Amino acid compositions and methods for treating cystic fibrosis

Abstract

This document provides amino acid compositions that can be used to increase the transmembrane conductance (CFTR) protein of cystic fibrosis from the cytoplasm to the plasma membrane, particularly in epithelial cells. Methods for increasing the concentration of CFTR in the plasma membrane, increasing chloride ion transport, and increasing water transport are also provided. These compositions and methods can be used to treat cystic fibrosis in subjects carrying one or more mutations in the said CFTR protein. The use of these compositions in the treatment of cystic fibrosis and in the preparation of pharmaceutical agents for the treatment of cystic fibrosis is also covered herein.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 752,847, filed October 30, 2018, which is incorporated herein by reference for all purposes.

[0003] sequence list

[0004] This application contains a sequence list, which is submitted electronically in ASCII format and is hereby incorporated in its entirety by reference. The ASCII copy was created on October 25, 2019, named 174821-013301PCT_SL.txt, and is 25,987 bytes in size. Technical Field

[0005] This article describes amino acid compositions that can be used to increase the translocation of cystic fibrosis transmembrane conductance (CFTR) proteins from the cytoplasm to the plasma membrane. Methods for increasing the concentration of CFTR in the plasma membrane, increasing chloride ion transport, and increasing water transport are also provided. The compositions and methods described herein can be used to treat cystic fibrosis in subjects carrying one or more mutations in the CFTR protein. The use of these compositions in the treatment of cystic fibrosis and in the preparation of pharmaceutical agents for the treatment of cystic fibrosis is also covered herein. Background Technology

[0006] Cystic fibrosis (CF) is a recessive genetic disorder caused by reduced or absent CFTR synthesis, protein misfolding, and / or channel dysfunction, resulting in decreased chloride secretion, increased sodium absorption (ENaC), and impaired fluid homeostasis in the epithelial cells of the airways, intestines, and pancreas. CF is the most common life-limiting inherited disease, affecting the lungs, liver, pancreas, kidneys, and intestines. CF is inherited in an autosomal recessive manner, where each parent carries a mutation in at least one allele of the gene encoding the cystic fibrosis transmembrane conductance (CFTR) protein, resulting in one in four offspring carrying two mutated copies of the CFTR gene (e.g., alleles). Therefore, by age 2, more than 75% of patients are diagnosed with cystic fibrosis.1 Although life expectancy has increased in recent years for people with cystic fibrosis, the current median survival age is approximately 40 years. Current treatments typically involve intensive supportive care designed to manage the increased risk of lung infections and poor nutritional status. In addition, although small molecule therapies for the underlying genetic causes of CF have recently been approved, these therapies are least effective against the most common CFTR mutation, Phe508del, and are expensive, costing up to $300,000 per patient per year.

[0007] Although over 2,000 CFTR mutations have been identified in patients with cystic fibrosis, the vast majority of cystic fibrosis diagnoses present with only one or more of a handful of mutations. The most common mutation, Phe508del, is a deletion of three nucleotides, resulting in the loss of a single codon for the amino acid phenylalanine (three-letter code: Phe, single-letter code: F). This mutation causes defective CFTR protein processing (e.g., folding and transport to the plasma membrane), resulting in almost no membrane expression of CFTR (a chloride ion transporter involved in the transmembrane transport of ions and water). Furthermore, the small number of Phe508del CFTRs that successfully translocate to the plasma membrane are typically functionally defective, characterized by impaired chloride ion transport. Generally, CFTR mutations cause dysregulation of the transmembrane ion gradient, leading to decreased osmotic pressure for water outflow from epithelial cells, manifested as a thick mucus layer covering the cell. Nutrient-rich, thick mucus provides an optimal environment for bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus to capture and grow, causing persistent infections that are often unresponsive to antibiotics. In addition, many CF patients develop other lung diseases, such as bronchopulmonary aspergillosis and bronchiectasis, leading to increased morbidity and mortality.

[0008] While targeted therapies for cystic fibrosis caused by specific mutations in CFTR have been developed, many therapies are only effective in patients with certain CFTR mutations, and adverse side effects have been reported.2 One example is ivacaftor (VX-770, KAYLDECO™) (a small molecule potentiator of CFTR),4 as described in U.S. Patent Application Publication US 2014 / 0221424 and International Patent Application No. PCT / US2015 / 036691. However, ivacaftor is for the treatment of CF in patients with a missense mutation Gly551Asp (G551D) in at least one allele of the CFTR gene, which covers approximately 4% to 5% of patients with CF.5 There is a clear unmet need for therapies to treat cystic fibrosis, particularly for patients carrying the most prevalent CFTR mutation, Phe508del CFTR. Attached Figure Description

[0009] Figure 1A The graphs (n=4) show the benzamir insensitive current (μA) in the absence of C18 for the control (basal Ringer's) solution and various individual amino acids (AA).

[0010] Figure 1B The graphs (n=4) show the benzamir insensitive current (μA) of the control (basic Ringer's) solution and various individual AA solutions in the presence of C18.

[0011] Figure 2A The graphs (n=4) show the benzamir insensitive current (μA) in the absence of C18 for the control (basic Ringer's) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4).

[0012] Figure 2B The graphs (n=4) show the benzamir insensitive current (μA) in the presence of C18 for the control (basic Ringer's) solution, the basic Ringer's solution containing DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4).

[0013] Figure 3A The graphs (n=4) show the benzamir sensitive current (μA) of the control (basal Ringer's) solution and various individual amino acids (AA) in the absence of C18.

[0014] Figure 3B The graphs (n=4) show the benzamir sensitive current (μA) of the control (basal Ringer's) solution and various individual amino acids (AA) in the presence of C18.

[0015] Figure 4A The graphs (n=4) show the benzamir sensitive current (μA) in the absence of C18 for the control (basic Ringer's) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4).

[0016] Figure 4B The graphs (n=4) show the benzamir sensitive current (μA) in the presence of C18 for the control (basic Ringer's) solution, the basic Ringer's solution containing DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4).

[0017] Figure 5 A graph depicting basal chlorine flux is shown. Unstimulated CF cells immersed in the medium do not secrete chlorine. However, CF cells immersed in CF-5AA-3 showed a significant increase in chlorine secretion (P<0.05; n=7).

[0018] Figure 6 A graph depicting basal chloride flux is shown. Compared with unstimulated normal HBECs, unstimulated CF cells immersed in the medium and CF-5AA-3 had significantly higher sodium uptake (P<0.001; n=6).

[0019] Figures 7A to 7F A graph depicting anion current and irritant chlorine flux is shown. Figure 7A The study showed that compared with normal HBECs, CF cells immersed in the medium had significantly lower benzamir insensitive currents (anion currents), but CF-5AA-3 increased the anion currents by >10 times (n=4). Figure 7B The study showed that bumetanide-sensitive current (chloride current) in CF cells immersed in CF-5AA-3 was significantly higher (n=4) compared to the medium, but did not reach the value of normal HBEC. Figure 7C , Figure 7D , Figure 7E and Figure 7F This study showed that CF cells immersed in CF-5AA-3 exhibited significantly higher anion peak currents. Figure 7E ) and total chlorine secretion ( Figure 7F Furthermore, stimulation with FSK and GLPG1837 did not contribute to the increase in AA in the presence of AA. Figure 7C , Figure 7D ).

[0020] Figure 8A and Figure 8B A graph depicting ENaC activity and the sodium flux of the blockage is shown. Figure 8A The study showed that CF cells immersed in the medium or CF-5AA-3 had significantly higher ENaC activity (benzamir-sensitive current) compared to normal HBECs, but the ENaC activity in CF cells immersed in CF-5AA-3 was significantly lower than that in the medium (n=4). Figure 8B Similarly, it was shown that sodium uptake was significantly increased in CF cells, while sodium uptake was moderately decreased in CF cells immersed in CF-5AA-3 (n=7).

[0021] Figure 9 A graph depicting the effect of the AA formulation shown on ENaC activity in CF cells is presented. CF cells immersed in CF-4AA-3 showed a further reduction in ENaC activity compared to the medium and CF-5AA-3 (n=4).

[0022] Figure 10A and Figure 10BFigures depicting the effects of C18 (corrector; similar to Lumacaftor) and / or VX661 (corrector; tezacaftor)) on CFTRΔF508 cells in the presence of forskolin and synergists are presented. Ivacaftor: GLPG1837 (reversible synergist); Symdeko: VX661 (tezacaftor) / C18. CF5AA-3 increases the current to a greater extent than C18 or Symdeko.

[0023] Figure 11 The image depicts a primary HBEC with CFTRΔF508. 36 Chlorine flux studies show a graph of increased chlorine secretion in response to CF-5AA-3.

[0024] Figure 12 A graph showing the increase in anion current via CFTR in a primary HBEC with CFTRΔF508 is presented. The anion current through CF5AA-3 is significantly higher and sustained compared to the triple combination.

[0025] Figure 13 A graph showing the increase in anion current via Ano1 from CF5AA-3 is presented. The Ano1-mediated current through CF5AA-3 is significantly higher and more sustained compared to the current observed with the triplet combination.

[0026] Figure 14 The figure shows that CF5AA-3 increases apical anion secretion to a degree similar to that of the correctors and synergists shown. Summary of the Invention

[0027] The embodiments covered are defined by the claims, not by the Detailed Description of the Invention. The Detailed Description provides a high-level overview of various aspects and introduces concepts that will be further described in the Detailed Description section below. The Detailed Description is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by referring to the appropriate portions of the specification throughout, any or all of the accompanying drawings, and each claim.

[0028] This disclosure recognizes the need for more cost-effective and effective treatment options for patients with cystic fibrosis. The amino acid compositions described herein are particularly useful for treating CF patients carrying at least one Phe508del mutation in an allele of the CFTR gene. When the CFTR gene is homozygous for the Phe508del mutation, very little CFTR is transported to the plasma membrane, making treatment with CFTR potentiators (such as ivacapator), which enhances the function of CFTR present in the plasma membrane, almost ineffective. This disclosure provides compositions that demonstrate enhanced translocation of both wild-type and Phe508del CFTR proteins from the cytoplasm to the plasma membrane. Specifically, the compositions described herein are particularly effective in increasing the amount of mutant CFTR protein on the plasma membrane. Furthermore, methods for treating diseases with CFTR dysfunction, such as cystic fibrosis, are also provided herein.

[0029] Formulation – Amino Acids

[0030] In a first aspect, the present invention provides a formulation comprising: cysteine ​​and proline as free amino acids, and at least one other free amino acid selected from the group consisting of glycine, tyrosine and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0031] In one implementation, the free amino acid is an L-amino acid.

[0032] In one embodiment, the formulation does not contain any free amino acids other than cysteine, proline, and at least one other amino acid selected from the group consisting of glycine, tyrosine, and lysine.

[0033] In one embodiment, the formulation is substantially composed of cysteine ​​and proline as free amino acids, and one or more other free amino acids selected from the group consisting of glycine, tyrosine and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0034] In one embodiment, the formulation comprises: cysteine ​​and proline as free amino acids, and one or more other free amino acids selected from the group consisting of glycine, tyrosine and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0035] In one embodiment, the formulation comprises, is composed of, or substantially composed of, free amino acids cysteine, proline, and glycine, as well as one or more other free amino acids selected from the group consisting of tyrosine and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0036] In one embodiment, the formulation comprises, is composed of or substantially comprises, cysteine, proline and glycine in the form of 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.

[0037] In one embodiment, the formulation comprises, consists of, or is substantially composed of, free amino acids cysteine, proline, and tyrosine, as well as one or more other free amino acids selected from the group consisting of glycine and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0038] In one embodiment, the formulation comprises, consists of, or is substantially composed of, the free amino acids cysteine, proline, and tyrosine, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0039] In one embodiment, the formulation comprises, consists of, or is substantially composed of, free amino acids cysteine, proline, and lysine, as well as one or more other free amino acids selected from the group consisting of glycine and tyrosine, provided that at least one of the free amino acids is an L-amino acid.

[0040] In one embodiment, the formulation comprises, consists of, or is substantially composed of, the free amino acids cysteine, proline, and lysine, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0041] In one embodiment, the formulation comprises, consists of or substantially consists of, the free amino acids cysteine, proline, glycine and tyrosine, and optionally additionally comprises the free amino acid lysine, provided that at least one of the free amino acids is an L-amino acid.

[0042] In one embodiment, the formulation comprises, or is substantially composed of, the free amino acids cysteine, proline, glycine, and tyrosine, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0043] In one embodiment, the formulation comprises, consists of, or is substantially composed of, the free amino acids cysteine, proline, glycine, and lysine, and optionally additionally comprises the free amino acid tyrosine, provided that at least one of the free amino acids is an L-amino acid.

[0044] In one embodiment, the formulation comprises, is composed of or substantially comprises, free amino acids cysteine, proline, glycine and lysine, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0045] In one embodiment, the formulation comprises, consists of or substantially consists of, the free amino acids cysteine, proline, lysine and tyrosine, and optionally additionally comprises the free amino acid glycine, provided that at least one of the free amino acids is an L-amino acid.

[0046] In one embodiment, the formulation comprises, is composed of or substantially comprises, free amino acids cysteine, proline, lysine and tyrosine, provided that at least one of the free amino acids is an L-amino acid and that no other free amino acids are present.

[0047] In one embodiment, the formulation comprises, or is substantially composed of, free amino acids cysteine, proline, glycine, tyrosine, and lysine, provided that at least one of the free amino acids is an L-amino acid.

[0048] In one implementation, the formulation does not contain other free amino acids.

[0049] In a second aspect, the present invention provides a formulation comprising: cysteine ​​and proline as free amino acids, and at least one other free amino acid selected from the group consisting of glycine, tyrosine, lysine and valine, provided that at least one of the free amino acids is an L-amino acid.

[0050] In one embodiment of the second aspect, the free amino acid is an L-amino acid.

[0051] In one embodiment of the second aspect, the formulation does not contain any free amino acids other than cysteine, proline, and at least one other amino acid selected from the group consisting of glycine, tyrosine, lysine, and valine.

[0052] In one embodiment of the second aspect, the formulation comprises, or is substantially composed of, cysteine, proline, and valine as free amino acids, provided that at least one of the free amino acids is an L-amino acid.

[0053] In one embodiment of the second aspect, the formulation comprises, or is substantially composed of, cysteine, proline, valine, and glycine in the form of free amino acids, provided that at least one of the free amino acids is an L-amino acid.

[0054] In one embodiment of the second aspect, the formulation comprises, or is substantially composed of, free amino acids cysteine, proline, valine, and tyrosine, provided that at least one of the free amino acids is an L-amino acid.

[0055] In one implementation, neither of the formulations in the second aspect contains other free amino acids.

[0056] Formulations – Excipients, etc.

[0057] In one embodiment, the formulation also includes water.

[0058] In one implementation, the formulation is a pharmaceutical formulation.

[0059] In one embodiment, the formulation further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient.

[0060] In one embodiment, the formulation is sterile.

[0061] In one embodiment, the formulation is prepared for administration via the intestinal, pulmonary, inhalation, intranasal, or sublingual routes.

[0062] In one implementation, each free amino acid is present in a therapeutically effective amount.

[0063] Treatment

[0064] In another aspect, the present invention provides a formulation as described above, which is used as a pharmaceutical agent.

[0065] In one embodiment of the present invention, the formulation described above is used to treat cystic fibrosis.

[0066] The present invention also provides the use of the formulation described above in the manufacture of a medicament for treating cystic fibrosis.

[0067] In another aspect, the present invention provides a method for treating a subject suffering from cystic fibrosis, the method comprising administering to the subject suffering from cystic fibrosis an agent as described above, wherein the administration relieves at least one symptom of cystic fibrosis.

[0068] In one embodiment, a 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 both wild-type CFTR and a mutant CFTR. In one embodiment, the subject has 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, wherein the wild-type CFTR sequence comprises SEQ ID NO:1. In one embodiment, the mutation in the CFTR comprises Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or a combination thereof, wherein X is any amino acid. In one embodiment, the CFTR is a Phe508del CFTR mutant.

[0069] In one implementation, the formulation described above is used in combination with another therapeutic agent.

[0070] In one embodiment, the additional therapeutic agent includes 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 readthrough agent, or a combination thereof. In one embodiment, the additional therapeutic agent is a CFTR enhancer. In one embodiment, the CFTR enhancer is ivacathotocin.

[0071] Other aspects of the invention

[0072] In another aspect of the invention, a kit is provided comprising: a pharmaceutical preparation as described above; and instructions for administering the preparation to a subject or for contacting a biological sample with the preparation.

[0073] In another aspect of the invention, a method is provided for increasing the amount of cystic fibrosis transmembrane conductance regulator (CFTR) protein present on the plasma membrane of at least one cell, the method comprising: contacting the at least one cell with an effective amount of a pharmaceutical preparation as described above, wherein the contact promotes at least one of CFTR folding or CFTR translocation to the plasma membrane, thereby increasing the amount of CFTR protein present on the cell plasma membrane.

[0074] In one embodiment, the number of wild-type CFTR proteins on the plasma membrane is increased. In one embodiment, the number of mutant CFTR proteins on the plasma membrane is increased. In one 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 on the plasma membrane is increased, wherein the 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 combinations thereof, wherein X is any amino acid. In one embodiment, one or more of the CFTR proteins comprise the Phe508del mutation. In one embodiment, the increase in the number of CFTR proteins present on the plasma membrane is detected by an increase in chloride ions expelled from the cell. In one embodiment, the increase in the number of CFTR proteins present on the plasma membrane is associated with an increase in water expelled from 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 lung epithelial cells are bronchial epithelial cells. In another embodiment, bronchial epithelial cells are isolated from a subject with cystic fibrosis.

[0075] In one particular aspect, a pharmaceutical preparation is provided comprising: therapeutically effective amounts of cysteine ​​and proline as free amino acids, and therapeutically effective amounts of at least one other free amino acid from the group consisting of glycine, tyrosine, or lysine, provided that at least one of the free amino acids is an L-amino acid, wherein the pharmaceutical preparation is substantially composed of cysteine, proline, and at least one other free amino acid.

[0076] In a specific embodiment of the pharmaceutical preparation, the free amino acid is an L-amino acid. In another specific embodiment, the pharmaceutical preparation further comprises water. In another specific embodiment, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In another specific embodiment, the pharmaceutical preparation is sterile. In another specific embodiment, the pharmaceutical preparation is formulated for administration via the enteric, pulmonary, inhalation, intranasal, or sublingual routes.

[0077] In one specific embodiment of the pharmaceutical formulation, the formulation comprises a therapeutically effective amount of cysteine ​​and proline as free amino acids, and a therapeutically effective amount of at least one other free amino acid, substantially composed of, or composed of, the group consisting of glycine, tyrosine, or lysine, and contains no other free amino acids. The combination of said amino acids includes, for example: cysteine, proline, and glycine; cysteine, proline, and tyrosine; cysteine, proline, and lysine; cysteine, proline, glycine, and tyrosine; cysteine, proline, tyrosine, and lysine; and cysteine, proline, glycine, tyrosine, and lysine.

[0078] In another specific embodiment of the pharmaceutical formulation, the formulation comprises a therapeutically effective amount of cysteine ​​and proline as free amino acids, and a therapeutically effective amount of at least one other free amino acid, substantially composed of or consisting of, the group consisting of glycine, tyrosine, lysine, or valine, and contains no other free amino acids. The combinations of said amino acids include, for example: cysteine, proline, and valine; cysteine, proline, valine, and glycine; and cysteine, proline, valine, and tyrosine.

[0079] In another specific embodiment of the pharmaceutical preparation, the pharmaceutical preparation comprises a therapeutically effective amount of each of the free amino acids cysteine, proline, glycine, tyrosine, and lysine, and contains no other free amino acids. In another specific embodiment of the pharmaceutical preparation, the pharmaceutical preparation is substantially composed of or constitutes, in a therapeutically effective amount of each of the free amino acids cysteine, proline, glycine, tyrosine, and lysine.

[0080] In a particular embodiment, the pharmaceutical preparation is used in a therapy. In a more particular embodiment, the pharmaceutical preparation is used to treat cystic fibrosis. In yet another particular embodiment, the pharmaceutical preparation is used to manufacture an agent for treating cystic fibrosis.

[0081] In another aspect, a method for treating a subject with cystic fibrosis is provided, the method comprising administering a pharmaceutical preparation described herein to the subject with cystic fibrosis, wherein the administration alleviates at least one symptom of cystic fibrosis. In one 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 both wild-type CFTR and mutant CFTR. In another particular embodiment, the subject has cystic fibrosis, wherein 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, wherein the wild-type CFTR sequence comprises SEQ ID NO:1. In another particular embodiment, the mutation in the CFTR comprises Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or combinations thereof, wherein X is any amino acid. In another particular embodiment, the CFTR is a Phe508del CFTR mutant. In another specific embodiment of the method, the method further includes administering an additional therapeutic agent. In another specific embodiment of the method, the additional therapeutic agent includes at least one of a small molecule drug, a protein drug, a nucleic acid drug, or a combination thereof. In another specific embodiment of the method, the additional therapeutic agent is at least one of a CFTR enhancer, a CFTR corrector, a CFTR readthrough agent, or a combination thereof. In another specific embodiment of the method, the additional therapeutic agent is a CFTR enhancer. In another specific embodiment of the method, the CFTR enhancer is ivacathotoxin.

[0082] In one specific embodiment of the method, the pharmaceutical preparation comprises a therapeutically effective amount of cysteine ​​and proline as free amino acids, and a therapeutically effective amount of at least one other free amino acid, substantially composed of, or composed of, the group consisting of glycine, tyrosine, or lysine, and contains no other free amino acids. The combinations of said amino acids include, for example: cysteine, proline, and glycine; cysteine, proline, and tyrosine; cysteine, proline, and lysine; cysteine, proline, glycine, and tyrosine; cysteine, proline, tyrosine, and lysine; and cysteine, proline, glycine, tyrosine, and lysine.

[0083] In another specific embodiment of the method, the pharmaceutical preparation comprises a therapeutically effective amount of each of the free amino acids cysteine, proline, glycine, tyrosine, and lysine, and contains no other free amino acids. In another specific embodiment of the method, the pharmaceutical preparation consists essentially of or is composed of a therapeutically effective amount of each of the free amino acids cysteine, proline, glycine, tyrosine, and lysine.

[0084] In another aspect, a method is provided for increasing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of at least one cell, the method comprising: contacting the at least one cell with an effective amount of a pharmaceutical preparation described herein, wherein the contact promotes at least one of CFTR folding or CFTR translocation to the plasma membrane, thereby increasing the number of CFTR proteins present on the cell membrane. In one particular embodiment of the method, the number of wild-type CFTR proteins on the plasma membrane is increased. In another particular embodiment of the method, the number of mutant CFTR proteins on the plasma membrane is increased. In another particular embodiment of the method, 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 CFTRs on the plasma membrane is increased, wherein the wild-type CFTR comprises SEQ ID NO:1. In another particular embodiment of the method, one or more of the CFTR proteins comprise Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or combinations thereof, wherein X is any amino acid. In another specific embodiment of the method, one or more of the CFTR proteins contain the Phe508del mutation. In another specific embodiment of the method, an increase in the number of CFTR proteins present on the cell membrane is detected by an increase in chloride ions expelled from the cell. In another specific embodiment of the method, an increase in the number of CFTR proteins present on the cell membrane is associated with an increase in water expelled from the cell. In another specific embodiment of the method, the cells are epithelial cells. In a more specific embodiment of the method, the epithelial cells are lung epithelial cells. In yet another more specific embodiment of the method, the lung epithelial cells are bronchial epithelial cells. In another specific embodiment of the method, bronchial epithelial cells are isolated from a subject with cystic fibrosis, and the results of in vitro assays of such cells provide guidance on which therapeutic agents and / or combinations thereof will confer therapeutic efficacy to the subject from whom the isolated cells were isolated. In another specific embodiment of the method, the method further includes the administration of additional therapeutic agents. In another specific embodiment of the method, the additional therapeutic agents include at least one of small molecule drugs, protein drugs, nucleic acid drugs, or combinations thereof. In another specific embodiment of the method, the additional therapeutic agents are at least one of CFTR enhancers, CFTR correctors, CFTR readout agents, or combinations thereof. In another specific embodiment of the method, the additional therapeutic agent is a CFTR enhancer. In another specific embodiment of the method, the CFTR enhancer is ivacathotocin.

[0085] In one specific embodiment of the method, the pharmaceutical preparation comprises a therapeutically effective amount of cysteine ​​and proline as free amino acids, and a therapeutically effective amount of at least one other free amino acid, substantially composed of, or composed of, the group consisting of glycine, tyrosine, or lysine, and contains no other free amino acids. The combinations of said amino acids include, for example: cysteine, proline, and glycine; cysteine, proline, and tyrosine; cysteine, proline, and lysine; cysteine, proline, glycine, and tyrosine; cysteine, proline, tyrosine, and lysine; and cysteine, proline, glycine, tyrosine, and lysine.

[0086] In another specific embodiment of the method, the pharmaceutical preparation comprises a therapeutically effective amount of each of the free amino acids cysteine, proline, glycine, tyrosine, and lysine, and contains no other free amino acids. In another specific embodiment of the method, the pharmaceutical preparation consists essentially of or is composed of a therapeutically effective amount of each of the free amino acids cysteine, proline, glycine, tyrosine, and lysine.

[0087] In another particular embodiment, a kit is provided comprising: a pharmaceutical preparation comprising, substantially comprising, or consisting of, the free amino acids cysteine, proline, glycine, tyrosine, and lysine; and instructions for administration to a subject or for contacting a biological sample with the composition.

[0088] In another aspect, this disclosure provides compositions for treating cystic fibrosis, said compositions comprising, substantially comprising, or comprising of cysteine, proline, glycine, tyrosine, and lysine. In another aspect, the invention provides the use of amino acid compositions in treating cystic fibrosis in subjects with this need. In another aspect, the invention provides the use of amino acid compositions in the preparation of medicaments for treating cystic fibrosis in subjects with this need.

[0089] In one aspect, this disclosure provides compositions comprising, substantially comprising, or consisting of cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the amino acid is an L-amino acid. In some embodiments, the composition further comprises water. In another aspect, the composition further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In some embodiments, the composition is sterile. In another aspect, the composition is formulated for administration via the enteric, pulmonary, inhalation, intranasal, or sublingual routes.

[0090] In some embodiments, the subject has cystic fibrosis in which wild-type CFTR is present. In one aspect, the subject has a mutation in the CFTR gene. In another aspect, the subject has cystic fibrosis in which both wild-type and mutant CFTR are present. In yet another aspect, the subject has 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 in amino acid sequence to the wild-type CFTR provided by SEQ ID NO:1. In still other aspects, the CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del CFTR mutant, where X is any amino acid. In some embodiments, the CFTR is a Phe508del CFTR mutant.

[0091] This document also provides a method for increasing the amount of cystic fibrosis transmembrane conductance regulator (CFTR) protein present on the cell membrane, the method comprising contacting a cell with an effective amount of a composition comprising, substantially comprise, or comprise of cysteine, proline, glycine, tyrosine, and lysine. In some aspects, the effective amount increases the amount of CFTR protein present on the cell membrane. In other aspects, the amount of wild-type CFTR protein on the plasma membrane increases. In still other aspects, the amount of mutant CFTR protein on the plasma membrane increases. In some aspects, the amount of CFTR protein on the plasma membrane that is identical to at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence of the 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, wherein X is any amino acid. In other embodiments, one or more of the CFTR proteins are Phe508del CFTR mutants. In some embodiments, chloride ion excretion from the cells is increased. In other embodiments, water excretion from the cells is increased. In other embodiments, the cells are epithelial cells. In some embodiments, the epithelial cells are lung epithelial cells. In some embodiments, the lung epithelial cells are bronchial epithelial cells. In other embodiments, bronchial epithelial cells are obtained from a subject with cystic fibrosis. The cells may be in vitro, in vivo, or ex vivo.

[0092] In some aspects, the methods provided herein also include the administration of additional therapeutic agents. 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 other embodiments, the additional therapeutic agent is a CFTR enhancer, corrector, or readability agent. In other embodiments, the additional therapeutic agent is a CFTR enhancer. In some aspects, the CFTR enhancer is ivacaprot. In some embodiments, the additional therapeutic agent is a CFTR corrector. In some embodiments, the CFTR corrector is rumacaprot. The additional therapeutic agent may be included in any of the compositions described herein (e.g., the composition further comprises the additional therapeutic agent). The additional therapeutic agent may be administered concurrently with, before, or after the administration of any of the compositions described herein (e.g., combination therapy).

[0093] On the other hand, this disclosure provides a method for treating cystic fibrosis, the method comprising administering the composition described herein to a subject in need of treatment, wherein the composition is formulated for administration via the intestine,

[0094] The composition may be administered via the lung, inhalation, nasal, or sublingual routes. In some embodiments, the composition is formulated for pulmonary administration to a subject. In some embodiments, the composition is formulated for intestinal administration to a subject. In some embodiments, the composition is formulated for oral administration to a subject.

[0095] In another aspect, this disclosure provides a method for treating cystic fibrosis, the method comprising administering the composition described herein to a subject in need of treatment. In some embodiments, the subject has cystic fibrosis, wherein the subject has a mutation in the CFTR gene. In some embodiments, the subject has cystic fibrosis, wherein the subject is heterozygous for both wild-type CFTR and mutant CFTR. In some embodiments, the subject has cystic fibrosis, wherein the subject is heterozygous for a first mutant CFTR and a second mutant CFTR (e.g., wherein the mutations are different). In some embodiments, the subject has cystic fibrosis, wherein the subject is homozygous for the mutant CFTR (e.g., both alleles have the same mutation). In some embodiments, the subject has cystic fibrosis, wherein the subject is homozygous for wild-type CFTR (e.g., neither allele is mutated). In some embodiments, the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del CFTR mutant, wherein 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 in amino acid sequence to the wild-type CFTR 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 in amino acid sequence to the Phe508del CFTR provided by SEQ ID NO:2.

[0096] Information on the diagnosis and treatment of various diseases (including cystic fibrosis and related lung diseases) can be found in Longo, D. et al. (eds.), Harrison's Principles of Internal Medicine, 18th edition; McGraw-Hill Professional, 2011. Information on various therapeutic agents and human diseases (including lung 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 (July 2009). All patents, patent applications, books, papers, documents, databases, websites, publications, references, etc., mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict between this specification and any incorporated references, this specification (including any amendments thereof) shall prevail. The applicant reserves the right to modify this specification and / or correct obvious errors based on, for example, any incorporated material. Nothing in the incorporated material limits the invention. Unless otherwise stated, terminology is used herein in its standard, art-recognized sense. Standard abbreviations for various terms are used herein.

[0097] In some respects, the compositions provided herein comprise, are substantially composed of, or consist of, one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine, and contain no other free amino acids.

[0098] This article also provides the use of the composition in treating cystic fibrosis in subjects with this need, wherein the composition comprises cysteine, proline, glycine, tyrosine, and lysine.

[0099] This document also provides a kit comprising a composition containing, substantially composed of, or consisting of, cysteine, proline, glycine, tyrosine, and lysine; and instructions for administration to a subject or for contacting a biological sample with the composition.

[0100] All combinations of implementation schemes described individually are envisioned.

[0101] The details of certain embodiments of the invention are set forth in the following detailed description of some embodiments. Other features, objects, and advantages of the invention will become apparent from the definitions, examples, drawings, and claims.

[0102] definition

[0103] For convenience, this document contains descriptions and certain information relating to the various terms used in this disclosure.

[0104] As used herein, the term "agent" refers 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, peptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotides, aptamers), lipids, polysaccharides, etc. Generally, any suitable method known in the art can be used to obtain an agent. Those skilled in the art will select an appropriate method based on, for example, the properties of the agent. The agent may be at least partially purified. In some embodiments, the agent may be provided as part of a composition, which, in addition to the agent, may also contain, for example, counterions, aqueous or non-aqueous diluents or carriers, buffers, preservatives, or other components. In some embodiments, the agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments, the agent is cell-permeable within the range of typical agents for cellular absorption, and acts intracellularly (e.g., in mammalian cells) to produce a biological effect. Certain compounds may exist in specific geometric or stereoisomeric forms. Unless otherwise indicated, this disclosure covers 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) in various embodiments.

[0105] Certain compounds may exist in multiple or protonated states, have multiple configurations, exist as solvates [e.g., with water (i.e., hydrates) or common solvents], and / or may have different crystalline forms (e.g., polymorphs) or different tautomer forms. Where applicable, this disclosure covers embodiments exhibiting such alternative protonated states, configurations, solvates, and forms. The term "agent" may also cover "therapeutic agent." The terms "compound" and "agent" are used interchangeably.

[0106] For example, when delivered to cells or an organism according to a selected form, route, and / or regimen of administration, the “effective amount” or “effective dose” of an agent (or a composition containing such an agent) refers to an amount sufficient to achieve the desired biological and / or pharmacological action. The phrase “effective amount” is used interchangeably with “therapeutic effective amount.” As will be understood by those skilled in the art, the absolute amount of an effective particular agent or composition can vary depending on factors such as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. Those skilled in the art will further understand that, in various embodiments, the “effective amount” may be applied to a subject through contact with cells or in a single dose or by using multiple doses. In some embodiments, the effective amount is the amount that increases the transport of CFTR to the cell membrane. In some embodiments, the effective amount is the amount that increases the translocation of CFTR from the cytoplasm to the cell membrane. In some embodiments, the effective amount is the amount that increases the excretion of chloride ions from the cells. In some embodiments, the effective amount is the amount that increases the excretion of water from the cells. In some embodiments, the effective amount is the amount that alleviates the symptoms of lung disease and / or treats lung disease. In some implementations, the effective amount is the amount that relieves the symptoms of cystic fibrosis and / or treats cystic fibrosis.

[0107] As used herein, the term "consistently composed of..." limits the scope of ingredients and steps to those specified materials or steps that do not materially affect one or more essential and novel features of the invention, such as compositions for treating cystic fibrosis and their use, as well as methods for treating cystic fibrosis. For example, by using "consistently composed of...", a therapeutic composition does not contain 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 cystic fibrosis. Furthermore, by using the term "consistently composed of...", a composition may contain substances that have no therapeutic effect on cystic fibrosis; such ingredients include carriers, excipients, adjuvants, flavoring agents, etc., that do not affect the health or function of the lung epithelium.

[0108] The description of any aspect or embodiment using terms such as “comprising,” “having,” “including,” or “containing” with respect to one or more elements herein is intended to support the notion that a similar aspect or embodiment “consists of one or more specific elements,” “consists substantially of one or more specific elements,” or “generally contains” one or more specific elements, unless otherwise stated or clearly contradicted by the context (e.g., a composition described herein as containing a specific element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by the context).

[0109] The term "gene" refers to a locus (e.g., region) of DNA containing nucleotides. Typically, a gene contains multiple regions, including one or more upstream or downstream regulatory sequences (e.g., enhancers / silencers, promoters, 5' non-coding sequences, 3' non-coding sequences) usually required to initiate transcription, and an open reading frame containing one or more exons and one or more introns. An "exon" is any part of a gene that encodes a portion of the final mature RNA that will be translated into a protein sequence. An "intron" is any part of a gene that is removed by RNA splicing during the maturation of the final mature RNA. A "cryptic exon" is an exon that may introduce an early termination codon into the mature RNA or cause atypical splicing patterns. The term "gene" can also refer to a nucleic acid segment that expresses a protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences). A "natural gene" is a gene found in nature that has its own regulatory sequences. Therefore, chimeric genes or chimeric constructs can contain regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from those found in nature. "Endogenous genes" refer to naturally occurring genes located in their natural positions within an organism's genome. "Exotic" genes are genes that are not normally found in a host organism but are introduced into it through gene transfer. Exotic genes can include naturally occurring genes or chimeric genes inserted into non-natural organisms.

[0110] The term "gene product" (also referred to herein as "gene expression product" or "expression product") includes products resulting from gene expression, such as RNA transcribed from a gene and polypeptides produced by the translation of such RNA. It should be understood that some gene products may be processed or modified, for example, within the cell. For instance, RNA transcripts may be spliced, polyadenylated, etc., before mRNA translation, and / or polypeptides may undergo co-translational or post-translational processing, such as removal of secretion signaling sequences, removal of organelle targeting sequences, or modifications such as phosphorylation, fatty acylation, etc. The term "gene product" encompasses forms of such processing or modification. Genomes, mRNA, and polypeptide sequences from various species, including humans, are known in the art and are available in publicly accessible databases such as the National Center for Biotechnology Information (www.ncbi.nih.gov) or the Universal Protein Resource (www.uniprot.org).

[0111] Databases include, for example, GenBank, RefSeq, Gene, UniProtKB / SwissProt, UniProtKB / Trembl, etc. Generally, sequences (e.g., mRNA and peptide sequences) in the NCBI Reference Sequence Database can be used as gene product sequences of the gene of interest. It should be understood that multiple alleles of a gene can exist among individuals of the same species. For example, differences in one or more nucleotides of the nucleic acid encoding a particular protein (e.g., as many as approximately 1%, 2%, 3% to 5% of nucleotides) may exist among individuals of a given species. Due to the degeneracy of the genetic code, although DNA polymorphisms may exist that lead to changes in the sequence encoding a protein, such variations typically do not alter the encoded amino acid sequence. Examples of polymorphic variants can be found, for example, in the Single Nucleotide Polymorphism Database (dbSNP), which is available from the NCBI website. 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,

[0112] 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 isotypes of certain proteins may exist, for example, due to alternative RNA splicing or editing. Generally, where aspects of this disclosure relate to genes or gene products, unless otherwise indicated, embodiments involving allele variants or isotypes are covered where applicable. Some embodiments may target one or more specific sequences, such as one or more specific alleles or one or more isotypes.

[0113] The term "amino acid" encompasses all known amino acids, which include an amine (-NH2) functional group, a carboxyl (-COOH) functional group, and a side chain ("R") group characteristic of each amino acid. "Amino acid" encompasses the 21 amino acids encoded by the human genome (i.e., proteogenic amino acids), amino acids encoded or produced by bacteria or single-celled organisms, and amino acids of natural origin. For the purposes of this disclosure, unless otherwise stated, the conjugate acid form of amino acids with basic side chains (arginine, lysine, and histidine) or the conjugate base form of amino acids with acidic side chains (aspartic acid and glutamic acid) are substantially the same. "Amino acid" also encompasses derivatives thereof that maintain substantially the same or better activity in enhancing the effects of the compositions of the present invention (e.g., increasing the amount of CFTR protein in the plasma membrane, increasing chloride ion expulsion from cells, and treating cystic fibrosis). Derivatives may be, for example, enantiomers and include both D- and L-forms of amino acids. Derivatives may be derivatives of “natural” or “non-natural” amino acids (e.g., selenocysteine, pyrrolidone, iodinated tyrosine, or leucine, or valine) (e.g., β-amino acids, high-amino acids, proline derivatives, pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, cyclic-substituted tyrosine derivatives, cyclic-substituted phenylalanine derivatives, linear core amino acids, and N-methyl amino acids). Other amino acid derivatives include, but are not limited to, those synthesized by, for example, 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 in the form of 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). Free amino acids are free in solution but may associate with salts or other components in solution.

[0114] 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 term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide may refer to a single protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl, isofarnesyl, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Proteins, peptides, or polypeptides may also be monomolecules or multimolecular complexes. In some embodiments, the protein comprises a homodimer or a heterodimer. A protein, peptide, or polypeptide may simply be a fragment of a naturally occurring protein or peptide. Proteins, peptides, or polypeptides may be naturally occurring, recombinant, synthetic, or any combination thereof. Proteins may contain different domains, such as nucleic acid-binding domains (e.g., the gRNA-binding domain of Cas9 that guides protein binding to a target site) and nucleic acid cleavage domains. In some embodiments, the protein comprises a protein moiety (e.g., an amino acid sequence constituting 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, the protein is complexed or associated with a nucleic acid (e.g., RNA). In some embodiments, the protein comprises a ligand-binding domain. In some embodiments, the protein comprises an active site (e.g., a site of biological or enzymatic activity). In some embodiments, the protein comprises an allosteric site (e.g., a protein site that can bind ligands that may be located away from the active site). Any protein provided herein can 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 for 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.

[0115] "Identity" or "identity percentage" is a measure of how similar the sequences of two or more nucleic acids or polypeptides are. The identity percentage between sequence A of interest and sequence B can be calculated by the following steps: aligning the sequences, allowing gaps to be introduced to maximize identity, determining the number of residues (nucleotides or amino acids) opposite to the same residues, dividing by the minimum of the TGA and TGB (where TGA and TGB are the sum of the number of residues in sequences A and B in the alignment and the number of internal gap sites), and multiplying by 100. When calculating the number of identical residues required to achieve a specific identity percentage, the decimal should be rounded to the nearest integer. Sequences can be aligned using a variety of 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 identity percentages. The algorithm of Karlin and Altschul, modified as described in Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873-5877, 1993 (Karlin and Altschul, Proc Natl Acad Sci USA, 87:22264-2268, 1990), was incorporated into the NBLAST and XBLAST procedures of Altschul et al. [Altschul et al. (1990) J Mol Biol, 215:403-410]. In some implementations, Gapped BLAST is used to obtain gap alignments for comparison purposes, as described by Altschul et al. [Altschul et al. (1997) Nucleic Acids Res, 25:3389-3402]. When using the BLAST and Gapped BLAST procedures, the default parameters of the respective procedures can be used. See the website with the URL www.ncbi.nlm.nih.gov and / or McGinnis, S. and Madden, TL, W20–W25, Nucleic Acids Research, 2004, Vol. 32, Web server issue.Other suitable procedures 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 SysBiol, 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]). The percentage of identity can be evaluated on the evaluation window. In some embodiments, the length of the evaluation window may be 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, such as 100%, of the shortest sequence length of the compared sequences. In some embodiments, the evaluation window 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 implementations, gaps occupy no more than 20%, 10%, 5%, or 1% of the positions in any one or two sequences within the evaluation window.

[0116] A "variant" of a specific polypeptide or polynucleotide has 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). 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 a "fragment." A "fragment" is a continuous portion of a polypeptide or polynucleotide that is shorter than the original polypeptide. In some embodiments, the variant comprises or is composed of a fragment. In some embodiments, the fragment or variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or longer than the length of the original polypeptide or polynucleotide. The fragment may be N-terminal, C-terminal, or an internal fragment. In some embodiments, the variant polypeptide comprises or is composed of at least one domain of the original polypeptide. In some embodiments, the 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 or constitutes at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the original polypeptide or polynucleotide. In some embodiments, the variant polypeptide or polynucleotide comprises or consists of a polypeptide or polynucleotide comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the original polypeptide or polynucleotide. In some embodiments, the sequence of the variant polypeptide comprises or consists of a sequence that differs from the original sequence by N amino acids, where N is any integer representing at most 1%, 2%, 5% or 10% of the number of amino acids in the original polypeptide, and where “amino acid difference” refers to the substitution, insertion or deletion of amino acids. In some embodiments, the substitution is a conservative substitution. Conservative substitutions may be made, for example, based on the similarity of the side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilicity of the residues involved. In some implementations, conservative substitutions can be made according to Table A, wherein amino acids in the same box in the second column and in the same row in the third column can substitute for each other in conservative substitutions. Some conservative substitutions involve replacing an amino acid in the row of the third column corresponding to a box in the second column with an amino acid in the other row of the third column that is in the same box in the second column.

[0117] Table A

[0118]

[0119] In some embodiments, proline (P), cysteine ​​(C), or both are considered to be in separate groups. Within a particular group, certain substitutions may be of particular interest in some embodiments, for example, leucine is replaced by isoleucine (or vice versa), serine is replaced by threonine (or vice versa), or alanine is replaced by glycine (or vice versa).

[0120] In some embodiments, the variant is a bioactive variant, meaning that the variant at least partially retains at least one activity of the original polypeptide or polynucleotide. In some embodiments, the variant at least partially retains one or more, or substantially all, known biologically important activities of the original polypeptide or polynucleotide. Activities may include, for example, catalytic activity, binding activity, the ability to perform or participate in biological structures or processes, etc. In some embodiments, the activity of the variant may 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, up to about 100%, about 125% or about 150% of the activity of the original polypeptide or polynucleotide. In some embodiments, the variant (e.g., a bioactive variant) comprises or consists of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 88%, 99%, 99.5%, or 100% of the same as or greater than the original polypeptide. In some embodiments, changes (e.g., substitutions or deletions) in the functional variant do not alter or omit amino acids or nucleotides known or predicted to be important for activity, such as known or predicted catalytic residues or residues involved in binding substrates or cofactors. The variant can be tested in one or more suitable assays to assess activity.

[0121] As used herein, the term "small molecule" refers to an organic molecule with a mass less than about 2 kilodaltons (kDa). In some embodiments, the small molecule is less than about 1.5 kDa or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Typically, the small molecule has a mass of at least 50 Da. In some embodiments, the small molecule is non-polymerized. In some embodiments, the small molecule is not an amino acid. In some embodiments, the small molecule is not a nucleotide. In some embodiments, the small molecule is not a carbohydrate. In some embodiments, the small molecule contains multiple carbon-carbon bonds and may contain one or more heteroatoms and / or one or more functional groups, such as amine, carbonyl, hydroxyl, or carboxyl groups, which are important for structural interactions with proteins (e.g., hydrogen bonding), and in some embodiments, contains at least two functional groups. Small molecules typically contain one or more cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, optionally substituted with one or more of the aforementioned 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 Federal Regulations (CFR)). The small molecule may also complex with one or more metal atoms and / or metal ions. In this case, the small molecule is also referred to as an "organometallic small molecule." Preferred small molecules are biologically active because they produce biological effects in animals (preferably mammals, 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, although not required, a drug is one that is deemed safe and effective in humans or animals by an appropriate government or regulatory agency. For example, drugs approved for human use are listed by the FDA in 21 CFR § 330.5, 331 to 361, and 440 to 460, which are incorporated herein by reference. According to the invention, all listed drugs are considered acceptable.

[0122] The terms “composition” and “formulation” are used interchangeably.

[0123] The term “administer (administering or administration)” means to implant, absorb, ingest, inject, inhale or otherwise introduce the compound or a combination thereof described herein into or onto a subject.

[0124] In various embodiments, the “subject” can be any vertebrate. The subject can be an individual to whom an agent is administered, for example, for experimental, diagnostic, and / or therapeutic purposes, or from whom a sample is obtained, or on whom a procedure is performed. In some embodiments, the subject is a mammal, such as a human, a non-human primate, or a rodent (e.g., a mouse, rat, rabbit). Mammal species that may benefit from the disclosed treatment methods include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domesticated animals, such as dogs, cats; livestock, such as horses, cattle, pigs, sheep, goats, chickens; and animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a human. A human can be of either sex and may be at any stage of development. In some embodiments, the subject has been diagnosed with cystic fibrosis. In some embodiments, the subject has been diagnosed with cystic fibrosis caused by a CFTR mutation. In some embodiments, the subject has been diagnosed with cystic fibrosis caused by a Phe508delCFTR mutation.

[0125] Animal models of human CF are known in the art and are described herein. See, for example, 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 2016, Vol. 5258727, p. 14), the entire contents of which are incorporated herein by reference. For example, Lavelle et al. describe mouse and pig models of CF.

[0126] As used herein in the context of treating a subject, the terms “treatment” and similar terms refer to the provision of medical and / or surgical care to the subject. Treatment may include, but is not limited to, the administration of agents or compositions (e.g., pharmaceutical compositions) to the subject. As used herein, the term “treatment” or any grammatical variations thereof (e.g., treatment, treating, and treatment) includes, but is not limited to, the relief of symptoms of a disease or condition; and / or the reduction, suppression, inhibition, mitigation, or influence of the progression, severity, and / or extent of a disease or condition.

[0127] Therapeutic effects may also include reducing the likelihood of disease onset or recurrence, or alleviating one or more symptoms or manifestations of the disease. Therapeutic agents may be administered to subjects who have the disease or who are at increased risk of developing the disease relative to members of the general population. In some embodiments, therapeutic agents may be administered to subjects who already have the disease but no longer show evidence of it. The agent may be administered, for example, to reduce the likelihood of disease recurrence. The agent may be administered preventively, i.e., before any symptoms or manifestations of the disease develop.

[0128] "Preventive treatment" refers to providing medical and / or surgical management to subjects who have not developed a disease or have not shown evidence of disease, in order to, for example, reduce the likelihood of developing a disease or reduce the severity of a disease. Subjects may have been identified as being at risk of developing a disease (e.g., at an increased risk relative to the general population, or having risk factors that increase the likelihood of developing a disease).

[0129] As used herein, the term “improvement” or any grammatical variations thereof (e.g., improvement (ameliorate, ameliorating, and amelioration)) includes, but is not limited to, delaying the onset of a disease or symptom (e.g., cystic fibrosis or its complications) or reducing the severity of said disease or symptom. As used herein, improvement does not necessarily mean the complete absence of symptoms.

[0130] The terms “symptoms,” “disease,” and “symptoms” are used interchangeably.

[0131] The term "effective amount" of the compounds described herein refers to an amount sufficient to elicit the desired biological response. In some embodiments, the effective amount is an amount sufficient to increase chloride ion transport. In some embodiments, the effective amount is an amount sufficient to modulate (e.g., enhance) the function of cystic fibrosis transmembrane conductance regulator (CFTR) proteins (e.g., wild-type CFTR or mutant CFTR). In some embodiments, the effective amount is an amount sufficient to modulate (e.g., enhance) the function of Phe508del CFTR. In some embodiments, the effective amount is an amount that increases the translocation of CFTR from the cytoplasm to the plasma membrane. The effective amount of the compounds described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the route of administration, and the age and health of the subject. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is for prophylactic treatment. In some embodiments, the effective amount is the amount of the compounds described herein in a single dose. In some embodiments, the effective amount is a combined amount of the compounds described herein in multiple doses.

[0132] The "therapeuticly effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a symptom or sufficient to delay or minimize one or more symptoms associated with the symptom. A therapeutically effective amount of a compound means an amount that provides a therapeutic benefit in the treatment of a symptom, either alone or in combination with other therapies. The term "therapeuticly effective amount" may include amounts that improve overall therapy, reduce or avoid symptoms, signs, or causes of the symptom, and / or enhance the therapeutic efficacy of another therapeutic agent. In some embodiments, a therapeutically effective amount is an amount sufficient to increase chloride ion transport (e.g., increase chloride excretion from epithelial cells). In some embodiments, a therapeutically effective amount is an amount sufficient to increase water transport (e.g., increase water excretion from epithelial cells). In some embodiments, a therapeutically effective amount is an amount sufficient to modulate (e.g., enhance) the function of cystic fibrosis transmembrane conductance regulator (CFTR) proteins (e.g., wild-type CFTR or mutant CFTR). In some embodiments, a therapeutically effective amount is an amount sufficient to modulate (e.g., enhance) the function of Phe508del CFTR. In some embodiments, the therapeutically effective amount is the amount that increases the translocation of CFTR from the cytoplasm to the plasma membrane. In some embodiments, the therapeutically effective amount is an amount sufficient to treat lung disease. In some embodiments, the therapeutically effective amount is an amount sufficient to treat cystic fibrosis.

[0133] As used herein, the term “salt” means any and all salts, including pharmaceutically acceptable salts.

[0134] The term "carrier" can refer to any diluent, adjuvant, excipient, or medium applied with the compositions disclosed herein. Examples of suitable drug carriers are described in Remington's Essentials of Pharmaceuticals, 21st edition, edited by Felton, 2012, which is incorporated herein by reference.

[0135] 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.

[0136] Pharmaceutically acceptable excipients used to manufacture the provided pharmaceutical compositions include inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents and aromatizers may also be present in the composition. The exact amount of the composition containing the amino acids required to achieve an effective amount will vary from subject to subject, depending on factors such as the subject's species, age and general condition, severity of side effects or illness, characteristics of the particular compound, route of administration, etc. An effective amount may be included in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In some embodiments, when multiple doses are administered to a subject or applied to tissues or cells, any two doses comprise different or substantially the same amount of the compound described herein. In some embodiments, when multiple doses are administered to a subject or applied to tissues or cells, the frequency of administering multiple doses to the subject or applying multiple doses to tissues or cells is three doses per day, two doses per day, one dose per day, one dose every other day, one dose every three days, one dose per week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to tissues or cells is one dose per day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to tissues or cells is two doses per day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to tissues or cells is three doses per day. In some embodiments, when multiple doses are administered to a subject or applied to tissues or cells, the duration between the first and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifespan of the subject, tissue, or cell. In some embodiments, the duration between the first and last doses of the multiple doses is three months, six months, or one year. In some embodiments, the duration between the first and last doses of the multiple doses is the lifespan of a subject, tissue, or cell. In some embodiments, the doses described herein (e.g., a single dose or any of multiple doses) independently comprise a composition containing the amino acids described herein in amounts between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g (inclusive).In some embodiments, the dosage described herein independently comprises a composition containing the amino acids described herein, ranging from 1 mg to 3 mg (inclusive). In some embodiments, the dosage described herein independently comprises a composition containing the amino acids described herein, ranging from 3 mg to 10 mg (inclusive). In some embodiments, the dosage described herein independently comprises a composition containing the amino acids described herein, ranging from 10 mg to 30 mg (inclusive). In some embodiments, the dosage described herein independently comprises a composition containing the amino acids described herein, ranging from 30 mg to 100 mg (inclusive).

[0137] The dosage ranges described herein provide guidance for administering the provided pharmaceutical compositions to adults. The dosage administered, for example, to children or adolescents, may be determined by a medical practitioner or person skilled in the art and may be less than or equal to the dosage administered to adults.

[0138] The composition may be administered concurrently with, before, or after the administration of one or more other pharmaceutical or therapeutic agents that may be used as, for example, in combination therapy. Pharmaceutical agents include therapeutically active agents.

[0139] The agent also includes a preventative active agent. The agent includes small organic molecules, such as pharmaceutical compounds (e.g., compounds approved by the U.S. Food and Drug Administration for human or veterinary use as provided in Federal Regulations (CFR), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In some embodiments, additional agents are those that can be used to treat and / or prevent diseases (e.g., proliferative diseases, lung diseases, gastrointestinal diseases, blood diseases, neurological diseases, pain symptoms, mental illnesses, or metabolic disorders). In some embodiments, additional therapeutic agents are those that can be used to treat lung diseases.

[0140] In some embodiments, the additional therapeutic agent is an agent that can be used to treat cystic fibrosis. In some embodiments, the agent that can be used to treat cystic fibrosis may be ivacathotoxin. Rumacato Ataluren or tezacator. In some embodiments, the additional therapeutic agent is ivacator. In some embodiments, the additional therapeutic agent is rumacator. In some embodiments, the additional therapeutic agent is an agent that can be used to treat cystic fibrosis, manage symptoms associated with cystic fibrosis, or treat diseases or infections (e.g., bacterial infections, viral infections, bronchiolitis, asthma, etc.) occurring concurrently with cystic fibrosis. Other therapeutic agents that can be used for the purposes of this disclosure include, but are not limited to, systemic dornase alfa systemic (Pulmozyme), azithromycin (e.g., Zithromax, Zmax), aztreonam (Cayston, Azactam), and tobramycin (e.g., Nebcin, Bethkis, Kitabis Pak, TOBIPodhaler), amikacin, pancreatic lipases (Creon, Zenpep, Pancreaze, Viokase, Pertzye, Ultresa, Pangestyme EC, Panocaps), gentamicin, and pancreatic enzymes. Each additional agent may be administered at a dose and / or schedule determined for that agent. Additional agents may also be administered together with each other and / or with the compounds or compositions described herein, either in a single dose or individually at different doses. Specific combinations employed in the regimen will take into account the compatibility of the compounds described herein with one or more other agents and / or the desired therapeutic and / or preventative effects to be achieved. Generally, it is desirable to use one or more other agents in combination at levels not exceeding those used alone. In some embodiments, the combined level will be lower than the level used alone. In some embodiments, the compounds or pharmaceutical compositions described herein may be administered in combination with anticancer therapies, including but not limited to surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.

[0141] 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 related symptoms, including: bronchiectasis, bronchitis, asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, and pulmonary fibrosis.

[0142] This disclosure also includes kits (e.g., pharmaceutical kits). The provided kits may include the pharmaceutical compositions or compounds described herein and containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser packages or other suitable containers). In some embodiments, the provided kits may optionally include a second container containing pharmaceutical excipients for diluting or suspending the pharmaceutical compositions or compounds described herein. In some embodiments, the pharmaceutical compositions or compounds described herein provided in the first and second containers are combined to form a unit dosage form. In some embodiments, the kits described herein also include instructions for using the kit. Detailed Implementation

[0143] Cystic fibrosis (CF) is the most common fatal recessive genetic disorder in individuals of European descent, affecting 1 in 2,500 to 1 in 3,500 newborns annually. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to reduced or absent CFTR synthesis, protein misfolding, and / or channel dysfunction. This results in decreased chloride secretion, increased sodium uptake (ENaC), and impaired fluid homeostasis in the airway, intestinal, and pancreatic epithelial cells. The CFTR gene is expressed in multiple tissues. CF is characterized by multisystem pathology, including the respiratory, gastrointestinal, pancreatic, and hepatobiliary systems. Primary morbidity and mortality are associated with chronic lung infections and inflammation.

[0144] CF mutations are classified as follows: Class I (22%) = no CFTR expression; Class II (88%) = CFTR protein misfolding; Class III (6%) = CFTR channel not open; Class IV (6%) = CFTR channel malfunction; Class V (5%) = too little CFTR. Mutations can also span more than one class. Drugs such as rumacapto are used to address Class II mutations (folding); drugs such as tezacotto or ivacapotto are 'synergists' used to address Class III through V mutations.

[0145] 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 the CFTRΔF508 mutation. The inventors have identified a combination of selected amino acids (AAs) that stimulates chloride secretion and reduces ENaC activity, thereby increasing apical chloride channel activity and reducing sodium uptake at the apical membrane. Therefore, the selected AA combination and its composition are presented as therapeutic agents for treating subjects with CF, alone and in combination with other CF therapeutic agents, such as rumamacalorie, tezacotto, and / or ivacalorie. This document also covers the use of the selected AA combination for treating CF or in the preparation of pharmaceutical agents for treating CF, wherein said use is alone and / or in combination with other CF therapeutic agents, such as rumamacalorie, tezacotto, and / or ivacalorie.

[0146] As described herein, the inventors have experimentally compared fully differentiated primary normal human bronchial epithelial cells with homozygous CFTRΔF508 (type II mutation) human bronchial epithelial cells. Briefly, the transepithelial short-circuit current (Ig) was measured in a Ussing chamber. sc ), resistance (R) and 22 Na and 36 unidirectional flux of Cl (J) ms &J sm ) and net flux (J net When CF cells were stimulated with saliva and GLPG1837 (an synergist for ivacato) in the absence of CF-5AA-3 (an amino acid preparation used to treat CF) (control) or in the presence of CF-5AA-3 (control), the peak current ( Figure 7E ) and chlorine secretion ( Figure 7F The effect was significantly improved in the presence of CF-5AA-3 (also referred to herein as CF5AA-3). For further details of the study, see Example 2 below.

[0147] In example Figures 5 to 9 The results presented indicate that, by correcting and / or modifying plasma membrane channel function, the selected amino acid combination (e.g., CF-5AA-3) improved dysfunctional chloride and sodium channel activity in fully differentiated primary normal human bronchial epithelial cells (HBECs) (homozygous for CFTRΔF508). Therefore, the selected amino acid formulation (e.g., CF-5AA-3) is proposed to supplement the existing standard of care for patients with the CFTRΔF508 mutation.

[0148] In the presence of the corrector (C18; VX661, tezacator) and synergist (GLPG1837, a substitute for ivacato), the peak current was more than double the baseline, but still lower than the base peak current of CF-5AA-3 when used alone. Adding the same triple mixture to CF-5AA-3 increased the current by another 26% compared to CF-5AA-3 alone. Figure 10A and Figure 10B ).

[0149] Furthermore, it was further demonstrated that CF-5AA-3 significantly increased chlorine flux compared to the triple mixture due to its direct effect on CFTR and ANO1 channels. Figure 11 CF-5AA-3 showed minimal effect on ENaC. For further details of the study, see Example 2 below.

[0150] In summary, in vitro electrophysiological studies using primary human bronchial epithelial cells with the CFTRΔF508 mutation (class II) showed that, similar to common correctors and enhancers [column AA DMSO BASAL versus column C18+GLPG], CF-5AA-3 alone quantitatively increased chloride secretion (negative flux) in F508del human bronchial cells, and when combined with commonly used correctors (C18) and enhancers (GLPG) alone, CF-5AA-3 doubled chloride secretion (negative flux) compared to correctors and enhancers alone.

[0151] The results presented in this study indicate that other chloride channels (e.g., SLC26A9) may also be active in HBECs. The activation of chloride channels other than CFTR suggests that CF-5AA-3 can alleviate all five CF class mutations by inducing chloride secretion in a CFTR-independent manner. This is particularly noteworthy for subjects with CFTR class I mutations who currently have no drug treatment available.

[0152] Compositions containing amino acids that increase CFTR translocation to the plasma membrane

[0153] The compositions described herein increase the translocation of both wild-type and mutant CFTR proteins from the cytoplasm to the plasma membrane. Specifically, the compositions described herein are particularly effective in increasing the amount of Phe508del CFTR protein on the plasma membrane, leading to increased secretion of chloride ions and water by epithelial cells, thereby combating the formation of thick mucus (a hallmark of cystic fibrosis).

[0154] In one aspect, the composition comprises, is substantially composed of, or is composed of: one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the composition further comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In another aspect, the composition comprises, is substantially composed 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 pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In another aspect, the composition comprises, is substantially composed 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 pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0155] In one aspect, the composition comprises, is substantially composed of, or is composed of: one or more free amino acids selected from threonine and lysine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids threonine and lysine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids threonine and lysine. In some embodiments, the composition also comprises, is substantially composed of, or is composed of, one or more free amino acids selected from threonine and lysine.

[0156] In one aspect, the composition comprises, is substantially composed of, or is composed of: one or more free amino acids selected from cysteine, proline, and valine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, and valine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, and valine. In some embodiments, the composition further comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, and valine. On the other hand, the composition comprises, is substantially composed of, or consists of two or more or all three free amino acids selected from cysteine, proline, and valine; and optionally a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0157] In one aspect, the composition comprises, is substantially composed of, or is composed of: one or more free amino acids selected from cysteine, proline, glycine, and valine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, glycine, and valine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, glycine, and valine. In some embodiments, the composition further comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, glycine, and valine. On the other hand, the composition comprises, is substantially composed 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 pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0158] In one aspect, the composition comprises, is substantially composed of, or is composed of: one or more free amino acids selected from cysteine, proline, valine, and tyrosine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, valine, and tyrosine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids cysteine, proline, valine, and tyrosine. In some embodiments, the composition further comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, valine, and tyrosine. On the other hand, the composition comprises, is substantially composed 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 pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0159] In one aspect, the composition comprises, is substantially composed of, or is composed of: one or more free amino acids selected from glycine, leucine, and lysine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids glycine, leucine, and lysine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids glycine, leucine, and lysine. In some embodiments, the composition further comprises, is substantially composed of, or is composed of, one or more free amino acids selected from glycine, leucine, and lysine. On the other hand, the composition comprises, is substantially composed of, or consists of two or more or all three free amino acids selected from glycine, leucine, and lysine; and optionally a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0160] In one aspect, the composition comprises, is substantially composed of, or is composed of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine; and optionally, a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In some embodiments, the composition is substantially composed of, or is composed only of, the specified free amino acids, and contains no other free amino acids, or contains negligible amounts of other free amino acids. In some embodiments, the composition comprises an amino acid derivative as a “natural” or “non-natural” amino acid derivative. In some embodiments, the composition comprises a salt of an amino acid and / or a prodrug. In some embodiments, the composition comprises, is substantially composed of, or is composed of, the free amino acids glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In some embodiments, the composition further comprises, is substantially composed of, or is composed of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In another embodiment, the composition comprises, is substantially composed of, or is composed 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 pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, and / or additional therapeutic agent.

[0161] Table B below provides non-limiting examples of compositions containing amino acids that increase CFTR translocation to the plasma membrane.

[0162] Table B: Compositions containing amino acids

[0163]

[0164]

[0165] If present in the composition, each free amino acid may be present, for example, in the following concentrations: proline at about 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 0.9 to about 1.1 g / L, or about 1.5 to about 1.7 g / L; glutamic acid at about 0.7 to about 1.7 g / L, about 0.9 to about 1.5 g / L, about 1.1 to about 1.3 g / L, or about 1.5 g / L.

[0166] From about 1.7 g / L; glutamine from about 0.6 to about 1.6 g / L, from about 0.8 to about 1.4 g / L, from about 1.0 to about 1.2 g / L, or from about 1.5 to about 1.7 g / L; leucine from about 0.05 to about 0.4 g / L, from about 0.1 to about 0.3 g / L, or from about 1.5 to about 1.7 g / L; alanine from about 0.4 to about 1.5 g / L, from about 0.7 to about 1.3 g / L, from about 1.1 to about 2.1 g / L, or from about 1.3 to about 1. 9 g / L or about 1.5 to about 1.7 g / L; aspartic acid about 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 1.1 to about 2.1 g / L, about 1.3 to about 1.9 g / L or about 1.5 to about 1.7 g / L; phenylalanine about 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 1.1 to about 2.1 g / L, about 1.3 to about 1.9 g / L or about 1.5 to about 1.7 g / L; histidine 0 g / L. 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 1.1 to about 2.1 g / L, about 1.3 to about 1.9 g / L, or about 1.5 to about 1.7 g / L; threonine is 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 0.9 to about 1.1 g / L, or about 1.5 to about 1.7 g / L; isoleucine is 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 1.1 to about 2.1 g / L, or about 1.5 to about 1.7 g / L. 1.3 to about 1.9 g / L or about 1.5 to about 1.7 g / L; asparagine 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 1.1 to about 2.1 g / L, about 1.3 to about 1.9 g / L or about 1.5 to about 1.7 g / L; tryptophan 0.4 to about 1.5 g / L, about 0.7 to about 1.3 g / L, about 1.1 to about 2.1 g / L, about 1.3 to about 1.9 g / L or about 1.5 to about 1.7 g / L.

[0167] In some embodiments, the amino acids in the composition are free amino acids. In some embodiments, the amino acids in the composition are L-amino acids. In some embodiments, the amino acids in the composition are D-amino acids. In some embodiments, the amino acids in the composition are a combination of D-amino acids and L-amino acids.

[0168] In some embodiments, the amino acids in the compositions described herein may be prodrugs containing free amino acids. The term "prodrug" refers to a compound having a cleavable group and being converted into the compound described herein (which has pharmaceutical activity in vivo) by solvent decomposition or under physiological conditions.

[0169] In some embodiments, the amino acids in the compositions described herein may be salts of amino acids (i.e., amino acid salts). Amino acids may be in salt form with: cations (e.g., amino acid salts having negatively charged side chains in solution (e.g., glutamic acid and aspartic acid)), anions (amino acid salts having positively charged side chains in solution (e.g., lysine, arginine, histidine)), and inorganic compounds. Exemplary amino acid salts are listed in Fleck M and Petrosyan AM, Salts of AminoAcids, 1st edition; Springer International Publishing, 2014, which are incorporated herein by reference.

[0170] In some embodiments, the composition also contains water.

[0171] In some embodiments, the composition further comprises a buffer. Exemplary buffers include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium acetopropionate, valeric acid, dicalcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

[0172] In some embodiments, phosphate ions (such as H₂PO₄⁻, HPO₄²⁻, and PO₄³⁻) are used to buffer the compositions of the present invention. In some embodiments, the therapeutic compositions use HCO₃⁻ or CO₃²⁻ as buffers. In other embodiments, the therapeutic compositions do not use HCO₃⁻ or CO₃²⁻ as buffers.

[0173] In some embodiments, the composition comprises one or more electrolytes selected from, for example, the following: Na+; K+; HCO3-; CO32-; Ca2+; Mg2+; Fe2+; Cl-; phosphate ions such as H2PO4-, HPO42-, and PO43-; zinc; iodine; copper; iron; selenium; chromium; and molybdenum. In an alternative embodiment, the composition does not contain HCO3- or CO32-. In another alternative embodiment, the composition comprises HCO3- and CO32- in a total concentration of less than 5 mg / L or a concentration of less than 5 mg / L. 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 does not contain one or more of the following: Na+; K+; HCO3-; CO32-; Ca2+; Mg2+; Fe2+; Cl-; phosphate ions such as H2PO4-, HPO42-, and PO43-; zinc; iodine; copper; iron; selenium; chromium; and molybdenum.

[0174] In some embodiments, the composition further comprises sugar, vitamin, electrolyte, mineral, protein, or lipid. In some embodiments, the composition further comprises sugar. In some embodiments, the composition further comprises vitamin. In some embodiments, the composition further comprises electrolyte. In some embodiments, the composition further comprises mineral. In some embodiments, the composition further comprises protein. In some embodiments, the composition further comprises lipid.

[0175] In some embodiments, the composition does not contain one or more of the following ingredients: oligosaccharides, polysaccharides and carbohydrates; oligopeptides or polypeptides or proteins; lipids; small-chain fatty acids, medium-chain fatty acids and / or long-chain fatty acids; and / or foods containing one or more of the above nutrients.

[0176] The composition may have a pH ranging from about 2.5 to about 8.5. In some embodiments, the pH range of the composition is from about 2.5 to about 6.5, from about 3.0 to about 6.0, from about 3.5 to about 5.5, from about 3.9 to about 5.0, or from about 4.2 to about 4.6. In other embodiments, the pH range of the composition is from about 6.5 to about 8.5, from about 7.0 to about 8.0, or from about 7.2 to about 7.8. In some embodiments, the composition has a pH of, for example, from about 2.5 to about 8.5. In some embodiments, the pH of the composition is from about 2.5 to about 6.5, from about 2.5 to about 6.0, from about 3.0 to about 6.0, from about 3.5 to about 6.0, from about 3.9 to about 6.0, from about 4.2 to about 6.0, from about 3.5 to about 5.5, from about 3.9 to about 5.0, or from 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.

[0177] In some embodiments, the total osmotic pressure of the composition is from about 100 mosm to 280 mosm or any value between therewith. In some embodiments, the total osmotic pressure is from about 150 mosm to 260 mosm. In another embodiment, the total osmotic pressure of the composition is any value below 280 mosm.

[0178] In some embodiments, the composition is sterile.

[0179] The compositions described herein can be prepared by any method known in the field of pharmacology. Generally, such preparation methods involve associating the compounds (i.e., one or more free amino acids) of the compositions described herein with a carrier or excipient and / or one or more other auxiliary components, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single- or multi-dose units.

[0180] The relative amounts of one or more active ingredients, pharmaceutically acceptable excipients, and / or any other ingredients in the pharmaceutical compositions described herein will vary depending on the identity, body type, and / or condition of the treated subject and further depending on the route of administration of the composition. The composition may contain between 0.1% and 100% (by weight) of the active ingredient.

[0181] As described herein, compounds or compositions may be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutic and / or preventative agents). Compounds or compositions may be administered in combination with additional pharmaceutical agents that improve their activity in treating, preventing, reducing the risk of disease development in a subject with a need, and / or increasing CFTR activity in a subject or cells (e.g., activity (e.g., potency and / or efficacy), improving bioavailability, improving safety, reducing resistance, reducing and / or regulating metabolism, inhibiting excretion, and / or altering distribution in a subject or cells). It should also be understood that the treatments employed may achieve the desired effect for the same condition, and / or may achieve different effects. In some embodiments, pharmaceutical compositions comprising the compounds described herein and additional pharmaceutical agents exhibit a synergistic effect not present in pharmaceutical compositions comprising one, but not both, of the compounds and additional pharmaceutical agents.

[0182] The compositions described herein may be administered concurrently with, before, or after the administration of one or more additional agents or therapeutics that are different from the compounds or compositions and suitable for, for example, combination therapy. Agents include therapeutic agents. Agents also include preventative agents. Agents include small organic molecules, such as pharmaceutical compounds (e.g., compounds approved by the U.S. Food and Drug Administration for human or veterinary use as provided in Federal Regulations (CFR), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In some embodiments, additional agents are agents that can be used to treat and / or prevent diseases (e.g., proliferative diseases, blood disorders, neurological diseases, painful symptoms, mental illnesses, lung diseases, or metabolic disorders). In some embodiments, additional agents can be used to treat lung diseases. In some embodiments, additional agents can be used to treat cystic fibrosis. Each additional agent may be administered at a dose and / or schedule determined for that agent. Additional agents may also be administered together with each other and / or with the compounds or compositions described herein, either in a single dose or individually at different doses. Specific combinations employed in the regimen will take into account the compatibility of the compounds described herein with one or more other agents and / or the desired therapeutic and / or preventative effects to be achieved. Generally, it is desirable to use one or more other agents in combination at levels not exceeding those achieved when used alone. In some embodiments, the combined level will be lower than the level achieved when used alone.

[0183] In some embodiments, the composition is administered concurrently with, before, or after the administration of one or more additional therapeutic agents, wherein the additional therapeutic agents are CFTR potentiators, correctors, or readthrough agents. A “CFTR potentiator” is a compound that enhances the function of CFTR on the cell membrane. These compounds can be used to treat cystic fibrosis mutations that result in the proper transport of CFTR protein to the cell membrane but impaired function (e.g., channel gating defects, reduced or absent ATP binding, and / or reduced chloride transport). In some embodiments, the CFTR potentiator is ivacartocin (VX-770). "CFTR corrector" is a compound that improves the intracellular processing and translocation of mutant CFTR, thereby allowing more protein to reach the plasma membrane. In some embodiments, the CFTR corrector is rumacotto (VX-809). In some implementations, the CFTR corrector is tezacotto (VX-661). A “CFTR readthrough” is a compound that promotes transcription in the presence of a premature stop codon (PTC) mutation caused by a point mutation in the CFTR gene sequence.

[0184] Typically, PTC causes truncation of the CFTR protein, which cannot be properly processed and / or malfunctions (e.g., channel gating defects, reduced or absent ATP binding, and / or reduced chloride transport). In some embodiments, the CFTR readthrough agent is atalulren (TRANSLARNA™). In some embodiments, a composition containing the amino acids described herein is administered in combination with ivacato. In some embodiments, a composition containing the amino acids described herein is administered in combination with rumacaprol. In some embodiments, a composition containing the amino acids described herein is administered in combination with atalulren. In some embodiments, a composition containing the amino acids described herein is administered in combination with both ivacato and rumacaprol. In some embodiments, a composition containing the amino acids described herein is administered in combination with both ivacato and atalulren. In some embodiments, a composition containing the amino acids described herein is administered in combination with both rumacaprol and atalulren. In some embodiments, the composition is administered alone, i.e., not simultaneously, before, or after the administration of one or more other agents or therapeutics. In some embodiments, the composition is applied in the absence of CFTR synergists, correctors, or readability agents.

[0185] 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, pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and / or the following substances: (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption enhancers, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin and bentonite; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include buffers.

[0186] In some embodiments, compositions comprising the amino acids described herein may be provided in powder form and reconstituted for administration to a subject. The pharmaceutical compositions described herein may be prepared, packaged, and / or sold in formulations suitable for administration via the buccal cavity to the lungs. Such formulations may comprise dry particles containing the active ingredient and having a diameter ranging from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are conveniently presented in dry powder form for administration using devices including dry powder reservoirs (in which a propellant flow can be directed to disperse the powder) and / or using self-propelled solvent / powder dispensing containers (such as devices containing the active ingredient dissolved and / or suspended in a low-boiling-point propellant in a sealed container). Such powders comprise particles in which at least 98% by weight of the particles have a diameter greater than 0.5 nm, and at least 95% by number of the particles have a diameter less than 7 nm. Alternatively, at least 95% by weight of the particles have a diameter greater than 1 nm, and at least 90% by number of the particles have a diameter less than 6 nm. Dry powder compositions may include solid fine powder diluents, such as sugar, and are conveniently provided in unit dosage forms.

[0187] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters and mixtures thereof of sorbitol. In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers. In some embodiments for parenteral administration, the conjugates described herein are combined with solubilizers (such as...) Mixtures of alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and mixtures thereof.

[0188] The pharmaceutical compositions described herein, formulated for pulmonary delivery, may deliver the active ingredient in the form of solutions and / or suspension droplets. Such formulations may be prepared, packaged, and / or sold as optional sterile aqueous and / or dilute alcoholic solutions and / or suspensions containing the active ingredient, and may be conveniently administered using any spray and / or nebulizer. Such formulations may also contain 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 methylparaben). The average diameter of the droplets delivered via this route of administration may range from about 0.1 nanometers to about 200 nanometers.

[0189] The formulations described herein that can be used for lung delivery can also be used 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 micrometers to 500 micrometers. This formulation is administered by rapid inhalation through the nasal passage from a powder container held near the nostrils.

[0190] Formulations for nasal application may, for example, contain from as low as about 0.1% (by weight) to as high as 100% (by weight) of the active ingredient, and may contain one or more of the additional ingredients described herein. The pharmaceutical compositions described herein may be prepared, packaged, and / or sold in buccal formulations. Such formulations may be in the form of tablets and / or lozenges, for example, prepared using conventional methods, and may contain, for example, 0.1% to 20% (by weight) of the active ingredient, the remainder comprising an orally soluble and / or biodegradable composition and optionally one or more of the additional ingredients described herein. Alternatively, formulations for buccal application may comprise powders and / or aerosols and / or nebulized solutions and / or suspensions containing the active ingredient. When dispersed, the average particle size and / or droplet size of such powders, aerosols, and / or nebulized formulations may be in the range of about 0.1 nanometers to about 200 nanometers, and may also contain one or more of the additional ingredients described herein.

[0191] Methods for increasing CFTR translocation to the plasma membrane

[0192] This disclosure provides compositions for increasing the translocation of cystic fibrosis transmembrane conductance regulator (CFTR) protein from the cytoplasm to the plasma membrane. Generally, without being bound by any particular theory, mutations in CFTR proteins (e.g., Phe508del) disrupt proper processing, folding, and / or transport to the plasma membrane, resulting in a reduction in the amount of CFTR protein on the plasma membrane and impaired chloride ion transport in cells expressing mutant CFTR proteins. Impaired chloride ion transport leads to an osmotic imbalance, preventing water from being drawn out of cells expressing mutant CFTR proteins via osmosis due to abnormally high intracellular chloride ion concentrations. This results in the formation of a thick mucus layer covering the cell, a hallmark of cystic fibrosis.

[0193] Therefore, in one aspect, this disclosure provides a method for increasing the amount of CFTR protein on a cell membrane, the method comprising contacting cells with an effective amount of a composition comprising the amino acids disclosed herein. In some embodiments, the cells are epithelial cells. In some embodiments, the epithelial cells are small intestinal epithelial cells or lung epithelial cells. In some embodiments, the epithelial cells are lung epithelial cells. In some embodiments, the lung epithelial cells are bronchial epithelial cells. The bronchial epithelial cells may be normal human bronchial epithelial cells (NHBE) or diseased human bronchial epithelial cells (DHBE). Diseased human bronchial epithelial cells may be obtained from a donor diagnosed with a lung disease (e.g., asthma, COPD, cystic fibrosis). In some embodiments, the bronchial epithelial cells express wild-type CFTR. In some embodiments, the bronchial epithelial cells express mutant CFTR. In some embodiments, bronchial epithelial cells express both wild-type and mutant CFTR (i.e., one allele of the CFTR gene encodes wild-type CFTR, while the second allele contains a mutation (e.g., Phe508delCFTR)). In some embodiments, bronchial epithelial cells express only the Phe508del mutant CFTR. The cells may be present in vitro, in vivo, or ex vivo.

[0194] In some embodiments, the amount of wild-type CFTR protein on the plasma membrane is increased. In some embodiments, the amount of mutant CFTR protein on the plasma membrane is increased. In some embodiments, the amount of Gly542X mutant CFTR protein on the plasma membrane is increased, where X is any amino acid. In some embodiments, the amount of Gly551Asp mutant CFTR protein on the plasma membrane is increased. In some embodiments, the amount of Arg553X mutant CFTR protein on the plasma membrane is increased, where X is any amino acid. In some embodiments, the amount of Arg117His mutant CFTR protein on the plasma membrane is increased. In some embodiments, the amount of 120del23 mutant CFTR protein on the plasma membrane is increased. In some embodiments, the amount of Phe508del mutant CFTR protein on the plasma membrane is increased. In some embodiments, the amount of CFTR protein on the plasma membrane 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 is increased. In some embodiments, the number of CFTR proteins on the plasma 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 Phe508del CFTR provided by SEQ ID NO:2 is increased.

[0195] In some embodiments, this disclosure provides a method for increasing the amount of CFTR protein on a cell membrane, the method comprising contacting a cell with an effective amount of the composition described herein. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from threonine and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, and valine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, glycine, and valine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, valine, and tyrosine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from glycine, leucine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In another embodiment, the composition comprises, is substantially composed of, or is composed of the following: free amino acids cysteine ​​and proline, and at least one other free amino acid selected from glycine, tyrosine, and / or lysine; and optionally a buffer, electrolyte, adjuvant, and / or excipient.

[0196] "Negligible amount" means that the presence of amino acids has no effect on the CFTR protein. Alternatively, in some embodiments, even if amino acids are present in the composition, their presence in any amount will not affect the translocation of CFTR to the plasma membrane, chloride ion transport, or the therapeutic effect on subjects requiring such translocation. 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 should be understood that a negligible amount is an amount greater than zero.

[0197] In some embodiments, the compositions of this disclosure comprise one or more free amino acids that are necessary (e.g., essential) for increasing the translocation of CFTR to the plasma membrane. In some embodiments, the compositions of this disclosure may comprise one or more free amino acids that are not necessary for increasing the translocation of CFTR to the plasma membrane (i.e., do not directly stabilize CFTR), but rather provide alternative beneficial properties of the composition (e.g., maintaining a specific pH or osmotic pressure).

[0198] In one aspect, a method for increasing the amount of CFTR protein on the cell membrane involves increasing the translocation of CFTR from the cytoplasm of the cell to the cell membrane, said method comprising contacting the cells with an effective amount of a composition containing the amino acids disclosed herein. In one embodiment, said method results in an increase in the amount of CFTR protein on the plasma membrane of the treated cells. The increase in the amount of CFTR protein on the plasma membrane can be determined by comparing cells contacted with a composition containing the amino acids described herein with untreated cells (e.g., control cells). For example, Western blotting can be used to compare the presence and amount of CFTR in membrane vesicles isolated from epithelial cells contacted with a composition containing the amino acids described herein with the amount of CFTR in membrane vesicles isolated from untreated epithelial cells (e.g., control cells). Performing the Western blotting analysis described herein is within the capabilities of those skilled in the art. Other related techniques that can be used to determine the expression level of CFTR protein in a sample include dot blot analysis, immunohistochemistry, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA).

[0199] CFTR protein is an ABC transporter that functions as an ATP-gated ion channel. When activated, CFTR allows chloride ions (Cl-) and other negatively charged ions (such as thiocyanate ([SCN]-)) to flow down their electrochemical gradient (e.g., passive diffusion or passive transport). Mutations in CFTR (e.g., like Gly551X, where X represents any amino acid, such as Gly551Asp) result in a defect in the ion channel gating function of the CFTR protein. The most common CFTR mutation, Phe508del, results in the CFTR protein lacking the codon for phenylalanine 508 and failing to fold or transport properly to the plasma membrane. Therefore, in one aspect, this disclosure provides a method for increasing the amount of CFTR protein on the cell membrane and increasing the transport of chloride ions across the cell membrane (e.g., chloride ion expulsion from the cell), said method comprising contacting the cell with a composition comprising amino acids as described herein.

[0200] Therefore, in one aspect, this disclosure provides a method for increasing chloride ion excretion from cells, the method comprising contacting cells with an effective amount of the composition described herein. In some embodiments, the composition comprises, substantially constitutes, or consists of one or more free amino acids selected from threonine and lysine. In some embodiments, the composition comprises, substantially constitutes, or consists of one or more free amino acids selected from cysteine, proline, and valine. In some embodiments, the composition comprises, substantially constitutes, or consists of one or more free amino acids selected from cysteine, proline, glycine, and valine. In some embodiments, the composition comprises, substantially constitutes, or consists of one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In some embodiments, the composition comprises, substantially constitutes, or consists of one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the composition comprises, substantially constitutes, or consists of one or more free amino acids selected from glycine, leucine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In another embodiment, the composition comprises, is substantially composed of, or is composed of the following: free amino acids cysteine ​​and proline, and at least one other free amino acid selected from glycine, tyrosine, and / or lysine; and optionally a buffer, electrolyte, adjuvant, and / or excipient.

[0201] In some embodiments, chloride efflux from cells is further enhanced by contacting cells with a CFTR potentiator, corrector, or passivator. In some embodiments, chloride efflux from cells is increased by contacting cells with a combination therapy comprising the composition described herein and a CFTR potentiator. In some embodiments, chloride efflux from cells is increased by contacting cells with a combination therapy comprising the composition described herein and a CFTR corrector. In some embodiments, chloride efflux from cells is increased by contacting cells with a combination therapy comprising the composition described herein and a CFTR passivator. In some embodiments, chloride efflux from cells is increased by contacting cells with a combination therapy comprising the composition described herein and ivacastor.

[0202] The flow of ions (such as chloride ions) across membranes is a biological mechanism used to regulate the flow of water across semipermeable membranes (e.g., cell membranes). Osmosis describes the process by which solvent (e.g., water) molecules flow from regions of low solute concentration to regions of higher solute concentration to balance the concentration on each side of the semipermeable membrane (e.g., passive diffusion of water). When the chloride ion concentration in a cell increases due to a malfunction of the CFTR, water molecules do not flow out of the cell and into the surrounding mucus membrane, resulting in the formation of thick mucus. Therefore, in another aspect, this disclosure provides methods and compositions for increasing the outflow of water from cells (e.g., osmosis), said methods comprising contacting cells with a composition comprising amino acids as described herein.

[0203] Therefore, in one aspect, this disclosure provides a method for increasing water excretion from cells, the method comprising contacting cells with a composition described herein. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from threonine and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, and valine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, glycine, and valine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, valine, and tyrosine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, a free amino acid selected from glycine, leucine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In another embodiment, the composition comprises, is substantially composed of, or is composed of the following: free amino acids cysteine ​​and proline, and at least one other free amino acid selected from glycine, tyrosine, and / or lysine; and optionally a buffer, electrolyte, adjuvant, and / or excipient.

[0204] In some embodiments, water excretion from cells is further enhanced by contacting cells with a CFTR potentiator, corrector, or transducer. In some embodiments, water excretion from cells is increased by contacting cells with a combination therapy comprising the composition described herein and a CFTR potentiator. In some embodiments, water excretion from cells is increased by contacting cells with a combination therapy comprising the composition described herein and a CFTR corrector. In some embodiments, water excretion from cells is increased by contacting cells with a combination therapy comprising the composition described herein and a CFTR transducer. In some embodiments, water excretion from cells is increased by contacting cells with a combination therapy comprising the composition described herein and ivacastor.

[0205] Methods for treating cystic fibrosis

[0206] As described above, the flow of chloride ions through cystic fibrosis transmembrane conductance regulator (CFTR) proteins is crucial for maintaining fluid levels in cells and surrounding mucous membranes, particularly the lungs and small intestine. Mutations in CFTR proteins that disrupt CFTR processing, folding, and transport to the plasma membrane (e.g., Phe508del) reduce the number of functional CFTR proteins on the plasma membrane. The biological result is the formation of a thick mucus layer covering the epithelial cell layer, which promotes bacterial growth and prevents epithelial cells from obtaining nutrients from the surrounding fluids. Ultimately, patients carrying one or more CFTR gene mutations may develop respiratory and pulmonary diseases such as cystic fibrosis.

[0207] Subjects may be, for example, individuals with lung disease. In some embodiments, the subject has cystic fibrosis. In some embodiments, the underlying genetic cause of cystic fibrosis may be a Phe508del mutation in one or more alleles of the CFTR gene. Individuals may also suffer from other complications that often occur concurrently with cystic fibrosis, such as bacterial infections, viral infections, asthma, and chronic respiratory failure. Therefore, compositions comprising the amino acids disclosed herein may also be suitable for managing symptoms and other complications in subjects with cystic fibrosis.

[0208] In some embodiments, the methods described herein increase survival in patients with lung diseases (e.g., cystic fibrosis). The methods and compositions described herein can also be used to improve treatment outcomes in patients with cystic fibrosis.

[0209] Therefore, in one aspect, this disclosure provides compositions for treating cystic fibrosis, wherein said compositions are administered to a subject in need of treatment (e.g., a subject with cystic fibrosis). In some embodiments, the subject has cystic fibrosis and the wild-type CFTR of SEQ ID NO:1 is present. In some embodiments, the subject has cystic fibrosis and the CFTR protein is present, having an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to that of the wild-type CFTR provided by SEQ ID NO:1. In some embodiments, the subject has cystic fibrosis and the mutant CFTR is present. In some embodiments, the subject has cystic fibrosis and the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, or Phe508del mutant, where X represents any amino acid. In some embodiments, the subject has cystic fibrosis and both wild-type CFTR and mutant CFTR are present. In some embodiments, the subject has 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 in amino acid sequence to the Phe508del CFTR provided by SEQ ID NO:2. In some embodiments, the subject has cystic fibrosis in which the mutant CFTR is the Phe508del CFTR of SEQ ID NO:2. In some embodiments, the subject has cystic fibrosis in which both the wild-type CFTR of SEQ ID NO:1 and the mutant Phe508del CFTR of SEQ ID NO:2 are present. The compositions described herein may be administered in combination with one or more other therapeutic agents, such as combination therapies, to further enhance the therapeutic benefit of the compositions described herein.

[0210] In another aspect, this disclosure provides a method for treating cystic fibrosis, the method comprising administering the composition described herein to a subject in need. In some embodiments, the composition comprises, is substantially composed of, or is composed of, one or more free amino acids selected from threonine and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, and valine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, valine, and tyrosine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, one or more free amino acids selected from cysteine, proline, glycine, tyrosine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of, one or more free amino acids selected from glycine, leucine, and lysine. In some embodiments, the composition comprises, is substantially composed of, or is composed of one or more free amino acids selected from glycine, leucine, lysine, tyrosine, arginine, and isoleucine. In another embodiment, the composition comprises, is substantially composed of, or is composed of the following: free amino acids cysteine ​​and proline, and at least one other free amino acid selected from glycine, tyrosine, and / or lysine; and optionally a buffer, electrolyte, adjuvant, and / or excipient.

[0211] In some embodiments, the composition further comprises a CFTR synergist, corrector, or readability agent. In some embodiments, the composition further comprises a CFTR synergist. In some embodiments, the composition further comprises a CFTR corrector. In some embodiments, the composition further comprises a CFTR readability agent. In some embodiments, the composition does not further comprise a CFTR synergist, corrector, or readability agent.

[0212] Tezacotto / evaccato and evaccato are indicated for the treatment of patients aged 6 years and older with cystic fibrosis (CF) who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that responds to tezacotto / evaccato based on in vitro data and / or clinical evidence. For patients aged 6–11 years and weighing <30 kg, the typical dose is 50 mg tezacotto + 75 mg evaccato in the morning and 75 mg evaccato 12 hours later. For patients aged 6–11 years and weighing ≥30 kg, or for patients aged 12 years and older, the typical dose is 100 mg tezacotto + 150 mg evaccato in the morning and 150 mg evaccato 12 hours later.

[0213] (Rumacapator / Ivacapator) is indicated for cystic fibrosis (CF) in patients homozygous for the F508del mutation in the CFTR gene. For patients 6 to 11 years of age: 2 tablets every 12 hours (each tablet contains 100 mg of rumamacapator / 125 mg of ivacapator). For patients 12 years of age and older: 2 tablets every 12 hours (each tablet contains 200 mg of rumamacapator / 125 mg of ivacapator).

[0214] Ivacapor is a cystic fibrosis transmembrane conductance regulator (CFTR) enhancer indicated for the treatment of cystic fibrosis (CF) patients 6 months and older who have a mutation in the CFTR gene that responds to ivacapor enhancement based on clinical and / or in vitro assay data. For patients 6 months to <6 years of age, the recommended dose is based on body weight: 5 kg to <7 kg: 25 mg every 12 hours; 7 kg to <14 kg: 50 mg every 12 hours; ≥14 kg: 75 mg every 12 hours.

[0215] Atalulus can be used at a dose of 40 mg / kg / day.

[0216] It should be understood that typical dosages may vary when used in combination with the pharmaceutical formulations described herein. In a particular embodiment of such combination therapy, when administered in combination with the pharmaceutical formulations described herein, for example... The dose of any of the atalulenes may be reduced relative to the dose and / or frequency of administration.

[0217] CFTR gene and its product and CFTR mutants

[0218] As described above, compositions containing amino acids as described herein effectively increase the translocation of CFTR to the plasma membrane. Specifically, compositions containing amino acids as described herein effectively increase the translocation of mutant (e.g., Phe508del) CFTR to the plasma membrane. The compositions result in an increase in the amount of CFTR protein on the plasma membrane, thereby providing a method for increasing chloride ion transport across the epithelial cell membrane to maintain membrane hyperpolarization and transmembrane water transport. These compositions can be used to treat lung diseases (such as cystic fibrosis) or diseases caused by dysregulation of humoral transport in other epithelial cells (such as those in the pancreas and small intestine).

[0219] While sequences of non-human mammalian homologs may be used in some embodiments, the sequences of the CFTR gene products of interest herein typically contain or consist of sequences encoded by or composed of human CFTR genes. Generally, sequences of CFTR proteins or CFTR RNA typically contain or consist of sequences of human CFTR. In some embodiments, the sequences of the gene products of the CFTR gene contain or consist of naturally occurring sequences. It should be understood that a genetic locus may have more than one sequence or allele in an individual population. In some embodiments, the naturally occurring sequence is the standard sequence. Unless otherwise indicated, sequences listed as reference sequences for proteins in the RefSeq database (represented herein by specific names, abbreviations, or symbols) are considered "standard sequences." If a sequence has been updated since the time of this disclosure, the current version or a later version thereof may be used in some embodiments. It should be understood that a genetic locus may have more than one sequence or allele in an individual population. In some embodiments, the naturally occurring sequence differs from the standard sequence at one or more amino acid positions. Naturally occurring polynucleotides or polypeptides that differ from the standard sequence and perform one or more normal functions of the polynucleotide or polypeptide may be referred to as having a "normal sequence."

[0220] The CFTR gene is approximately 189 kb in length and consists of 27 exons and 26 introns. In some embodiments, the CFTR protein is a full-length wild-type CFTR. CFTR can be a mammalian (e.g., human) CFTR. In some embodiments, the sequence of the 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. The biologically active variant of the androgen receptor protein may contain one or more additions, substitutions, and / or deletions relative to the naturally occurring CFTR protein sequence. In some embodiments, the sequence of the CFTR protein comprises the standard CFTR sequence. The full-length CFTR protein is 1480 amino acids in length and has five domains: two transmembrane domains, an intracellular nucleotide-binding domain (NBD) linked to each transmembrane domain, and an intracellular regulatory “R” domain. The full-length wild-type CFTR has the following standard amino acid sequence (GenBank and NCBI Reference Sequence Accession Number: NG_016465.4):

[0221]

[0222]

[0223] In some embodiments, this disclosure provides compositions comprising amino acids that can be used to increase the amount of CFTR protein on the plasma membrane. In some embodiments, the CFTR protein is sequence-identical to at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher (e.g., 100%) the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is sequence-identical to at least 70% the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is sequence-identical to at least 80% the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is sequence-identical to at least 90% the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is sequence-identical to at least 95% the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is sequence-identical to at least 96% the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is at least 97% sequence identical to the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is at least 98% sequence identical to the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is at least 99% sequence identical to the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is at least 99.5% sequence identical to the wild-type CFTR of SEQ ID NO:1. In some embodiments, the CFTR protein is at least 100% sequence identical to the wild-type CFTR of SEQ ID NO:1.

[0224] In some embodiments, the CFTR protein is a variant or fragment of the full-length CFTR of SEQ ID NO:1. The term "variant" also covers splice variants of the CFTR resulting from alternative splicing of the CFTR gene. In some embodiments, the CFTR variant comprises or is composed 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.

[0225] In some embodiments, the CFTR variant comprises or consists of at least 50% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 60% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 70% of the full-length CFTR of SEQ ID NO:1. In some embodiments, 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% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 95% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 96% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 97% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 98% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 99% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 99.5% of the full-length CFTR of SEQ ID NO:1. In some embodiments, the CFTR variant comprises or consists of at least 100% of the full-length CFTR of SEQ ID NO:1. These variants may be bioactive variants of wild-type CFTR, such that the ion channel gating function and / or chloride transport function of the CFTR is retained in the variant. These variants may be bioinactive variants of wild-type CFTR, such that the ion channel gating function and / or chloride transport function of the CFTR is eliminated in the variant (e.g., does not function).

[0226] In some embodiments, the CFTR protein is a mutant CFTR protein, for example, the sequence of the protein includes a sequence in the form of a naturally occurring mutant CFTR. The mutant CFTR can be a mammalian (e.g., human) CFTR mutant. The mutant CFTR can be caused by nonsense frameshift or mRNA splicing mutations. More than 2,000 mutations in the CFTR gene have been identified, many of which are clinically relevant and / or cause disease phenotypes (e.g., cystic fibrosis). See Bobadilla JL et al., 2002, HumanMutation, 19; pp. 575-606, which is incorporated herein by reference, regarding other CFTR gene mutations that demonstrate their usefulness in application to the compositions of this disclosure. In some embodiments, 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 has 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 has a CFTR mutation in at least two alleles of the gene encoding the CFTR protein. In some embodiments, the human subject has the same mutation in at least two alleles of the gene encoding the CFTR protein (e.g., both alleles encode the Phe508del mutant, i.e., homozygous). In some embodiments, the human subject has different mutations in at least two alleles of the gene encoding the CFTR protein (e.g., one allele encodes the Phe508del mutant and one allele encodes the Gly551Asp mutant, i.e., heterozygous). In some embodiments, the cell has 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 cell has a CFTR mutation in at least two alleles of the gene encoding the CFTR protein.In some embodiments, the cells have the same mutation in at least two alleles of the gene encoding the CFTR protein (e.g., both alleles encode the Phe508del mutant, i.e., homozygous). In some embodiments, the cells have different mutations in at least two alleles of the gene encoding the CFTR protein (e.g., one allele encodes the Phe508del mutant and one allele encodes the Gly551Asp mutant, i.e., heterozygous). In some embodiments, the cells are epithelial cells. In some embodiments, the epithelial cells are lung epithelial cells. In some embodiments, the lung epithelial cells are bronchial epithelial cells. In some embodiments, the bronchial epithelial cells are derived from a patient with cystic fibrosis.

[0227] The Phe508del mutant is the most common mutant in patients with cystic fibrosis. The Phe508del mutant is produced by a deletion mutation in which the codon for phenylalanine at amino acid position 508 is missing, resulting in the CFTR protein lacking residue 508 (e.g., a length of 1479 amino acids). The Phe508del CFTR has the following standard amino acid sequence (GenBank and NCBI reference sequence accession number: (NM_000492.3(CFTR):c.1521_1523delCTT):

[0228]

[0229] In some embodiments, the CFTR protein is a mutant CFTR protein, for example, the sequence of the protein includes the sequence of a naturally occurring mutant form of CFTR. In some embodiments, the mutant CFTR protein is sequence-identical to at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher (e.g., 100%) of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is sequence-identical to at least 70% of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is sequence-identical to at least 80% of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is sequence-identical to at least 90% of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is sequence-identical to at least 95% of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 96% sequence identical to Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 97% sequence identical to Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 98% sequence identical to Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 99% sequence identical to Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 99.5% sequence identical to Phe508del CFTR of SEQ ID NO:2. In some embodiments, the mutant CFTR protein is at least 100% sequence identical to Phe508del CFTR of SEQ ID NO:2.

[0230] In some embodiments, the CFTR protein cannot be properly processed by conventional cellular mechanisms in the endoplasmic reticulum (ER). In some embodiments, the CFTR protein fails to fold correctly. In some embodiments, the CFTR protein cannot be properly transported to the plasma membrane (e.g., retained in the cytoplasm or ER). In some embodiments, the CFTR protein exhibits impaired ion channel gating function, i.e., it cannot properly open to allow chloride ions to be transported out of the cell.

[0231] In some embodiments, the CFTR protein is a variant or fragment of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or is composed of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or is composed of at least 50% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or is composed of at least 60% of the polypeptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or is composed 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% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 90% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 95% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 96% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 97% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 98% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variant comprises or consists of at least 99% of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variants comprise or consist of at least 99.5% of or more of the peptide of Phe508del CFTR of SEQ ID NO:2. In some embodiments, the CFTR variants comprise or consist of at least 100% of or more of the peptide of Phe508del CFTR of SEQ ID NO:2. These variants may be bioactive variants of Phe508del CFTR, such that the ion channel gating function and / or chloride transport function of the CFTR is retained in the variant. These variants may be bioinactive variants of Phe508del CFTR, such that the ion channel gating function and / or chloride transport function of the CFTR is eliminated in the variant (e.g., does not function).

[0232] In some embodiments, the mammalian nucleic acid sequence (e.g., a human nucleic acid sequence, such as a human DNA sequence encoding the CFTR protein (e.g., wild-type CFTR, Phe508del CFTR)) may be codon-optimized to increase expression in cells. In some embodiments, the sequence encoding the CFTR protein may be codon-optimized to increase expression in epithelial cells. In some embodiments, the epithelial cells are small intestinal epithelial cells. In some embodiments, the epithelial cells are lung epithelial cells. In some embodiments, the lung epithelial cells are bronchial epithelial cells. The bronchial epithelial cells may be derived from a subject suffering from cystic fibrosis.

[0233] In some embodiments, CFTR protein is provided in purified form. In some embodiments, CFTR protein is provided in the form of cell lysate. In some embodiments, CFTR protein is provided in the form of tissue homogenate.

[0234] Example

[0235] To enable a fuller understanding of the invention described herein, the following embodiments are set forth. The embodiments described herein are provided to illustrate the methods, compositions, and systems provided herein, and are not to be construed as limiting their scope in any way.

[0236] General Experiment

[0237] Us chamber equipment for cell culture: EM-CSYS-8 Us chamber system, P2300 chamber, P2302 slider, VCCMC8 multichannel voltage / current clamp, P2020 electrode and DM MC6 single-channel electrode input module and virtual membrane (Physiologic Instruments, San Diego, CA).

[0238] Electrode: The silver / silver chloride (Ag / AgCl) electrode is placed in the electrode tip containing 4% agar-Ringer's buffer.

[0239] Ringer's solution: 115mM NaCl, 25mM NaHCO3 3- 2.4 mM K₂HPO₄, 0.4 mM KH₂PO₄, 1.2 mM MgCl₂, 1.2 mM CaCl₂, and 20 mM HEPES. The pH was adjusted to 7.4 with NaOH. The osmotic pressure was 290-300 mOsm.

[0240] Example 1

[0241] This article describes the selected amino acids (AAs) or combinations thereof in the formulation, which stimulate chloride secretion and reduce ENaC activity by increasing CFTR and decreasing ENaC expression and activity on the apical membrane. The AA formulation was tested in primary human bronchial epithelial cells (HBECs) with the mutant CFTRΔF508. Cells were obtained from both CF and non-CF patients. Cells were grown to 80% confluence in culture dishes and then transferred to a snapwell permeable insert. Cells were grown to maturity in a permeable cell culture support and differentiated at the air-medium interface for approximately 30 days. Cells were then studied in a Ussen chamber to measure transepithelial currents and resistances.

[0242] Short-circuit current (Isc) and resistance (R) were measured in fully differentiated primary homozygous CFTRΔF508 HBECs cultured at the air-liquid interface for 28 to 42 days after exposure to selected AA formulations (CF3AA, CF4AA-1 / 2 / 4, CF5AA), CF4AA-3 (negative control), and a mediator (control) in a Ussen chamber. Changes in Isc were measured after inhibition of ENaC with 6 μM benzamidar, activation of cAMP-activated CFTR channel activity with 10 μM fenestration and 1 μM PG01 (synergist), blocking of CFTR channels with 20 μM CFTR(inh)-172, and blocking of basal Ca-activated chloride channel (CaCC) activity with 10 μM CaCCinh-A01. Similar experiments were repeated in CFTRΔF508 HBECs treated for 24 hours, followed by experiments with 6 μM C18 (a CFTR misfolding corrector). The expression of CFTR and ENaC proteins in cells was analyzed using Western blotting, and the protein localization was confirmed using immunofluorescence microscopy.

[0243] Figure 1A The graphs (n=4) show the benzamir insensitive current (μA) in the absence of C18 for the control (basal Ringer's) solution and various individual amino acids (AA). Figure 1B A graph (n=4) shows the benzamir insensitive current (μA) of the control (basic Ringer's) solution and various individual AAs in the absence of C18. Benzamir is an ENaC blocker, therefore the benzamir insensitive current represents the current remaining after blocking ENaC. For Figure 1A and Figure 1B The optimal amino acid is selected from a single amino acid based on the Δ change of the selected current.

[0244] Figure 2AThe graph (n=4) shows the benzamir insensitive current (μA) in the absence of C18 for the control (basic Ringer's) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3, and CF4AA-4). CF5AA-3 produced the highest benzamir insensitive current, and CF4AA-3 served as the negative control. Figure 2B The graph (n=4) shows the benzamir insensitive current (μA) in the absence of C18 for the control (basic Ringer's) solution, the basic Ringer's solution containing DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3, and CF4AA-4). CF5AA-3 still produces the highest benzamir insensitive current, and CF4AA-3 is the negative control.

[0245] Figure 3A The graphs (n=4) show the benzamir sensitive current (μA) of the control (basal Ringer's) solution and various individual amino acids (AA) in the absence of C18. Figure 3B A graph (n=4) shows the benzamir-sensitive current (μA) of the control (basal Ringer's) solution and various individual amino acids (AA) in the presence of C18. A higher benzamir-sensitive current indicates more retained ENaC activity. AA solutions that inhibit ENaC activity show low benzamir-sensitive currents.

[0246] Figure 4A The graphs (n=4) show the benzamir sensitive current (μA) in the absence of C18 for the control (basic Ringer's) solution and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3 and CF4AA-4). Figure 4B The graph (n=4) shows the benzamir sensitive current (μA) in the presence of C18 for the control (basic Ringer's) solution, the basic Ringer's solution containing DMSO, and various AA combinations (CF2AA, CF3AA-2, CF4AA, CF4AA-2, CF5AA-3, CF4AA-3, and CF4AA-4). CF5AA-3 produces the largest decrease in ENaC current.

[0247] Overall, the fundamental resistance of a cell is between 350 ohms and 500 ohms. For example... Figure 4A As shown, CF4AA-4 and CF5AA-3 reduced ENaC activity (benzamil-sensitive current) compared to the control. In cells treated with AA agents (except CF4AA-3), the benzamildil-insensitive current, representing chloride secretion, was higher, and C18 did not cause a significant change in Isc (…). Figure 2A and Figure 2BWhen compared with control and CF4AA-3, trichomoniasis and PG01 further increased Isc in cells exposed to the AA formulation. CFTR is a major factor in chloride secretion in HBECs treated with the AA formulation instead of CaCC. Pretreatment with C18 showed less increase in Isc after trichomoniasis and PG01, and less inhibition after CFTR(inh)-172, compared with the corresponding control. In HBECs treated with the AA formulation, CFTR protein expression was increased and ENaC was decreased, with most CFTR located in the apical membrane of the cell.

[0248] In summary, formulations based on a selected set of amino acids can improve the activity of dysfunctional chloride and sodium ion channels in CFTRΔF508 HBECs by modifying the expression and function of membrane channels. These formulations can be used alone or in combination with existing standards of care.

[0249] Example 2

[0250] Materials and methods

[0251] Cell model: Fully differentiated primary normal human bronchial epithelial cells (HBEC) and homozygous CFTRΔF508HBEC (CF cells) were cultured on snapwell at the air-liquid interface for 28 to 40 days.

[0252] Uss chamber: Measurement of transepithelial short-circuit current (Ig) in normal HBECs and CF cells when immersed in the medium or CF-5AA-3 and CF-4AA-3. sc ), resistance (R) and 22 Na and 36 unidirectional flux of Cl (J) ms &J sm ) and net flux (J net Chloride secretion was stimulated with follicular ketone (FSK, 10 μM at the tip and lateral base) and the synergist GLPG1837 (3 μM at the tip), and benzamir (6 μM at the tip) and bumetanide (20 μM at the tip and lateral base) were used to block ENaC and NKCC.

[0253] Statistical analysis: Analysis of variance (OriginPlus 2016) was used to calculate the statistical difference between the carrier and the AA formulation. P < 0.05 was considered statistically significant.

[0254] Especially with Figure 10 and Figure 11 Further experimental details are as follows:

[0255] The effects of C18 and / or VX661 on primary human bronchial epithelial cells with CFTRΔF508 were investigated by incubating cells with DMSO containing 6 μM C18 and / or 3 μM VX661 for 24 hours prior to the experiment. The experiment was repeated with or without GLPG (N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-5-carboxamide). Cells were immersed in basal Ringer's or amino acid (AA) solutions and bubbled with 5% CO2 and 95% O2, respectively. The basal Ringer's or AA solution containing 5 mM glucose was placed on the outer side of the basal layer.

[0256] The following steps will be taken:

[0257] 1.30min baseline current

[0258] 2.6 μM benzamidar (1 μL, M) – 15 min

[0259] 3. With or without 10 μM laryngin (1 μL, M / S) + 3 μM GLPG1837 (1 μL, M) – 15 min

[0260] 4.20uM CFTR Inh 172 (2μL, M / S)–15min

[0261] 5.10uM CaCC Inh AO1 (1μL, M)–10min

[0262] 6.20 μM bumetanide (2 μL, S) – 15 min

[0263] Rumacapto: C18; Symdeko: VX661 (tezacotto) / C18; Ivacapto: GLPG1837 (reversible synergist)

[0264] result

[0265] Figures 5 to 9 The results showed that, by correcting and / or modifying plasma membrane channel function, the selected AA combination improved the activity of dysfunctional chloride and sodium ion channels in the CFTRΔF508HBEC. These formulations successfully complemented the existing standard of care for patients with the CFTRΔF508 mutation.

[0266] Figure 10A and Figure 10B The results showed that CF5AA-3 was more effective than the corrector and more effective than one or more correctors plus a synergist. Figure 10A and Figure 10B It was also shown that CF5AA-3 works synergistically with one or more correctors and synergists.

[0267] Figure 11 The results showed that, compared to C18 (the corrector) alone, CF-5AA-3 induced a statistically significant increase in chlorine secretion with respect to basal or stimulated chlorine flux.

[0268] Figure 12 The graph shows the increase in anion current of CF5AA-3 via CFTR. The anion current of CF5AA-3 is significantly higher and more sustained compared to the triple combination.

[0269] Figure 12 The results show that CF5AA-3 stimulates CFTR channels better than the corrector and superior to the corrector plus synergist. These results provide evidence that CF5AA-3 directly affects CFTR channels.

[0270] Figure 13 The results show that CF5AA-3 stimulates CaCC channels better than the corrector and superior to the corrector plus synergist. These results provide evidence that CF5AA-3 directly affects CaCC channels.

[0271] Example 3

[0272] Background: In patients with cystic fibrosis, homozygous F508del mutations cause misfolding, defective transport, and aberrant gating of the CFTR protein, resulting in reduced chloride secretion and consequently chronic airway inflammation and infection. Small molecule combinations that correct CFTR misfolding / transport (VX809 and VX661) and enhance CFTR gating (VX770) have been approved for the treatment of patients with F508del who have poor drug efficacy and adverse reactions. The inventors compared the efficiency or additive efficiency of transporting defective CFTR and increasing chloride secretion in primary human bronchial epithelial cells with homozygous F508del (HBEC-F508del) with treatment using CF5AA-3 alone or VX809 / VX770 or VX661 / VX770.

[0273] Methods: Transepithelial short-circuit current and AA were measured in differentiated HBEC-F508del samples immersed in a medium (AA-free), AA test preparation (CF5AA-3), or AA negative control (NC; CF4AA-3) in a Ussen chamber. 36 Cl net flux (J) netCells were treated with C18 (VX809 analogue), VX661, or DMSO for 24 hours. After blocking ENaC with benzamidar, chloride secretion was stimulated with or without GLPG1837 (VX770 analogue). CFTRinh-172, CaCCinh-A01, or bumetanide were added to differentiate CFTR-, TMEM16A-, and NKCC1-sensitive chloride currents, respectively. Western blotting of HBEC-F508del membrane fractions was performed to determine apical CFTR expression.

[0274] Results: HBEC-F508del immersed in AA showed significantly higher basal anion currents compared to the mediator 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 resulted in a slight increase in current, with a peak of 12.7±0.4 μA in C18-AA. In HBEC-F508del pretreated with C18 and VX661 and stimulated with saliva-containing acetylcholine and GLPG1837, the anion current was significantly higher, with a peak of 14.8 ± 0.4 μA in VX661-AA compared to a peak of 7.9 ± 0.7 μA in VX661-mediated acetylcholine. In the presence of AA, CFTR contributed the most to the stimulated current (60%), while AA also activated TMEM16A (16%) and other bumetanide-sensitive chloride channels (24%). 36 Flux studies of Cl confirmed that HBEC-F508del immersed in AA showed increased chlorine secretion compared to previously used media or NC, regardless of treatment. Figure 14 CFTR membrane expression (c band) increased with and without the corrector AA: from 6 (arbitrary units) (DMSO-mediated) to 9 (C18-mediated) and from 8 (DMSO-AA) to 12 (C18-AA), respectively. See also Figure 14 .

[0275] Conclusion: CF5AA-3 increases the secretion of apical anions, similar to a corrector and synergist, suggesting that CF5AA-3 is a promising active pharmaceutical ingredient as a standalone treatment option for CF patients or as an addition to combination therapies already used to treat CF patients following further preclinical and clinical studies.

[0276] Example 4

[0277] CF animal models

[0278] This protocol utilizes a genetically modified rat model. This model has alterations to its cystic fibrosis transmembrane conductance regulator (CFTR) gene. The rat model possesses the human form of the CFTR gene with the common patient mutation G551D inserted, resulting in the production of a humanized CFTR protein (hCFTR) with the G551D mutation (class III). This rat model is referred to herein and in the art as hG551D. As with other cystic fibrosis animal models, approximately 40% of hG551D rats exhibit intestinal obstruction by 6 weeks of age. Some cases of intestinal obstruction were prevented by administering Go-LYTELY with water and by adding DietGel to their diet for hydration. Furthermore, CF knockout (KO) rats exhibit dental abnormalities, including malocclusion, which may make chewing more difficult. Therefore, the rats will be fed softened rat food three times a week. The affected rats will also be monitored for the need for dental orthodontic treatment to promote feeding and prevent morbidity or death.

[0279] The hG551D rat strain was designed to mimic the human disease cystic fibrosis. This disease is characterized by a lack of chloride secretion through epithelial cell layers (e.g., the nasal epithelium). The airways of hG551D rats have been shown to exhibit characteristics of cystic fibrosis lung disease. Specifically, the submucosal glands expressed in the airways of these rats are similar to those in human patients, and thus provide a pathology consistent with that seen in human cystic fibrosis patients.

[0280] To test chloride secretion and drug response in hG551D rats, a nasal potential difference (NPD) procedure was performed. For the NPD procedure, the rat's tail was gently rubbed, and the rat was placed in lactated Ringer's solution and connected to a high-impedance voltage follower (VF 1; World Precision Instruments) via a calomel cell. A probe was established by connecting an Ag / AgCl electrode (wire) bridge to a syringe pumping the solution at a rate of 180 μl / h. After approximately five minutes, the rat was appropriately hypnotized to allow insertion of a PE 10 cannula with a tip pulled to approximately 0.15 mm in diameter into the nostril. The perfusion solution consisted of Ringer's lactated solution plus amiloride, a low [Cl] solution containing K₂HPO₄, KH₂PO₄, sodium gluconate, NaHCO₃, calcium gluconate, and trichomoniasis. Each perfusion solution was administered at a rate of approximately 2 mL / min, with a total of less than 80 μL entering the nasal cavity per mouse. Each hyperperfusion condition was studied for 6 to 10 minutes. Rats were allowed to recover from anesthesia and were then given atipamazole to reverse the process.

[0281] For example, animals in this study will be treated with one of four compounds: the test formulation (CF5AA-3), a positive control (evicato), a negative control (CF4AA-3), or a carrier. Evacarto will be administered orally by gavage; the other compounds will be administered via aerosol spray. Animals will be treated once daily for 7 days, followed by a nasal potential difference procedure to test drug efficacy. The nasal potential difference 24 hours after the final dose of evicato or VS-009 will be compared. Once completed, animals will be euthanized for tissue collection and subsequent analysis.

[0282] Table C provides exemplary dosages of the agents shown.

[0283] Program or purpose Agent dose way frequency CFTR adjustment Ivakato 40mg / kg / day oral gavage Every day, for seven consecutive days CFTR adjustment CF5AA-3 3mM spray Every day, for seven consecutive days CFTR adjustment CF4AA-3 3mM spray Every day, for seven consecutive days CFTR adjustment vehicle not applicable spray Every day, for seven consecutive days

[0284] Equivalent schemes and scope

[0285] Unless otherwise indicated or apparent from the context, articles such as "a / an" and "described" in the claims may mean one or more. Unless otherwise indicated or apparent from the context, a claim or description including "or" among one or more members of the group will be considered satisfactory if one, more, or all members of the group are present, used, or otherwise associated with a given product or process. The invention includes embodiments in which exactly one member of the group is present, used, or otherwise associated with a given product or process. The invention includes embodiments in which one or more members of the group are present, used, or otherwise associated with a given product or process.

[0286] Furthermore, this invention covers all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim relying on another claim may be modified to include one or more limitations seen in any other claim relying on the same basic claim. Where elements are provided in list form (e.g., in Markush group form), each subgroup of elements is also disclosed, and any or any element may be removed from the group. It should be understood that, in general, when referred to as including a particular element and / or feature in this invention or an aspect thereof, certain embodiments of this invention or an aspect thereof consist of or are substantially composed of such elements and / or features. For simplicity, these embodiments are not specifically described herein in the same words. It should also be noted that the terms “comprising” and “containing” are intended to be open-ended and allow for the inclusion of additional elements or steps.

[0287] This application relates to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference in their entirety. In the event of any conflict between any incorporated reference and this specification, this specification shall prevail. Furthermore, any particular embodiment of the invention falling within the scope of the prior art is expressly excluded from any one or more of the claims. Because such embodiments are considered to be known to those skilled in the art, they are excluded even if not expressly stated herein. Any particular embodiment of the invention may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0288] Those skilled in the art will recognize or be able to determine that many equivalents of the specific embodiments described herein can be achieved using only conventional experiments. The scope of the embodiments described herein is not intended to be limited to the above description, but rather as set forth in the appended claims. Those skilled in the art will understand that various changes and modifications may be made to this description without departing from the spirit or scope of the invention as defined in the appended claims. Where a claim or specification relates to a product (e.g., the composition of a substance), it should be understood that methods of making or using the product according to any method disclosed herein, and methods of using the product for any one or more purposes disclosed herein, are included in this disclosure where applicable, unless otherwise indicated or unless a contradiction or inconsistency would be apparent to those skilled in the art. Where a claim or specification relates to a method, it should be understood that one or more products (e.g., the composition of a substance), one or more devices, or one or more systems useful for performing one or more steps of the method are included in this disclosure where applicable, unless otherwise indicated or unless a contradiction or inconsistency would be apparent to those skilled in the art.

[0289] Where ranges are given herein, embodiments including endpoints, embodiments where both endpoints are excluded, and embodiments including one endpoint while excluding the other are provided. Unless otherwise indicated, it should be assumed that two endpoints are included. Furthermore, it should be understood that, unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, in different embodiments of the invention, values ​​expressed as ranges may be assumed to be any particular value or subrange within the range, not exceeding one-tenth of the lower limit of the range unless the context explicitly indicates otherwise. It should also be understood that where a series of numerical values ​​is stated herein, embodiments similarly related to any intermediate value or range defined by any two values ​​in that series are provided, and the lowest value may be considered the minimum and the highest value may be considered the maximum. Where phrases such as “at least,” “at most,” “not exceeding,” or similar phrases appear before a series of numbers herein, unless the context explicitly indicates otherwise, it should be understood that these phrases apply to each number in the list of various embodiments (it should be understood that, depending on the context, 100% of the value, e.g., a value expressed as a percentage, may be the upper limit). For example, in various embodiments, “at least 1, 2, or 3” should be understood to mean “at least 1, at least 2, or at least 3.” It should also be understood that, where applicable, any and all reasonable lower and upper limits are explicitly envisioned. For example, a person skilled in the art can select or determine a reasonable lower or upper limit based on factors such as convenience, cost, time, effort, availability (e.g., samples, reagents), statistical considerations, etc. In some embodiments, the upper or lower limit differs from a specific value by a factor of 2, 3, 5, or 10. As used herein, numerical values ​​include values ​​expressed as percentages. For each embodiment in which the numerical value begins with “about” or “approximately”, embodiments listing precise values ​​are provided. For each embodiment in which the numerical value does not begin with “about” or “approximately”, embodiments in which the value begins with “about” or “approximately” are provided. “About” or “approximately” generally includes numbers falling within the range of 1% in any direction (greater or less than the number), or within the range of 5% in some embodiments, or within the range of 10% in some embodiments, unless otherwise stated or obvious from the context (unless such a number is not permitted to exceed 100% of the possible value). It should be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one action, the order of the actions of the method is not necessarily limited to the order in which the actions of the method are stated, but the invention includes embodiments where the order is 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 jointly. In some embodiments, the method may be performed at least in part by requesting or authorizing another individual or entity to perform one, more, or all of the steps of the method.In some implementations, the method includes requesting two or more entities or individuals to each perform at least one step of the method. In some implementations, the execution of two or more steps is coordinated so that the method is performed jointly. Individuals or entities performing one or more different steps may or may not interact.

[0290] The chapter headings used herein are not to be construed as limiting in any way. It is explicitly envisioned that the topics presented under any chapter heading are applicable to any aspect or implementation described herein.

[0291] The embodiments or aspects described herein may be applied to any agent, composition, article, kit, and / or method described herein. It is contemplated that any one or more embodiments or aspects may be freely combined with any one or more other embodiments or aspects wherever appropriate. For example, any combination of two or more mutually non-contradictory agents, compositions, articles, kits, and / or methods may be provided. It should be understood that, unless otherwise indicated or explicitly stated, any description or example of a term anywhere in this document may be applied wherever that term appears herein (e.g., in any aspect or embodiment relating to that term).

[0292] References

[0293] 1.O'Sullivan BP and Freedman SD (2009) Cystic Fibrosis. Lancet, 373; pp.1891-904.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 5. Ramsey BW, Davies J, McElvaney NG, et al. (2011) ACFTR potentiatorinpatients with cystic fibrosis and the G551D mutation. NEngl JMed, 365; pp.1633-1672. sequence list <110> Vidyasagaar Sadasivan <120> Amino acid compositions and methods for treating cystic fibrosis <130> 174821-010301PCT <140> <141> <150> 62 / 752,847 <151> 2018-10-30 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1480 <212> PRT <213> Homo sapiens <400> 1 Met Gln Arg Ser Pro Leu Glu Lys Ala Ser Val Val Ser Lys Leu Phe 1 5 10 15 Phe Ser Trp Thr Arg Pro Ile Leu Arg Lys Gly Tyr Arg Gln Arg Leu 20 25 30 Glu Leu Ser Asp Ile Tyr Gln Ile Pro Ser Val Asp Ser Ala Asp Asn 35 40 45 Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Arg Glu Leu Ala Ser Lys 50 55 60 Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Arg Cys Phe Phe Trp Arg 65 70 75 80 Phe Met Phe Tyr Gly Ile Phe Leu Tyr Leu Gly Glu Val Thr Lys Ala 85 90 95 Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp 100 105 110 Asn Lys Glu Glu Arg Ser Ile Ala Ile Tyr Leu Gly Ile Gly Leu Cys 115 120 125 Leu Leu Phe Ile Val Arg Thr Leu Leu Leu His Pro Ala Ile Phe Gly 130 135 140 Leu His His Ile Gly Met Gln Met Arg Ile Ala Met Phe Ser Leu Ile 145 150 155 160 Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser 165 170 175 Ile Gly Gln Leu Val Ser Leu Leu Ser Asn Asn Leu Asn Lys Phe Asp 180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp Ile Ala Pro Leu Gln Val 195 200 205 Ala Leu Leu Met Gly Leu Ile Trp Glu Leu Leu Gln Ala Ser Ala Phe 210 215 220 Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Leu Phe Gln Ala Gly Leu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gln Arg Ala Gly Lys Ile Ser 245 250 255 Glu Arg Leu Val Ile Thr Ser Glu Met Ile Glu Asn Ile Gln Ser Val 260 265 270 Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Lys Met Ile Glu Asn Leu 275 280 285 Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr 290 295 300 Phe Asn Ser Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Ile Ile Leu Arg Lys Ile 325 330 335 Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Arg Met Ala Val Thr Arg 340 345 350 Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr Asp Ser Leu Gly Ala Ile 355 360 365 Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Glu Tyr Lys Thr Leu Glu 370 375 380 Tyr Asn Leu Thr Thr Thr Glu Val Val Met Glu Asn Val Thr Ala Phe 385 390 395 400 Trp Glu Glu Gly Phe Gly Glu Leu Phe Glu Lys Ala Lys Gln Asn Asn 405 410 415 Asn Asn Arg Lys Thr Ser Asn Gly Asp Asp Ser Leu Phe Phe Ser Asn 420 425 430 Phe Ser Leu Leu Gly Thr Pro Val Leu Lys Asp Ile Asn Phe Lys Ile 435 440 445 Glu Arg Gly Gln Leu Leu Ala Val Ala Gly Ser Thr Gly Ala Gly Lys 450 455 460 Thr Ser Leu Leu Met Val Ile Met Gly Glu Leu Glu Pro Ser Glu Gly 465 470 475 480 Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser Gln Phe Ser Trp 485 490 495 Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Ile Phe Gly Val Ser Tyr 500 505 510 Asp Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys Gln Leu Glu Glu 515 520 525 Asp Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile Val Leu Gly Glu Gly 530 535 540 Gly Ile Thr Leu Ser Gly Gly Gln Arg Ala Arg Ile Ser Leu Ala Arg 545 550 555 560 Ala Val Tyr Lys Asp Ala Asp Leu Tyr Leu Leu Asp Ser Pro Phe Gly 565 570 575 Tyr Leu Asp Val Leu Thr Glu Lys Glu Ile Phe Glu Ser Cys Val Cys 580 585 590 Lys Leu Met Ala Asn Lys Thr Arg Ile Leu Val Thr Ser Lys Met Glu 595 600 605 His Leu Lys Lys Ala Asp Lys Ile Leu Ile Leu His Glu Gly Ser Ser 610 615 620 Tyr Phe Tyr Gly Thr Phe Ser Glu Leu Gln Asn Leu Gln Pro Asp Phe 625 630 635 640 Ser Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gln Phe Ser Ala Glu 645 650 655 Arg Arg Asn Ser Ile Leu Thr Glu Thr Leu His Arg Phe Ser Leu Glu 660 665 670 Gly Asp Ala Pro Val Ser Trp Thr Glu Thr Lys Lys Gln Ser Phe Lys 675 680 685 Gln Thr Gly Glu Phe Gly Glu Lys Arg Lys Asn Ser Ile Leu Asn Pro 690 695 700 Ile Asn Ser Ile Arg Lys Phe Ser Ile Val Gln Lys Thr Pro Leu Gln 705 710 715 720 Met Asn Gly Ile Glu Glu Asp Ser Asp Glu Pro Leu Glu Arg Arg Leu 725 730 735 Ser Leu Val Pro Asp Ser Glu Gln Gly Glu Ala Ile Leu Pro Arg Ile 740 745 750 Ser Val Ile Ser Thr Gly Pro Thr Leu Gln Ala Arg Arg Arg Gln Ser 755 760 765 Val Leu Asn Leu Met Thr His Ser Val Asn Gln Gly Gln Asn Ile His 770 775 780 Arg Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala Pro Gln Ala 785 790 795 800 Asn Leu Thr Glu Leu Asp Ile Tyr Ser Arg Arg Leu Ser Gln Glu Thr 805 810 815 Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys 820 825 830 Phe Phe Asp Asp Met Glu Ser Ile Pro Ala Val Thr Thr Trp Asn Thr 835 840 845 Tyr Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe Val Leu Ile 850 855 860 Trp Cys Leu Val Ile Phe Leu Ala Glu Val Ala Ala Ser Leu Val Val 865 870 875 880 Leu Trp Leu Leu Gly Asn Thr Pro Leu Gln Asp Lys Gly Asn Ser Thr 885 890 895 His Ser Arg Asn Asn Ser Tyr Ala Val Ile Ile Thr Ser Thr Ser Ser 900 905 910 Tyr Tyr Val Phe Tyr Ile Tyr Val Gly Val Ala Asp Thr Leu Leu Ala 915 920 925 Met Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu Ile Thr Val 930 935 940 Ser Lys Ile Leu His His Lys Met Leu His Ser Val Leu Gln Ala Pro 945 950 955 960 Met Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu Asn Arg Phe 965 970 975 Ser Lys Asp Ile Ala Ile Leu Asp Asp Leu Leu Pro Leu Thr Ile Phe 980 985 990 Asp Phe Ile Gln Leu Leu Leu Ile Val Ile Gly Ala Ile Ala Val Val 995 1000 1005 Ala Val Leu Gln Pro Tyr Ile Phe Val Ala Thr Val Pro Val Ile 1010 1015 1020 Val Ala Phe Ile Met Leu Arg Ala Tyr Phe Leu Gln Thr Ser Gln 1025 1030 1035 Gln Leu Lys Gln Leu Glu Ser Glu Gly Arg Ser Pro Ile Phe Thr 1040 1045 1050 His Leu Val Thr Ser Leu Lys Gly Leu Trp Thr Leu Arg Ala Phe 1055 1060 1065 Gly Arg Gln Pro Tyr Phe Glu Thr Leu Phe His Lys Ala Leu Asn 1070 1075 1080 Leu His Thr Ala Asn Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp 1085 1090 1095 Phe Gln Met Arg Ile Glu Met Ile Phe Val Ile Phe Phe Ile Ala 1100 1105 1110 Val Thr Phe Ile Ser Ile Leu Thr Thr Gly Glu Gly Glu Gly Arg 1115 1120 1125 Val Gly Ile Ile Leu Thr Leu Ala Met Asn Ile Met Ser Thr Leu 1130 1135 1140 Gln Trp Ala Val Asn Ser Ser Ile Asp Val Asp Ser Leu Met Arg 1145 1150 1155 Ser Val Ser Arg Val Phe Lys Phe Ile Asp Met Pro Thr Glu Gly 1160 1165 1170 Lys Pro Thr Lys Ser Thr Lys Pro Tyr Lys Asn Gly Gln Leu Ser 1175 1180 1185 Lys Val Met Ile Ile Glu Asn Ser His Val Lys Lys Asp Asp Ile 1190 1195 1200 Trp Pro Ser Gly Gly Gln Met Thr Val Lys Asp Leu Thr Ala Lys 1205 1210 1215 Tyr Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn Ile Ser Phe Ser 1220 1225 1230 Ile Ser Pro Gly Gln Arg Val Gly Leu Leu Gly Arg Thr Gly Ser 1235 1240 1245 Gly Lys Ser Thr Leu Leu Ser Ala Phe Leu Arg Leu Leu Asn Thr 1250 1255 1260 Glu Gly Glu Ile Gln Ile Asp Gly Val Ser Trp Asp Ser Ile Thr 1265 1270 1275 Leu Gln Gln Trp Arg Lys Ala Phe Gly Val Ile Pro Gln Lys Val 1280 1285 1290 Phe Ile Phe Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Glu 1295 1300 1305 Gln Trp Ser Asp Gln Glu Ile Trp Lys Val Ala Asp Glu Val Gly 1310 1315 1320 Leu Arg Ser Val Ile Glu Gln Phe Pro Gly Lys Leu Asp Phe Val 1325 1330 1335 Leu Val Asp Gly Gly Cys Val Leu Ser His Gly His Lys Gln Leu 1340 1345 1350 Met Cys Leu Ala Arg Ser Val Leu Ser Lys Ala Lys Ile Leu Leu 1355 1360 1365 Leu Asp Glu Pro Ser Ala His Leu Asp Pro Val Thr Tyr Gln Ile 1370 1375 1380 Ile Arg Arg Thr Leu Lys Gln Ala Phe Ala Asp Cys Thr Val Ile 1385 1390 1395 Leu Cys Glu His Arg Ile Glu Ala Met Leu Glu Cys Gln Gln Phe 1400 1405 1410 Leo Val Ile Glu Glu Asn Lys Val Arg Gln Tyr Asp Ser Ile Gln 1415 1420 1425 Lys Leu Leu Asn Glu Arg Ser Leu Phe Arg Gln Ala Ile Ser Pro 1430 1435 1440 Ser Asp Arg Val Lys Leu Phe Pro His Arg Asn Ser Ser Lys Cys 1445 1450 1455 Lys Ser Lys Pro Gln Ile Ala Ala Leu Lys Glu Glu Thr Glu Glu 1460 1465 1470 Glu Val Gln Asp Thr Arg Leu 1475 1480 <210> 2 <211> 1479 <212> PRT[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp 100 105 110 Asn Lys Glu Glu Arg Ser Ile Ala Ile Tyr Leu Gly Ile Gly Leu Cys 115 120 125 Leu Leu Phe Ile Val Arg Thr Leu Leu Leu His Pro Ala Ile Phe Gly 130 135 140 Leu His His Ile Gly Met Gln Met Arg Ile Ala Met Phe Ser Leu Ile 145 150 155 160 Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser 165 170 175 Ile Gly Gln Leu Val Ser Leu Leu Ser Asn Asn Leu Asn Lys Phe Asp 180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp Ile Ala Pro Leu Gln Val 195 200 205 Ala Leu Leu Met Gly Leu Ile Trp Glu Leu Leu Gln Ala Ser Ala Phe 210 215 220 Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Leu Phe Gln Ala Gly Leu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gln Arg Ala Gly Lys Ile Ser 245 250 255 Glu Arg Leu Val Ile Thr Ser Glu Met Ile Glu Asn Ile Gln Ser Val 260 265 270 Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Lys Met Ile Glu Asn Leu 275 280 285 Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr 290 295 300 Phe Asn Ser Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Ile Ile Leu Arg Lys Ile 325 330 335 Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Arg Met Ala Val Thr Arg 340 345 350 Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr Asp Ser Leu Gly Ala Ile 355 360 365 Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Glu Tyr Lys Thr Leu Glu 370 375 380 Tyr Asn Leu Thr Thr Thr Glu Val Val Met Glu Asn Val Thr Ala Phe 385 390 395 400 Trp Glu Glu Gly Phe Gly Glu Leu Phe Glu Lys Ala Lys Gln Asn Asn 405 410 415 Asn Asn Arg Lys Thr Ser Asn Gly Asp Asp Ser Leu Phe Phe Ser Asn 420 425 430 Phe Ser Leu Leu Gly Thr Pro Val Leu Lys Asp Ile Asn Phe Lys Ile 435 440 445 Glu Arg Gly Gln Leu Leu Ala Val Ala Gly Ser Thr Gly Ala Gly Lys 450 455 460 Thr Ser Leu Leu Met Val Ile Met Gly Glu Leu Glu Pro Ser Glu Gly 465 470 475 480 Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser Gln Phe Ser Trp 485 490 495 Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Ile Gly Val Ser Tyr Asp 500 505 510 Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys Gln Leu Glu Glu Asp 515 520 525 Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile Val Leu Gly Glu Gly Gly 530 535 540 Ile Thr Leu Ser Gly Gly Gln Arg Ala Arg Ile Ser Leu Ala Arg Ala 545 550 555 560 Val Tyr Lys Asp Ala Asp Leu Tyr Leu Leu Asp Ser Pro Phe Gly Tyr 565 570 575 Leu Asp Val Leu Thr Glu Lys Glu Ile Phe Glu Ser Cys Val Cys Lys 580 585 590 Leu Met Ala Asn Lys Thr Arg Ile Leu Val Thr Ser Lys Met Glu His 595 600 605 Leu Lys Lys Ala Asp Lys Ile Leu Ile Leu His Glu Gly Ser Ser Tyr 610 615 620 Phe Tyr Gly Thr Phe Ser Glu Leu Gln Asn Leu Gln Pro Asp Phe Ser 625 630 635 640 Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gln Phe Ser Ala Glu Arg 645 650 655 Arg Asn Ser Ile Leu Thr Glu Thr Leu His Arg Phe Ser Leu Glu Gly 660 665 670 Asp Ala Pro Val Ser Trp Thr Glu Thr Lys Lys Gln Ser Phe Lys Gln 675 680 685 Thr Gly Glu Phe Gly Glu Lys Arg Lys Asn Ser Ile Leu Asn Pro Ile 690 695 700 Asn Ser Ile Arg Lys Phe Ser Ile Val Gln Lys Thr Pro Leu Gln Met 705 710 715 720 Asn Gly Ile Glu Glu Asp Ser Asp Glu Pro Leu Glu Arg Arg Leu Ser 725 730 735 Leu Val Pro Asp Ser Glu Gln Gly Glu Ala Ile Leu Pro Arg Ile Ser 740 745 750 Val Ile Ser Thr Gly Pro Thr Leu Gln Ala Arg Arg Arg Gln Ser Val 755 760 765 Leu Asn Leu Met Thr His Ser Val Asn Gln Gly Gln Asn Ile His Arg 770 775 780 Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala Pro Gln Ala Asn 785 790 795 800 Leu Thr Glu Leu Asp Ile Tyr Ser Arg Arg Leu Ser Gln Glu Thr Gly 805 810 815 Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys Phe 820 825 830 Phe Asp Asp Met Glu Ser Ile Pro Ala Val Thr Thr Trp Asn Thr Tyr 835 840 845 Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe Val Leu Ile Trp 850 855 860 Cys Leu Val Ile Phe Leu Ala Glu Val Ala Ala Ser Leu Val Val Leu 865 870 875 880 Trp Leu Leu Gly Asn Thr Pro Leu Gln Asp Lys Gly Asn Ser Thr His 885 890 895 Ser Arg Asn Asn Ser Tyr Ala Val Ile Ile Thr Ser Thr Ser Ser Tyr 900 905 910 Tyr Val Phe Tyr Ile Tyr Val Gly Val Ala Asp Thr Leu Leu Ala Met 915 920 925 Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu Ile Thr Val Ser 930 935 940 Lys Ile Leu His His Lys Met Leu His Ser Val Leu Gln Ala Pro Met 945 950 955 960 Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu Asn Arg Phe Ser 965 970 975 Lys Asp Ile Ala Ile Leu Asp Asp Leu Leu Pro Leu Thr Ile Phe Asp 980 985 990 Phe Ile Gln Leu Leu Leu Ile Val Ile Gly Ala Ile Ala Val Val Ala 995 1000 1005 Val Leu Gln Pro Tyr Ile Phe Val Ala Thr Val Pro Val Ile Val 1010 1015 1020 Ala Phe Ile Met Leu Arg Ala Tyr Phe Leu Gln Thr Ser Gln Gln 1025 1030 1035 Leu Lys Gln Leu Glu Ser Glu Gly Arg Ser Pro Ile Phe Thr His 1040 1045 1050 Leu Val Thr Ser Leu Lys Gly Leu Trp Thr Leu Arg Ala Phe Gly 1055 1060 1065 Arg Gln Pro Tyr Phe Glu Thr Leu Phe His Lys Ala Leu Asn Leu 1070 1075 1080 His Thr Ala Asn Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp Phe 1085 1090 1095 Gln Met Arg Ile Glu Met Ile Phe Val Ile Phe Phe Ile Ala Val 1100 1105 1110 Thr Phe Ile Ser Ile Leu Thr Thr Gly Glu Gly Glu Gly Arg Val 1115 1120 1125 Gly Ile Ile Leu Thr Leu Ala Met Asn Ile Met Ser Thr Leu Gln 1130 1135 1140 Trp Ala Val Asn Ser Ser Ile Asp Val Asp Ser Leu Met Arg Ser 1145 1150 1155 Val Ser Arg Val Phe Lys Phe Ile Asp Met Pro Thr Glu Gly Lys 1160 1165 1170 Pro Thr Lys Ser Thr Lys Pro Tyr Lys Asn Gly Gln Leu Ser Lys 1175 1180 1185 Val Met Ile Ile Glu Asn Ser His Val Lys Lys Asp Asp Ile Trp 1190 1195 1200 Pro Ser Gly Gly Gln Met Thr Val Lys Asp Leu Thr Ala Lys Tyr 1205 1210 1215 Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn Ile Ser Phe Ser Ile 1220 1225 1230 Ser Pro Gly Gln Arg Val Gly Leu Leu Gly Arg Thr Gly Ser Gly 1235 1240 1245 Lys Ser Thr Leu Leu Ser Ala Phe Leu Arg Leu Leu Asn Thr Glu 1250 1255 1260 Gly Glu Ile Gln Ile Asp Gly Val Ser Trp Asp Ser Ile Thr Leu 1265 1270 1275 Gln Gln Trp Arg Lys Ala Phe Gly Val Ile Pro Gln Lys Val Phe 1280 1285 1290 Ile Phe Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Glu Gln 1295 1300 1305 Trp Ser Asp Gln Glu Ile Trp Lys Val Ala Asp Glu Val Gly Leu 1310 1315 1320 Arg Ser Val Ile Glu Gln Phe Pro Gly Lys Leu Asp Phe Val Leu 1325 1330 1335 Val Asp Gly Gly Cys Val Leu Ser His Gly His Lys Gln Leu Met 1340 1345 1350 Cys Leu Ala Arg Ser Val Leu Ser Lys Ala Lys Ile Leu Leu Leu 1355 1360 1365 Asp Glu Pro Ser Ala His Leu Asp Pro Val Thr Tyr Gln Ile Ile 1370 1375 1380 Arg Arg Thr Leu Lys Gln Ala Phe Ala Asp Cys Thr Val Ile Leu 1385 1390 1395 Cys Glu His Arg Ile Glu Ala Met Leu Glu Cys Gln Gln Phe Leu 1400 1405 1410 Val Ile Glu Glu Asn Lys Val Arg Gln Tyr Asp Ser Ile Gln Lys 1415 1420 1425 Leu Leu Asn Glu Arg Ser Leu Phe Arg Gln Ala Ile Ser Pro Ser 1430 1435 1440 Asp Arg Val Lys Leu Phe Pro His Arg Asn Ser Ser Lys Cys Lys 1445 1450 1455 Ser Lys Pro Gln Ile Ala Ala Leu Lys Glu Glu Thr Glu Glu Glu 1460 1465 1470 Val Gln Asp Thr Arg Leu 1475

Claims

1. A formulation, wherein the formulation comprises: Cysteine, proline, glycine, tyrosine, and lysine are free amino acids; and Optionally, at least one component selected from the group consisting of pharmaceutically acceptable carriers, buffers, and electrolytes. The prerequisite is that at least one of the free amino acids is an L-amino acid.

2. The formulation of claim 1, wherein the pharmaceutically acceptable carrier comprises water.

3. The formulation of claim 1, wherein the pharmaceutically acceptable carrier comprises an adjuvant.

4. The formulation of claim 1, wherein the pharmaceutically acceptable carrier comprises an excipient.

5. The formulation of claim 1, wherein the formulation is sterile.

6. The formulation of claim 1, wherein the formulation is formulated for administration via the intestinal, pulmonary, or sublingual route.

7. The formulation of claim 1, wherein the formulation is formulated for administration via inhalation.

8. The formulation of claim 1, wherein the formulation is formulated for administration via the intranasal route.

9. The formulation as described in claim 1, which is used as a pharmaceutical agent.

10. Use of the formulation according to claim 1 in the manufacture of a medicament for treating a subject suffering from COPD.

11. Use of the formulation according to claim 1 in the manufacture of a medicament for treating a subject suffering from asthma.

12. The use as described in claim 11, wherein the subject suffers from intermittent asthma, mild persistent asthma, moderate persistent asthma, or severe persistent asthma.

13. Use of the formulation according to claim 1 in the manufacture of a medicament for treating a subject suffering from cystic fibrosis.

14. Use of the formulation according to claim 1 in the manufacture of a medicament for treating a subject suffering from bronchiectasis.

15. Use of the formulation according to claim 1 in the manufacture of a medicament for treating a subject suffering from bronchitis.

16. The use as claimed in claim 15, wherein the subject suffers from chronic bronchitis.

17. Use of the formulation according to claim 1 in the manufacture of a medicament for treating a subject suffering from pulmonary fibrosis.

18. A kit comprising: The formulation according to claim 1; and Instructions for use on subjects or to bring biological samples into contact with the formulation.

19. A non-therapeutic in vitro method for increasing the amount of cystic fibrosis transmembrane conductance regulator proteins present on the plasma membrane of at least one cell, the method comprising: Contact the at least one cell with an effective amount of the formulation according to claim 1. The contact promotes at least one of folding of cystic fibrosis transmembrane conductance regulators or translocation of cystic fibrosis transmembrane conductance regulators to the plasma membrane, thereby increasing the amount of cystic fibrosis transmembrane conductance regulator protein present on the cell membrane.

20. The method of claim 19, wherein the number of wild-type cystic fibrosis transmembrane conductance regulator proteins on the plasma membrane is increased.

21. The method of claim 19 or 20, wherein the number of mutant cystic fibrosis transmembrane conductance regulator proteins on the plasma membrane is increased.

22. The method of claim 19, wherein the number of cystic fibrosis transmembrane conductance regulator 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 the wild-type cystic fibrosis transmembrane conductance regulator on the plasma membrane is increased, wherein the wild-type cystic fibrosis transmembrane conductance regulator comprises SEQ ID NO:

1.

23. The method of claim 19, wherein one or more of the cystic fibrosis transmembrane conductance regulator proteins comprise Gly542X, Gly551Asp, Arg553X, Arg117His, 120del23, Phe508del, or a combination thereof, wherein X is any amino acid.

24. The method of claim 23, wherein one or more of the cystic fibrosis transmembrane conductance regulator proteins contain a Phe508del mutation.

25. The method of claim 19, wherein an increase in the number of cystic fibrosis transmembrane conductance regulator proteins present on the cell membrane is detected by an increase in chloride ions expelled from the cell.

26. The method of claim 19, wherein an increase in the number of cystic fibrosis transmembrane conductance regulator proteins present on the cell membrane is associated with an increase in water excretion from the cell.

27. The method of claim 19, wherein the cell is an epithelial cell.

28. The method of claim 27, wherein the epithelial cells are lung epithelial cells.

29. The method of claim 28, wherein the lung epithelial cells are bronchial epithelial cells.

30. The method of claim 29, wherein the bronchial epithelial cells are isolated from a subject suffering from cystic fibrosis.

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