, together with each other and / or the compounds or compositions described herein, or separately in different doses. The particular combination to be used in the dosing regimen will take into consideration the compatibility of the compounds described herein with the additional pharmaceutical agent(s) and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in the combination may be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in the combination will be lower than the levels utilized individually.

[0148] In some embodiments, the composition is administered simultaneously with, before, or after one or more additional therapeutic agents, the additional therapeutic agents being CFTR potentiators, correctors, or read-through agents. A "CFTR potentiator" is a compound that increases the function of CFTR on the cell membrane of a cell. These compounds may be useful to treat cystic fibrosis mutations that result in a CFTR protein that is correctly transported to the cell membrane but does not function properly (e.g., defective channel gating, reduced or absent ATP binding, and / or reduced chloride transport). In some embodiments, the CFTR potentiator is ivacaftor (VX-770, KALYDECO®). A "CFTR corrector" is a compound that improves intracellular processing and translocation of mutant CFTR, allowing more protein to reach the cell membrane. In some embodiments, the CFTR corrector is lumacaftor (VX-809, ORKAMBI®). In some embodiments, the CFTR corrector is tezacaftor (VX661). A "CFTR readthrough agent" is a compound that stimulates transcription in the presence of a premature termination codon (PTC) mutation resulting from a point mutation in the CFTR gene sequence.

[0149] Typically, PTC results in a truncated CFTR protein that is not properly processed and / or is dysfunctional (e.g., defective channel gating, reduced or absent ATP binding, and / or reduced chloride transport). In one embodiment, the CFTR read-through agent is ataluren (TRANSLARNA™). A composition comprising an amino acid described herein is administered in combination with ivacaftor. In one embodiment, a composition comprising an amino acid described herein is administered in combination with lumacaftor. In one embodiment, a composition comprising an amino acid described herein is administered in combination with ataluren. In one embodiment, a composition comprising an amino acid described herein is administered in combination with ivacaftor and lumacaftor. In one embodiment, a composition comprising an amino acid described herein is administered in combination with ivacaftor and ataluren. In one embodiment, a composition comprising an amino acid described herein is administered in combination with lumacaftor and ataluren. In one embodiment, the composition is administered alone, i.e., not administered simultaneously with, before, or after one or more additional pharmaceutical or therapeutic agents. In one embodiment, the composition is administered in the absence of a CFTR potentiator, corrector, or read-through agent.

[0150] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; (c) humectants, such as glycerol; (d) agar, calcium carbonate, potato; They are mixed with disintegrating agents such as potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain buffering agents.

[0151] In certain embodiments, compositions comprising the amino acids described herein may be provided in powder form and reconstituted for administration to a subject. Pharmaceutical compositions described herein may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers, or about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir into which a stream of propellant can be directed to disperse the powder, and / or using a self-propelled solvent / powder dispensing vessel, such as a device comprising the active ingredient dissolved and / or suspended in a low boiling propellant in a closed vessel. Such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dosage form.

[0152] Liquid dosage forms for oral and parenteral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and aromatic agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0153] The pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations contain the active ingredient, are optionally sterilized, and may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, and may be conveniently administered using any inhalation and / or nebulizer device. Such formulations may further include one or more additional ingredients, including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range of about 0.1 to about 200 nanometers.

[0154] The formulations described herein as useful for pulmonary delivery are useful for intranasal delivery of the pharmaceutical compositions described herein. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 μm. Such formulations are administered by rapid inhalation through the nasal passages from a container of the powder held close to the nostrils.

[0155] Formulations for nasal administration may contain, for example, as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein. The pharmaceutical compositions described herein may be prepared, packaged, and / or sold in formulations for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w / w) of the active ingredient, the remainder comprising a composition dissolvable and / or disintegrable in the mouth, and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

[0156] Methods for increasing translocation of CFTR to the cell membrane - Patent Application 20070229633 The present disclosure provides compositions for use in increasing the translocation of cystic fibrosis transmembrane conductance regulator (CFTR) protein from the cytoplasm to the cell membrane. In general, without wishing to be bound by any particular theory, mutations in CFTR protein (e.g., Phe508del) may interfere with correct processing, folding, and / or transport to the cell membrane, resulting in fewer CFTR proteins on the cell membrane and impaired chloride ion transport function in cells expressing mutated CFTR protein. Impaired chloride ion transport results in an unbalanced osmotic profile, so that water is not drawn out of cells expressing mutated CFTR protein via osmosis due to abnormally high intracellular concentration of chloride ion. This results in the formation of a thick mucus layer covering cells, which is a characteristic of cystic fibrosis.

[0157] Thus, in one aspect, the present disclosure provides a method for increasing the number of CFTR proteins on the cell membrane of a cell, comprising contacting the cell with an effective amount of a composition comprising an amino acid of the present disclosure. In an embodiment, the cell is an epithelial cell. In an embodiment, the epithelial cell is a small intestinal epithelial cell or a lung epithelial cell. In an embodiment, the epithelial cell is a lung epithelial cell. In an embodiment, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell can be a normal human bronchial epithelial cell (NHBE) or a diseased human bronchial epithelial cell (DHBE). The diseased human bronchial epithelial cell can be obtained from a human donor diagnosed with a lung disease (e.g., asthma, COPD, cystic fibrosis). In an embodiment, the bronchial epithelial cell expresses wild-type CFTR. In an embodiment, the bronchial epithelial cell expresses a mutant CFTR. In some embodiments, the bronchial epithelial cells express both wild-type and mutant CFTR (i.e., one allele in the CFTR gene encodes wild-type CFTR and the second allele contains a mutation (e.g., Phe508del CFTR)). In some embodiments, the bronchial epithelial cells express only Phe508del mutant CFTR. The cells may be in vitro, in vivo, or ex vivo.

[0158] In an embodiment, the number of wild type CFTR proteins on the cell membrane is increased. In an embodiment, the number of mutant CFTR proteins on the cell membrane is increased. In an embodiment, the number of Gly542X mutant CFTR proteins on the cell membrane is increased, where X is any amino acid. In an embodiment, the number of Gly551Asp mutant CFTR proteins on the cell membrane is increased. In an embodiment, the number of Arg553X mutant CFTR proteins on the cell membrane is increased, where X is any amino acid. In an embodiment, the number of Arg117His mutant CFTR proteins on the cell membrane is increased. In an embodiment, the number of 120del23 mutant CFTR proteins on the cell membrane is increased. In an embodiment, the number of Phe508del mutant CFTR proteins on the cell membrane is increased. In an embodiment, the number of CFTR proteins that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild type CFTR provided by SEQ ID NO:1 on the cell membrane is increased. In one embodiment, the number of CFTR proteins is increased by increasing the number of CFTR proteins that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of Phe508del CFTR provided by SEQ ID NO:2 on the cell membrane.

[0159] In an embodiment, the present disclosure provides a method for increasing the number of CFTR proteins on the cell membrane of a cell, comprising contacting the cell with an effective amount of a composition described herein. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from threonine and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, and lysine. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In yet another aspect, the composition comprises, consists essentially of, or consists of the free amino acids cysteine ​​and proline, and at least one additional free amino acid selected from glycine, tyrosine, and / or lysine, and optionally buffers, electrolytes, adjuvants, and / or excipients.

[0160] By "negligible amount" is meant that the amino acid present does not affect the CFTR protein. Alternatively, in some embodiments, even if the amino acid is present in the composition, it is not present in an amount that affects the translocation of CFTR to the cell membrane, chloride ion transport, or the therapeutic effect of treating a subject in need thereof. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, 0.1 mg / l, or 0.01 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 100 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 50 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 10 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 5 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 1 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 0.5 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 0.1 mg / l. In some embodiments, a negligible amount is an amount where the total concentration of amino acids is less than 0.01 mg / l. It is understood that a negligible amount is an amount greater than zero.

[0161] In certain embodiments, the compositions of the present disclosure include one or more free amino acids that are essential (e.g., required) to increase translocation of CFTR to the cell membrane. In certain embodiments, the compositions of the present disclosure include one or more free amino acids that are not essential to increase translocation of CFTR to the cell membrane (i.e., do not directly stabilize CFTR), but may instead provide an alternative beneficial property to the composition (e.g., maintain a particular pH or osmolality).

[0162] In one aspect, the method of increasing the number of CFTR proteins on the cell membrane of a cell includes increasing the translocation of CFTR from the cytoplasm of the cell to the cell membrane, the method includes contacting the cell with an effective amount of a composition comprising an amino acid of the present disclosure. In one embodiment, the method results in an increase in the number of CFTR proteins on the cell membrane of the treated cell. The increase in the number of CFTR proteins on the cell membrane can be determined by comparing cells contacted with a composition comprising an amino acid as described herein to untreated cells (e.g., control cells). For example, Western blots may be used to compare the presence and amount of CFTR in membrane vesicles isolated from epithelial cells contacted with a composition comprising an amino acid as described herein to the amount of CFTR in membrane vesicles isolated from untreated epithelial cells (e.g., control cells). It is within the capabilities of one of ordinary skill in the art to perform the Western blot analysis described herein. Further related techniques that can be used to determine the expression level of CFTR protein in a sample include, among others, dot blot analysis, immunohistochemistry, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA).

[0163] The CFTR protein is an ABC transporter protein that functions as an ATP-gated ion channel. When activated, CFTR transports chloride ions (Cl - ), and thiocyanate ([SCN] -CFTR allows other negatively charged ions, such as phenylalanine, arginine, cysteine, and arginine, to flow down their electrochemical gradient (e.g., passive diffusion or passive transport). Mutations in CFTR, such as Gly551X (wherein X represents any amino acid, e.g., Gly551Asp), result in CFTR proteins characterized by defective ion channel gating function. The most common CFTR mutation (Phe508del) results in a CFTR protein that lacks a codon for phenylalanine 508 and cannot properly fold or be transported to the cell membrane. Thus, in one aspect, the disclosure provides a method for increasing the number of CFTR proteins on the cell membrane of a cell and increasing chloride ion transport across the cell membrane (e.g., chloride ion export from the cell), comprising contacting the cell with a composition comprising an amino acid described herein.

[0164] Thus, in one aspect, the disclosure provides a method for increasing chloride ion efflux from a cell, comprising contacting the cell with an effective amount of a composition described herein. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from threonine and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, and lysine. In one embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In yet another embodiment, the composition comprises, consists essentially of, or consists of the free amino acids cysteine ​​and proline, and at least one additional free amino acid selected from glycine, tyrosine, and / or lysine, and optionally buffers, electrolytes, adjuvants, and / or excipients.

[0165] In some embodiments, chloride export from the cell is further enhanced by contacting the cell with a CFTR enhancer, corrector, or read-through agent. In some embodiments, chloride export from the cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR enhancer. In some embodiments, chloride export from the cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR corrector. In some embodiments, chloride export from the cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR read-through agent. In some embodiments, chloride export from the cell is increased by contacting the cell with a combination therapy comprising a composition described herein and an ivacaftor.

[0166] The flow of ions, such as chloride ions, across a membrane is one biological mechanism used to regulate the flow of water across a semipermeable membrane (e.g., cell membrane). Osmosis describes the process (e.g., passive diffusion of water) of solvent (e.g., water) molecules flowing from an area of ​​low solute concentration to an area of ​​higher solute concentration to equilibrate the concentration on each side of a semipermeable membrane. When the chloride ion concentration in a cell increases due to dysfunction of CFTR, water molecules do not flow out of the cell into the surrounding mucus membrane, and thick mucus is formed. Therefore, in a further aspect, the present disclosure provides methods and compositions for increasing the flow of water (e.g., osmosis) from a cell, comprising contacting the cell with a composition comprising an amino acid as described herein.

[0167] Thus, in one aspect, the disclosure provides a method for increasing water excretion from a cell, comprising contacting the cell with a composition described herein. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from threonine and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, and lysine. In one embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In yet another embodiment, the composition comprises, consists essentially of, or consists of the free amino acids cysteine ​​and proline, and at least one additional free amino acid selected from glycine, tyrosine, and / or lysine, and optionally buffers, electrolytes, adjuvants, and / or excipients.

[0168] In some embodiments, water export from cells is further enhanced by contacting the cells with a CFTR enhancer, corrector, or read-through agent. In some embodiments, water export from cells is increased by contacting the cells with a combination therapy comprising a composition described herein and a CFTR enhancer. In some embodiments, water export from cells is increased by contacting the cells with a combination therapy comprising a composition described herein and a CFTR corrector. In some embodiments, water export from cells is increased by contacting the cells with a combination therapy comprising a composition described herein and a CFTR read-through agent. In some embodiments, water export from cells is increased by contacting the cells with a combination therapy comprising a composition described herein and an ivacaftor.

[0169] Methods for the treatment of cystic fibrosis As mentioned above, the flow of chloride ions through the cystic fibrosis transmembrane conductance regulator (CFTR) protein is important for maintaining fluid levels in cells and surrounding mucosa, especially in the lungs and small intestine. Mutations in the CFTR protein (e.g., Phe508del) that disrupt the processing, folding, and transport of CFTR to the cell membrane result in fewer functional CFTR proteins on the cell membrane. The biological result is the accumulation of a thick mucus layer that covers the epithelial cell layer, promoting bacterial growth and preventing epithelial cells from obtaining nutrients from the surrounding fluid. Eventually, respiratory and pulmonary diseases such as cystic fibrosis may develop in patients carrying one or more CFTR gene mutations.

[0170] CFTR gene mutations The subject may be, for example, a human suffering from a pulmonary disease. In some embodiments, the subject suffers from cystic fibrosis. In some embodiments, the genetic cause of cystic fibrosis may be the Phe508del mutation in one or more alleles of the CFTR gene. Humans may also suffer from additional complications that often occur together with cystic fibrosis, such as bacterial infection, viral infection, asthma, and chronic respiratory failure, among others. Therefore, the compositions comprising the amino acids disclosed herein may also be useful in managing symptoms and other complications in subjects with cystic fibrosis.

[0171] In certain embodiments, the methods described herein result in increased survival in patients suffering from a pulmonary disease (e.g., cystic fibrosis). The methods and compositions described herein may also be useful for improving the treatment outcomes of patients with cystic fibrosis.

[0172] Thus, in one aspect, the disclosure provides a composition for use in treating cystic fibrosis, administered to a subject in need thereof (e.g., a subject with cystic fibrosis). In an embodiment, the subject is afflicted with cystic fibrosis, in which a wild-type CFTR of SEQ ID NO: 1 is present. In an embodiment, the subject is afflicted with cystic fibrosis, in which a CFTR protein is present that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of the wild-type CFTR provided by SEQ ID NO: 1. In an embodiment, the subject is afflicted with cystic fibrosis, in which a mutant CFTR is present. In an embodiment, the subject is afflicted with cystic fibrosis, in which the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del mutant, where X represents any amino acid. In some embodiments, the subject suffers from cystic fibrosis in which both wild-type and mutant CFTR are present. In some embodiments, the subject suffers from cystic fibrosis in which a CFTR protein is present that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of Phe508del CFTR provided by SEQ ID NO:2. In some embodiments, the subject suffers from cystic fibrosis in which the mutant CFTR is Phe508del CFTR of SEQ ID NO:2. In some embodiments, the subject suffers from cystic fibrosis in which both wild-type CFTR of SEQ ID NO:1 and Phe508del CFTR of SEQ ID NO:2 are present. The compositions described herein can be administered with one or more additional therapeutic agents, e.g., in combination therapy, to further increase the therapeutic benefit of the compositions described herein.

[0173] In a further aspect, the disclosure provides a method for treating cystic fibrosis, comprising administering to a subject in need thereof a composition described herein. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from threonine and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, and valine. In some embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In an embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, and lysine. In one embodiment, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In yet another embodiment, the composition comprises, consists essentially of, or consists of the free amino acids cysteine ​​and proline, and at least one additional free amino acid selected from glycine, tyrosine, and / or lysine, and optionally buffers, electrolytes, adjuvants, and / or excipients.

[0174] In some embodiments, the composition further comprises a CFTR enhancer, corrector, or read-through agent. In some embodiments, the composition further comprises a CFTR enhancer. In some embodiments, the composition further comprises a CFTR corrector. In some embodiments, the composition further comprises a CFTR read-through agent. In some embodiments, the composition further comprises ivacaftor. In some embodiments, the composition does not further comprise a CFTR enhancer, corrector, or read-through agent.

[0175] SYMDEKO® (tezacaftor / ivacaftor and ivacaftor) is indicated for the treatment of patients aged 6 years and older with cystic fibrosis (CF) who are homozygous for the F508del mutation or have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene who respond to tezacaftor / ivacaftor based on in vitro data and / or clinical evidence. For patients aged 6-11 years weighing less than 30 kg, a typical dose is 50 mg tezacaftor + 75 mg ivacaftor in the morning and 75 mg ivacaftor 12 hours later. For patients aged 6-11 years weighing 30 kg or more or patients aged 12 years and older, a typical dose is 100 mg tezacaftor + 150 mg ivacaftor in the morning and 150 mg ivacaftor 12 hours later.

[0176] ORKAMBI® (lumacaftor / ivacaftor) is indicated for the treatment of cystic fibrosis (CF) in patients who are homozygous for the F508del mutation in the CFTR gene. For patients 6 to 11 years of age: 2 tablets (each containing 100 mg lumacaftor / 125 mg ivacaftor) every 12 hours. For patients 12 years of age and older: 2 tablets (each containing 200 mg lumacaftor / 125 mg ivacaftor) every 12 hours.

[0177] KALYDECO® (ivacaftor) is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator indicated for the treatment of CF in patients 6 months of age or older who have a mutation in the CFTR gene that is responsive to ivacaftor potentiation based on clinical and / or in vitro assay data. For patients 6 months to less than 6 years of age, the recommended dose is based on body weight: 5 kg to less than 7 kg: one packet of 25 mg every 12 hours; 7 kg to less than 14 kg: one packet of 50 mg every 12 hours; 14 kg or more: one packet of 75 mg every 12 hours.

[0178] Ataluren may be used at a dose of 40 mg / kg / day.

[0179] It is understood that when used in combination with the pharmaceutical preparations described herein, the typical dosage may vary.In certain embodiments of such combination therapy, for example, the administration of any of SYMDEKO®, ORKAMBI®, KALYDECO®, or ataluren, when administered in combination with the pharmaceutical preparations described herein, may be reduced in terms of either the dosage administered and / or the frequency of administration.

[0180] The CFTR gene and gene products and CFTR variants As described above, compositions comprising the amino acids described herein effectively increase the translocation of CFTR to the cell membrane. In particular, compositions comprising the amino acids described herein can effectively increase the translocation of mutant (e.g., Phe508del) CFTR to the cell membrane. The compositions provide an increase in the number of CFTR proteins on the cell membrane, providing a method for increasing chloride ion transport across epithelial cell membranes to maintain membrane hyperpolarization and transmembrane water transport. These compositions are useful for treating lung diseases such as cystic fibrosis, or diseases resulting from dysregulation of fluid transport in other epithelial cells, such as cells in the pancreas and small intestine.

[0181] The sequence of the CFTR gene product of interest herein often comprises or consists of the sequence encoded by the human CFTR gene, although in some embodiments the sequence of a non-human mammalian homologue may be used. In general, the sequence of the CFTR protein or CFTR RNA often comprises or consists of the sequence of the human CFTR. In some embodiments, the sequence of the gene product of the CFTR gene comprises or consists of a naturally occurring sequence. It is understood that a locus may have one or more sequences or alleles in a population of individuals. In some embodiments, the naturally occurring sequence is a standard sequence. Unless otherwise indicated, a sequence listed in the Reference Sequence (RefSeq) database as the reference sequence for a protein referred to herein by a particular name, abbreviation, or symbol is considered to be a "standard sequence". If a sequence is updated after the time of this disclosure, the version as of the time of this disclosure or an updated version thereof may be used in some embodiments. It is understood that a locus may have two or more sequences or alleles in a population of individuals. In some embodiments, a naturally occurring sequence differs from a standard sequence at one or more amino acid positions. A naturally occurring polynucleotide or polypeptide whose sequence differs from a standard sequence and which carries out the normal function(s) of that polynucleotide or polypeptide may be said to have a "normal sequence."

[0182] The CFTR gene is approximately 189 kb in length and contains 27 exons and 26 introns. In some embodiments, the CFTR protein is full-length wild-type CFTR. The CFTR can be a mammalian (e.g., human) CFTR. In some embodiments, the sequence of CFTR or a variant thereof used in the compositions and methods described herein comprises the sequence of a naturally occurring CFTR protein or a biologically active variant thereof. A biologically active variant of the androgen receptor protein may contain one or more additions, substitutions, and / or deletions relative to the sequence of a naturally occurring CFTR protein. In some embodiments, the sequence of the CFTR protein comprises a canonical CFTR sequence. The full-length CFTR protein is 1480 amino acids in length and has five domains: two transmembrane domains, one intracellular nucleotide binding domain (NBD) linked to each transmembrane domain, and one intracellular regulatory "R" domain. The full-length wild-type CFTR has the following canonical amino acid sequence (GenBank and NCBI Reference Sequence Accession Number: NG_016465.4): MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWD RELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEE RSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISI GQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLA LFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQ TELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAV TRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFG ELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAG KTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKA CQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVL TEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPD FSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKR KNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGP TLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQET TRACKSEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAA SLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIF DFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIF THLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVI FFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFID MPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGN AILENISFSISPGQRVGLLGRTGSGKSTLLSAFRLLNTEGEIQIDGVSWDSITLQQWRK AFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVD GGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVIL CEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKC KSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 1).

[0183] In an embodiment, the present disclosure provides a composition comprising an amino acid useful for increasing the number of CFTR proteins on a cell membrane. In an embodiment, the CFTR protein is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more, e.g., 100%, identical in sequence to the wild-type CFTR of SEQ ID NO:1. In an embodiment, the CFTR protein is at least 70% identical in sequence to the wild-type CFTR of SEQ ID NO:1. In an embodiment, the CFTR protein is at least 80% identical in sequence to the wild-type CFTR of SEQ ID NO:1. In an embodiment, the CFTR protein is at least 90% identical in sequence to the wild-type CFTR of SEQ ID NO:1. In an embodiment, the CFTR protein is at least 95% identical in sequence to the wild-type CFTR of SEQ ID NO:1. In an embodiment, the CFTR protein is at least 96% identical in sequence to the wild-type CFTR of SEQ ID NO:1. In an embodiment, the CFTR protein is at least 97% identical in sequence to the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is at least 98% identical in sequence to the wild-type CFTR of SEQ ID NO: 1. In some embodiments, the CFTR protein is at least 99% identical in sequence to the wild-type CFTR of SEQ ID NO: 1. In some embodiments, the CFTR protein is at least 99.5% identical in sequence to the wild-type CFTR of SEQ ID NO: 1. In some embodiments, the CFTR protein is at least 100% identical in sequence to the wild-type CFTR of SEQ ID NO: 1.

[0184] In an embodiment, the CFTR protein is a variant or fragment of the full-length CFTR of SEQ ID NO: 1. The term "variant" also encompasses splice variants of CFTR resulting from alternative splicing of the CFTR gene. In an embodiment, the CFTR variant comprises or consists of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the full-length CFTR of SEQ ID NO: 1. In an embodiment, the CFTR variant comprises or consists of at least 50% of the full-length CFTR of SEQ ID NO: 1. In an embodiment, the CFTR variant comprises or consists of at least 60% of the full-length CFTR of SEQ ID NO: 1. In an embodiment, the CFTR variant comprises or consists of at least 70% of the full-length CFTR of SEQ ID NO: 1. In an embodiment, the CFTR variant comprises or consists of at least 80% of the full-length CFTR of SEQ ID NO: 1. In some embodiments, the CFTR variant comprises or consists of at least 90% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 95% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 96% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 97% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 98% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 99% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 99.5% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 100% or more of the polypeptide of the full-length CFTR of SEQ ID NO:1.The variants can be biologically active variants of wild-type CFTR, such that the ion channel gating function and / or chloride transport function of CFTR are retained in the variant. The variants can be biologically inactive mutants of wild-type CFTR, such that the ion channel gating function and / or chloride transport function of CFTR are abolished (e.g., non-functional) in the variant.

[0185] In an embodiment, the CFTR protein is a mutant CFTR protein, e.g., the sequence of the protein comprises the sequence of a naturally occurring mutant form of CFTR. The mutant CFTR can be a mammalian (e.g., human) CFTR mutant. The mutant CFTR can result from a nonsense mutation, a frameshift mutation, or an mRNA splicing mutation. Over 2,000 mutations have been discovered in the CFTR gene, many of which are clinically relevant and / or result in disease (e.g., cystic fibrosis) phenotypes. For additional CFTR gene mutations for which administration of the compositions of the present disclosure may prove useful, see Bobadilla JL et al., 2002, Human Mutation, 19; pp. 575-606, which is incorporated herein by reference. In an embodiment, the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del mutant, where X represents any amino acid. In some embodiments, the mutant CFTR is a Gly542X mutant, where X represents any amino acid. In some embodiments, the mutant CFTR is a Gly551Asp mutant. In some embodiments, the mutant CFTR is an Arg553X mutant, where X represents any amino acid. In some embodiments, the mutant CFTR is an Arg117His mutant. In some embodiments, the mutant CFTR is a 120del23 mutant. In some embodiments, the mutant CFTR is a Phe508del mutant. In some embodiments, the human subject carries a CFTR mutation in at least one allele of the gene encoding the CFTR protein (e.g., one allele encodes wild-type CFTR and one allele encodes Phe508del CFTR). In some embodiments, the human subject carries a CFTR mutation in at least two alleles of the gene encoding the CFTR protein. In one embodiment, the human subject carries the same mutation in at least two alleles of the gene encoding the CFTR protein (eg, both alleles encode the Phe508del variant, ie, homozygous).In some embodiments, the human subject carries different mutations in at least two alleles of the gene encoding the CFTR protein (e.g., one allele encodes the Phe508del variant and one allele encodes the Gly551Asp variant, i.e., heterozygous). In some embodiments, the cell carries a CFTR mutation in at least one allele of the gene encoding the CFTR protein (e.g., one allele encodes wild-type CFTR and one allele encodes the Phe508del CFTR). In some embodiments, the cell carries a CFTR mutation in at least two alleles of the gene encoding the CFTR protein. In some embodiments, the cell carries the same mutation in at least two alleles of the gene encoding the CFTR protein (e.g., both alleles encode the Phe508del variant, i.e., homozygous). In certain embodiments, the cell carries different mutations in at least two alleles of the gene encoding the CFTR protein (e.g., one allele encodes a Phe508del variant and one allele encodes a Gly551Asp variant, i.e., heterozygous). In certain embodiments, the cell is an epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain embodiments, the lung epithelial cell is a bronchial epithelial cell. In certain embodiments, the bronchial epithelial cell is from a patient with cystic fibrosis.

[0186] The Phe508del variant is the most common variant among patients with cystic fibrosis. The Phe508del variant results from a deletion mutation in which the codon for phenylalanine at amino acid position 508 is deleted, resulting in a CFTR protein lacking residue 508 (e.g., 1479 amino acids in length). Phe508del CFTR has the following standard amino acid sequence (GenBank and NCBI reference sequence accession number: (NM_000492.3(CFTR):c.1521_1523delCTT): MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWD RELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEE RSIAIYLGIGLLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISI GQLVSLLSNNNLNKFDEGLALAHFVWIAPQVALLMGLIWELLQASAFCGLFLIVLA LFQAGGLRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQ TELKLTRKAAYVRYFNSSAFFFSGFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAV TRQFPWAVQTVYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFG ELFEKAKQNNNNRKTSNGDDSLFFSNLGTPVLKDINFKIERGQLLAVAGSTGAG KTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIGVSYDEYRYRSVIKAC QLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLT EKEICESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFS SKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRK NSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPT LQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETG LEISEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIVFLAEVAAS LVVLWLGNTPQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDF IQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTH LVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFI AVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPT EGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAIL ENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQQWRKAF GVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGG CVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCE HRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKS KPQIAALKEETEEEVQDTRL (SEQ ID NO: 2).

[0187] In some embodiments, the CFTR protein is a mutant CFTR protein, e.g., the sequence of the protein comprises the sequence of a naturally occurring mutant form of CFTR. In some embodiments, the mutant CFTR protein is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more, e.g., 100%, identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 70% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 80% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 90% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 95% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In certain embodiments, the mutant CFTR protein is at least 96% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In certain embodiments, the mutant CFTR protein is at least 97% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In certain embodiments, the mutant CFTR protein is at least 98% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In certain embodiments, the mutant CFTR protein is at least 99% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In certain embodiments, the mutant CFTR protein is at least 99.5% identical in sequence to the Phe508del CFTR of SEQ ID NO:2. In certain embodiments, the mutant CFTR protein is at least 100% identical in sequence to the Phe508del CFTR of SEQ ID NO:2.

[0188] In some embodiments, the CFTR protein cannot be properly processed by traditional cellular machinery in the endoplasmic reticulum (ER). In some embodiments, the CFTR protein cannot be properly folded. In some embodiments, the CFTR protein cannot be properly transported to the cell membrane (e.g., remains in the cytoplasm or endoplasmic reticulum). In some embodiments, the CFTR protein exhibits impaired ion channel gating functionality, i.e., cannot properly open to allow chloride ions to be transported out of the cell.

[0189] In one embodiment, the CFTR protein is a variant or fragment of Phe508del CFTR of SEQ ID NO:2. In one embodiment, the CFTR variant comprises or consists of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more than 100% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In one embodiment, the CFTR variant comprises or consists of at least 50% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In one embodiment, the CFTR variant comprises or consists of at least 60% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In one embodiment, the CFTR variant comprises or consists of at least 70% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 80% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 90% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 95% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 96% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 97% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 98% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 99% or more of the polypeptide of the Phe508del CFTR of SEQ ID NO:2. In one embodiment, the CFTR variant comprises or consists of at least 99.5% of the polypeptide of Phe508del CFTR of SEQ ID NO:2.In certain embodiments, the CFTR variants comprise or consist of at least 100% or more of the polypeptide of Phe508del CFTR of SEQ ID NO: 2. These variants can be biologically active variants of Phe508del CFTR, such that the ion channel gating function and / or chloride transport function of CFTR is retained in the variant. These variants can be biologically inactive variants of Phe508del CFTR, such that the ion channel gating function and / or chloride transport function of CFTR is abolished (e.g., non-functional) in the variant.

[0190] In some embodiments, a mammalian nucleic acid sequence, e.g., a human nucleic acid sequence, e.g., a human DNA sequence encoding a CFTR protein (e.g., wild-type CFTR, Phe508del CFTR), can be codon-optimized for increased expression in cells. In some embodiments, the sequence encoding a CFTR protein can be codon-optimized for increased expression in epithelial cells. In some embodiments, the epithelial cell is a small intestine epithelial cell. In some embodiments, the epithelial cell is a lung epithelial cell. In some embodiments, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell can be derived from a subject with cystic fibrosis.

[0191] In some embodiments, the CFTR protein is provided in a purified form. In some embodiments, the CFTR protein is provided in the form of a cell lysate. In some embodiments, the CFTR protein is provided in the form of a tissue homogenate.

[0192] Working Example In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the methods, compositions, and systems provided herein and should not be construed in any way as limiting the scope thereof.

[0193] General Experiment Ussing chamber equipment for cell culture: EM-CSYS-8 Ussing chamber system, P2300 chamber, P2302 slider, VCC MC8 multichannel voltage / current clamp, P2020 electrode, and DM MC6 single channel electrode input module and dummy membrane (Physiologic Instruments, San Diego, CA).

[0194] Electrode: Silver / silver chloride (Ag / AgCl) electrode placed in an electrode tip containing 4% agar-Ringer's buffer

[0195] Ringer's solution: 115 mM NaCl, 25 mM NaHCO 3- , K 2.4 mM 2 HPO 4 , 0.4 mM KH 2 PO 4 , 1.2 mM MgCl 2 , 1.2 mM CaCl 2 , and 20 mM HEPES. NaOH to adjust pH to 7.4. Osmolality 290-300 mOsm.

[0196] Example 1 Described herein are selected amino acids (AA) or their combinations in a formulation that can stimulate chloride secretion and decrease ENaC activity by increasing CFTR and decreasing ENaC expression and activity on the apical membrane. The AA formulation was tested on primary human bronchial epithelial cells (HBEC) carrying the mutant CFTRΔF508. The cells were obtained from CF and non-CF patients. The cells were grown to 80% confluence in culture dishes and transferred to snapwell permeable inserts. The cells were allowed to fully grow and differentiate in permeable cell culture supports at the air-medium interface for about 30 days. The cells were then examined in Ussing chambers to measure transepithelial currents and resistance.

[0197] Short-circuit current (Isc) and resistance (R) were measured in fully differentiated primary homozygous CFTRΔF508 HBECs cultured at an air-liquid interface for 28-42 days after exposure to selected AA formulations (CF3AA, CF4AA-1 / 2 / 4, CF5AA), CF4AA-3 (negative control) and vehicle (control) in Ussing chambers. Changes in Isc were measured after inhibition of ENaC with 6 μM benzamil, activation of cAMP-activated CFTR channel activity with 10 μM forskolin, 1 μM PG01 (potentiator), block of CFTR channel with 20 μM CFTR(inh)-172, and block of basal Ca-activated chloride channel (CaCC) activity with 10 μM CaCCinh-A01, respectively. Similar experiments were repeated in CFTRΔF508 HBECs treated for 24 h prior to the experiment with 6 μM C18, a corrector for CFTR misfolding. Cellular CFTR and ENaC protein expression was analyzed using Western blots, and protein localization was demonstrated using immunofluorescence microscopy.

[0198] FIG. 1A shows a graph of benzamil insensitive current (μA) without C18 for the control (basic Ringer) solution and various individual amino acids (AA) (n=4). FIG. 1B shows a graph of benzamil insensitive current (μA) with C18 for the control (basic Ringer) solution and various individual AAs (n=4). Benzamil is an ENaC blocker, so the benzamil insensitive current therefore represents the current remaining after blocking ENaC. For both FIG. 1A and FIG. 1B, the best amino acid was selected from the single amino acids based on the delta change of the selected current.

[0199] FIG. 2A shows a graph of benzamil insensitive current (μA) without C18 for the control (basic Ringer) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4) (n=4). CF5AA-3 produced the highest benzamil insensitive current, and CF4AA-3 was the negative control. FIG. 2B shows a graph of benzamil insensitive current (μA) with C18 for the control (basic Ringer) solution, basal Ringer with DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4) (n=4). CF5AA-3 still produced the highest benzamil insensitive current, and CF4AA-3 was the negative control.

[0200] Figure 3A shows a graph of benzamil-sensitive current (μA) without C18 for a control (basic Ringer) solution and various individual amino acids (AA) (n=4). Figure 3B shows a graph of benzamil-sensitive current (μA) with C18 for a control (basic Ringer) solution and various individual amino acids (AA) (n=4). A higher benzamil-sensitive current means more ENaC activity remains. AA solutions that were able to inhibit ENaC activity show a low benzamil-sensitive current.

[0201] FIG. 4A shows a graph of benzamil-sensitive current (μA) without C18 for control (basic Ringer) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4) (n=4). FIG. 4B shows a graph of verapamil-sensitive current (μA) with C18 for control (basic Ringer) solution, basal Ringer with DMSO and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4) (n=4). CF5AA-3 produces the greatest reduction in ENaC current.

[0202] Overall, the basal resistance of the cells was 350-500 ohms. CF4AA-4 and CF5AA-3 reduced ENaC activity (benzamil-sensitive current) compared to the control, as shown in Figure 4A. Benzamil-insensitive currents, representing chloride secretion, were higher in cells treated with AA formulations, except for CF4AA-3, where C18 did not produce a significant change in Isc (Figures 2A and 2B). Forskolin and PG01 further increased Isc in cells exposed to AA formulations compared to the control and CF4AA-3. CFTR, but not CaCC, mainly contributed to chloride secretion in HBECs treated with AA formulations. Pretreatment with C18 showed a small increase in Isc after forskolin, PG01, and a small inhibition after CFTR(inh)-172 compared to the corresponding controls. CFTR protein expression was increased and ENaC was decreased in HBECs treated with AA formulations, with the majority of CFTR located in the apical membrane of the cells.

[0203] In conclusion, formulations based on a select set of AAs can improve dysfunctional chloride and sodium channel activity in CFTRΔF508 HBECs by modifying membrane channel expression and function. These formulations could be used alone or in combination with existing standards of care.

[0204] Example 2 Materials and Methods Cellular models: Fully differentiated primary normal human bronchial epithelial cells (HBECs) and homozygous CFTRΔF508 HBECs (CF cells) were cultured on snapwells at the air-liquid interface for 28-40 days.

[0205] Ussing chamber: Transepithelial short circuit current (I SC ), resistance (R), and unidirectional (J ms &J sm ) and 22 Na and 36 Net flux of Cl (J net) was measured in normal HBEC and CF cells bathed in vehicle, or CF-5AA-3 and CF-4AA-3. Chloride secretion was stimulated with forskolin (FSK, 10 μM apical and basal) and the potentiator GLPG1837 (3 μM apical), and ENaC and NKCC were blocked with benzamil (6 μM apical) and bumetanide (20 μM apical and basal).

[0206] Statistics: Statistical differences between vehicle and AA formulations were calculated using analysis of variance (OriginPlus 2016). P<0.05 was considered statistically significant.

[0207] In particular, additional experimental details related to Figures 10 and 11 are as follows:

[0208] The effects of C18 and / or VX661 on primary human bronchial epithelial cells harboring CFTRΔF508 were examined by incubating the cells with DMSO, 6 μM C18 in DMSO, and / or 3 μM VX661 for 24 h prior to the experiment. The experiment was repeated in the presence or absence of GLPG (N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-5-carboxamide). The cells were bathed in basal Ringer's solution or amino acid (AA) solution, respectively, and incubated at 37 °C for 24 h at 37 °C in 5% CO. 2 and 95% O 2 Basal Ringer's or AA solution in the base contained 5 mM glucose.

[0209] The treatment was as follows: 1.30 min baseline current 2.6 μM benzamil (1 μL, M) - 15 min 3. 10 μM forskolin (1 μL, M / S) + 3 μM GLPG1837 (1 μL, M) with or without - 15 min 4. 20 μM CFTR Inh 172 (2 μL, M / S) - 15 min 5. 10 μM CaCC Inh AO1 (1 μL, M) - 10 min 6. 20 μM bumetanide (2 μL, S) - 15 min

[0210] Lumacaftor: C18; Simdeco: VX661 (tezacaftor) / C18; Ivacaftor: GLPG1837 (reversible enhancer)

[0211] result Figures 5-9 show that select AA combinations can improve dysfunctional chloride and sodium channel activity in CFTRΔF508 HBECs by correcting and / or altering cell membrane channel function. These formulations could successfully complement the existing standard of care in patients with the CFTRΔF508 mutation.

[0212] Figures 10A and 10B show that CF5AA-3 is more effective than the correctors and more effective than the corrector(s) plus the potentiators. Figures 10A and 10B also show that CF5AA-3 plus the corrector(s) and the potentiators act synergistically.

[0213] FIG. 11 shows that CF-5AA-3 induces a statistically significant increase in chloride secretion compared to C18 (corrector) alone for either basal or stimulated chloride flux.

[0214] 12 shows a graph demonstrating that CF5AA-3 increased anion current through CFTR. The anion current with CF5AA-3 is significantly higher and sustained when compared to the triple combination.

[0215] Figure 12 shows that CF5AA-3 stimulates the CFTR channel better than the corrector and better than the corrector plus enhancer. These results provide evidence that CF5AA-3 directly affects the CFTR channel.

[0216] Figure 13 shows that CF5AA-3 stimulates CaCC channels better than the corrector and better than the corrector plus enhancer. These results provide evidence that CF5AA-3 directly affects CaCC channels.

[0217] Example 3 Background: In patients with cystic fibrosis, homozygous F508del mutation causes CFTR protein misfolding, defective trafficking and abnormal gating, reducing chloride secretion, which leads to chronic airway inflammation and infection. Combinations of small molecules that correct CFTR misfolding / trafficking (VX809 and VX661) and enhance CFTR gating (VX770) have been approved for the treatment of patients with F508del with suboptimal drug efficacy and side effects. We compared the efficiency or additive efficiency of CF5AA-3 with treatment with VX809 / VX770 or VX661 / VX770 alone in increasing defective CFTR trafficking and chloride secretion in primary human bronchial epithelial cells with homozygous F508del (HBEC-F50del).

[0218] Methods: Transepithelial short circuit current and 36 Cl net flux (J net ) was measured in differentiated HBEC-F508del bathed in vehicle (no AA), AA test formulation (CF5AA-3) or AA negative control (NC; CF4AA-3) in Ussing chambers. Cells were treated with C18 (VX809-analogue), VX661 or DMSO for 24 h. After blocking ENaC with benzamil, chloride secretion was stimulated with GLPG1837 (VX770-analogue) with or without forskolin. CFTR inh-172, CaCCinh-A01 or bumetanide were added to distinguish CFTR-, TMEM16A- and NKCC1-sensitive chloride currents, respectively. Western blots were performed on HBEC-F508del membrane fractions to determine apical CFTR expression.

[0219] result: HBEC-F508del soaked in AA exhibited significantly higher basal anion currents when compared to vehicle or NC, regardless of whether the cells were pretreated with DMSO (10.7 ± 0.2 μA vs. 1.4 ± 0.1 μA vs. 2.1 ± 0.1 μA), C18 (11.1 ± 0.2 μA vs. 1.6 ± 0.1 μA vs. 2.1 ± 0.1 μA), or VX661 (10.8 ± 0.3 μA vs. 1.5 ± 0.2 μA vs. 2.0 ± 0.2 μA). Stimulation with GLPG1837 caused a slight increase in current, peaking at 12.7 ± 0.4 μA with C18-AA. Anion currents were significantly higher in HBEC-F508del pretreated with C18 and VX661 and stimulated with forskolin and GLPG1837, peaking at 14.8±0.4 μA with VX661-AA compared with 7.9±0.7 μA with VX661-vehicle. In the presence of AA, CFTR contributed most to the stimulated current (60%), while AA also activated TMEM16A (16%) and other bumetanide-sensitive chloride channels (24%). 36 Flux studies using Cl confirmed increased chloride secretion in HBECF508del exposed to AA compared to vehicle or NC regardless of prior treatment (Figure 14). CFTR membrane expression (c-band) was increased in AA with or without corrective agents: from 6 (arbitrary units) (DMSO-vehicle) to 9 (C18-vehicle), and from 8 (DMSO-AA) to 12 (C18-AA), respectively. See Figure 14.

[0220] Conclusion: CF5AA-3 increases apical anion secretion as a corrector and enhancer, indicating that CF5AA-3 is a promising pharmaceutical active ingredient either as a stand-alone therapeutic option for CF patients or as an additive in combination therapies already implemented to treat CF patients after additional preclinical and clinical studies.

[0221] Example 4 Animal models of CF A genetically modified rat model is used in this protocol. This model has an alteration to its cystic fibrosis transmembrane conductance regulator (CFTR) gene. This rat model has a human version of the CFTR gene with the common patient mutation G551D inserted, which makes it a humanized CFTR protein (hCFTR) with the G551D mutation (class III). This rat model is referred to herein and in the art as hG551D. Similar to other animal models of cystic fibrosis, approximately 40% of hG551D rats exhibit ileus by 6 weeks of age. Some cases of ileus can be prevented with administration of Go-LYTELY with water and DietGel added to the food for hydration. Additionally, CF knockout (KO) rats develop dental abnormalities, including malocclusions that can make chewing more difficult. Thus, rats are fed softened rat chow three times a week. Affected rats are also monitored for the need to trim their teeth to facilitate feeding and prevent morbidity or mortality.

[0222] The hG551D rat strain was designed to mimic the human disease cystic fibrosis. This disease is characterized by the lack of chloride secretion through epithelial cell layers, such as the nasal epithelial layer. The inventors have shown that the rat airways of hG551D rats show characteristics of cystic fibrosis lung disease. In particular, these rats express submucosal glands in the airways, which are similar to those of human patients, thus providing pathology consistent with that seen in human cystic fibrosis patients.

[0223] Nasal potential difference (NPD) is performed to test chloride secretion and responses to drugs in hG551D rats. For the NPD procedure, the rat's tail is gently abraded, placed in lactated Ringer's solution and connected to a high impedance potential follower (VF1; World Precision Instruments) through a calomel cell. A probe is established by connecting an Ag / AgCl electrode (wire) bridge to a syringe that pumps the solution at a rate of 180 μl / h. After about 5 min, the rat is appropriately drowsy to allow cannulation of the nares with a PE10 cannula stretched to a tip diameter of about 0.15 mm. The perfusion solution is lactated Ringer's plus amiloride, K 2 HPO 4 , K.H. 2 PO 4 , Sodium Gluconate, NaHCO 3 , Ca gluconate, and a low [Cl] solution containing forskolin. Administer each perfusion solution intranasally at a rate of approximately 2 mL / min for a total of 80 µL per rat. Examine each superperfusion state for 6-10 min. Allow the rat to recover from anesthesia and administer atipamazole for recovery.

[0224] The animals in this study are treated with, for example, one of four compounds: test formulation (CF5AA-3), positive control (ivacaftor), negative control (CF4AA-3), or vehicle. Ivacaftor is given by oral gavage. The other compounds are given by aerosol spray. The animals are treated once daily for seven days, after which a nasal potential difference procedure is performed to test the efficacy of the drug. The nasal potential difference 24 hours after the last dose of ivacaftor or VS-009 is compared. Once this is complete, the animals are euthanized for tissue collection and subsequent analysis.

[0225] Table C shows exemplary dosing of the indicated medications. [Table 3]

[0226] Equivalence and Scope In the claims, articles such as "a," "an," and "the" can mean one or more, unless indicated to the contrary or otherwise clear from the context. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are provided in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which only one member of a group is provided in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are provided in, used in, or otherwise relevant to a given product or process.

[0227] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are presented as a list, for example in Markush group format, each subgroup of elements is also disclosed, and any element(s) can be excluded from the group. In general, when the invention, or aspects of the invention, are referred to as comprising certain elements and / or features, it should be understood that certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements and / or features. For the sake of brevity, these embodiments have not been specifically described herein as such. It should also be noted that the terms "comprise" and "contain" are intended to be open, allowing for the inclusion of additional elements or steps.

[0228] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification will control. In addition, any certain embodiments of the present invention that are within the scope of the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed to be known to those of skill in the art. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0229] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above description, but rather is set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the present invention, as defined in the following claims. When a claim or description relates to a product (e.g., the present composition), it should be understood that the claim or description includes, where applicable, methods of making or using the product according to any of the methods disclosed herein, and methods of using the product for any one or more purposes disclosed herein, unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one skilled in the art. Where a claim or description pertains to a method, it should be understood that product(s) useful for performing one or more steps of the method, e.g., the composition, device(s), or system(s), are encompassed by the disclosure where applicable, unless otherwise indicated or unless a contradiction or inconsistency would arise, apparent to one of ordinary skill in the art.

[0230] In the present specification, where ranges are given, embodiments are provided in which the endpoints are included, in which both endpoints are excluded, and in which one endpoint is included and the other endpoint is excluded. Both endpoints should be assumed to be included unless otherwise indicated. Furthermore, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any specific value or range within the range stated in different embodiments of the present invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It should also be understood that when a series of numerical values ​​is described herein, related embodiments are provided as well as any intervening values ​​or ranges defined by any two values ​​in the series, the lowest value may be taken as the minimum value and the highest value may be taken as the maximum value. When a phrase such as "at least one," "up to," "less than or equal to," or similar phrases precede a series of numbers herein, it should be understood that in various embodiments, the phrase applies to each number in the list unless the context clearly dictates otherwise (it should be understood that 100% of a value, e.g., a value expressed as a percentage, may be an upper limit, depending on the context). For example, "at least 1, 2, or 3" should be understood to mean "at least 1, at least 2, or at least 3" in various embodiments. It should also be understood that any and all reasonable lower and upper limits are expressly contemplated, where applicable. A reasonable lower or upper limit may be selected or determined by one of skill in the art based on factors such as, for example, convenience, cost, time, effort, availability (e.g., of samples, agents, or reagents), statistical considerations, and the like. In some embodiments, the upper or lower limit differs from a particular value by 2, 3, 5, or 10 fold. Numeric values ​​as used herein include values ​​expressed as percentages. For each embodiment in which a numerical value is prefaced by "about" or "approximately," an embodiment is provided in which the exact value is stated. For each embodiment in which a numerical value is not prefaced by "about" or "approximately," an embodiment is provided in which the value is prefaced by "about" or "approximately."Unless otherwise stated or otherwise clear from the context (unless such number would not unduly exceed 100% of the possible values), "approximately" or "about" generally includes values ​​within 1% of the number, or in some embodiments, within 5% of the number, or in some embodiments, within 10% of the number, in either direction. Unless expressly indicated to the contrary, in any method claimed herein that includes two or more operations, it should be understood that the order of the operations of the method is not necessarily limited to the order in which the operations of the method are described, but the invention includes embodiments in which the order is so limited. In some embodiments, the method may be performed by an individual or entity. In some embodiments, the steps of the method may be performed by two or more individuals or entities such that the method is performed collectively. In some embodiments, the method may be performed, at least in part, by requesting or authorizing another individual or entity to perform one, two or more, or all steps of the method. In some embodiments, the method includes requesting two or more entities or individuals to each perform at least one step of the method. In some embodiments, the performance of two or more steps is coordinated such that the method is performed collectively. The individuals or entities performing the different step(s) may or may not interact.

[0231] The section headings used herein should not be construed as limiting in any way, as it is expressly contemplated that subject matter presented under any section heading may be applicable to any aspect or embodiment described herein.

[0232] An embodiment or aspect of the present specification may be directed to any agent, composition, article, kit, and / or method described herein. It is contemplated that any one or more of the embodiments or aspects may be freely combined with any one or more of the other embodiments or aspects whenever appropriate. For example, any combination of two or more agents, compositions, articles, kits, and / or methods that are not mutually inconsistent is provided. Any description or illustration of a term anywhere in the present specification may be understood to apply whenever such term appears herein (e.g., in any aspect or embodiment to which such term is associated), unless otherwise indicated or otherwise clearly evident. References 1. O'Sullivan BP and Freedman SD (2009) Cystic Fibrosis. Lancet, 373; pp. 1891-904. 2. Brodlie M, Haq IJ, Roberts K, Elborn JS (2015) Targeted therapies to improve CFTR function in cystic fibrosis. Genome Medicine, 7; doi: 10.1186 / s13073-015-0223-6. 3. Corvol H, Thompson KE, Tabary O, et al. (2015) Translating the genetics of cystic fibrosis to personalized medicine. Transl Res; doi: 10.1016 / j.trsl.2015.04.008. 4. McPhail GL and Clancy JP (2013) Ivacaftor: the first therapy acting on the primary cause of cystic fibrosis. Drugs Today, 49; pp. 253-260. 5. Ramsey BW, Davies J, McElvaney NG, et al. (2011) A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med, 365; pp. 1633-1672.

Claims

1. A pharmaceutical formulation for the treatment of cystic fibrosis, comprising: a) with the proviso that at least one of the free amino acids is an L-amino acid; a therapeutically effective amount of each of cysteine ​​and proline as free amino acids; a therapeutically effective amount of at least one additional free amino acid selected from the group consisting of glycine, tyrosine, valine, and lysine; and as needed, b) at least one medicamentously inactive ingredient 13. A pharmaceutical formulation comprising:

2. 2. The pharmaceutical formulation of claim 1, wherein the free amino acid is an L-amino acid.

3. 3. The pharmaceutical formulation of claim 1 or claim 2, wherein the pharmaceutical formulation further comprises water as a medicamentously inactive ingredient.

4. The pharmaceutical formulation of any one of claims 1 to 3, wherein the at least one pharma- ceutical inactive ingredient comprises a pharma- ceutical acceptable carrier, buffer, electrolyte, adjuvant, or excipient.

5. with the proviso that at least one of the free amino acids is an L-amino acid. The medicament active ingredient comprises free amino acids cysteine, proline and glycine, A pharmaceutical formulation according to any one of claims 1 to 4, optionally comprising or consisting of one or more additional free amino acids selected from the group consisting of tyrosine, valine and lysine.

6. provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present; 6. The pharmaceutical formulation of claim 5, wherein the medicament active ingredient comprises or consists of cysteine, proline and glycine as free amino acids.

7. with the proviso that at least one of the free amino acids is an L-amino acid. The medicament active ingredient comprises cysteine, proline and tyrosine as free amino acids, A pharmaceutical formulation according to any one of claims 1 to 4, optionally comprising or consisting of one or more additional free amino acids selected from the group consisting of glycine, valine and lysine.

8. provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present; 8. The pharmaceutical formulation of claim 7, wherein the medicament active ingredient comprises or consists of cysteine, proline and tyrosine as free amino acids.

9. with the proviso that at least one of the free amino acids is an L-amino acid. The medicament active ingredient comprises cysteine, proline and lysine as free amino acids, A pharmaceutical formulation according to any one of claims 1 to 4, optionally comprising or consisting of one or more additional free amino acids selected from the group consisting of glycine, valine and tyrosine.

10. provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present; 10. The pharmaceutical formulation of claim 9, wherein the pharma- ceutical active ingredient comprises or consists of cysteine, proline and lysine as free amino acids.

11. with the proviso that at least one of the free amino acids is an L-amino acid. The pharmaceutical active ingredient comprises or consists of cysteine, proline, glycine and tyrosine as free amino acids; and The pharmaceutical formulation according to any one of claims 1 to 4, which optionally additionally comprises lysine as a free amino acid.

12. provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present; 12. The pharmaceutical formulation of claim 11, wherein the pharma- ceutical active ingredient comprises or consists of cysteine, proline, glycine and tyrosine as free amino acids.

13. with the proviso that at least one of the free amino acids is an L-amino acid. The pharmaceutical active ingredient comprises or consists of cysteine, proline, glycine and lysine as free amino acids; and A pharmaceutical formulation according to any one of claims 1 to 4, which optionally additionally contains tyrosine as a free amino acid.

14. provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present; 14. The pharmaceutical formulation of claim 13, wherein the pharma- ceutical active ingredient comprises or consists of cysteine, proline, glycine and lysine as free amino acids.

15. with the proviso that at least one of the free amino acids is an L-amino acid. The pharmaceutical active ingredient comprises or consists of cysteine, proline, lysine and tyrosine as free amino acids; and A pharmaceutical formulation according to any one of claims 1 to 4, optionally additionally comprising glycine as a free amino acid.

16. provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present; 16. The pharmaceutical formulation of claim 15, wherein the pharma- ceutical active ingredient comprises or consists of cysteine, proline, lysine and tyrosine as free amino acids.

17. with the proviso that at least one of the free amino acids is an L-amino acid.

5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the medicament active ingredient comprises or consists of cysteine, proline, glycine, tyrosine and lysine as free amino acids.

18. 18. The pharmaceutical formulation of claim 17, wherein the pharma- ceutical active ingredient is free of other free amino acids.

19. The pharmaceutical formulation according to any one of claims 1 to 18, wherein the pharmaceutical formulation is sterile.

20. The pharmaceutical formulation of any one of claims 1 to 19, wherein the pharmaceutical formulation is formulated for administration by enteral, pulmonary, inhalation, intranasal, or sublingual routes.

21. A pharmaceutical formulation according to any one of claims 1 to 20 for use as a medicament.

22. A pharmaceutical formulation according to any one of claims 1 to 21 for the treatment of a subject suffering from cystic fibrosis.

23. 23. The pharmaceutical preparation of claim 22, wherein the subject expresses wild-type CFTR.

24. 24. The pharmaceutical preparation of claim 22 or 23, wherein the subject has a mutation in the CFTR gene.

25. The pharmaceutical preparation of any one of claims 22 to 24, wherein the subject expresses wild-type and mutant CFTR.

26. 26. The pharmaceutical formulation of any one of claims 22-25, wherein the subject is afflicted with cystic fibrosis in which a CFTR protein is present that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a wild-type CFTR, wherein the wild-type CFTR sequence comprises SEQ ID NO:

1.

27. 27. The pharmaceutical formulation of any one of claims 24 to 26, wherein the mutations in CFTR comprise Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or combinations thereof, where X is any amino acid.

28. 28. The pharmaceutical preparation of claim 27, wherein the CFTR is a Phe508del CFTR mutant.

29. The pharmaceutical formulation of any one of claims 22 to 28, further comprising an additional therapeutic agent.

30. 30. The pharmaceutical formulation of claim 29, wherein the additional therapeutic agent comprises at least one of a small molecule drug, a protein drug, a nucleic acid drug, or a combination thereof.

31. 31. The pharmaceutical formulation of claim 29 or claim 30, wherein the additional therapeutic agent is at least one of a CFTR potentiator, a CFTR corrector, a CFTR readthrough agent, or a combination thereof.

32. 32. The pharmaceutical formulation of claim 31, wherein the additional therapeutic agent is a CFTR enhancer.

33. 33. The pharmaceutical formulation of claim 32, wherein the CFTR enhancer is ivacaftor.

34. A pharmaceutical formulation according to claim 19 or claim 20; and instructions for administering to a subject or contacting a biological sample with the formulation; Kit including:

35. Use of a pharmaceutical formulation according to any one of claims 1 to 20 in the manufacture of a medicament for the treatment of cystic fibrosis.

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