Synergistic stimulation of mucociliary clearance for treating mucous occlusion in cystic fibrosis and other mucous obstructive disorders

Abstract

The present disclosure provides a method of treating a mucosal obstructive disorder in a subject, the method comprising administering to the subject a b-adrenergic agonist or an adenylate cyclase activator in combination with a cholinergic agonist to treat a mucosal obstructive disorder in a subject.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 408,596, filed on September 21, 2022, which is incorporated herein by reference in its entirety. Background of the Invention

[0003] Cystic fibrosis (CF) is a multi - organ syndrome, and its most critical clinical phenotypes are airway mucus obstruction, chronic pulmonary infection, and neutrophilic inflammation. Unless curbed, the resulting tissue damage leads to a lifelong decline in lung function. CF is caused by loss - of - function mutations in the gene CFTR (cystic fibrosis transmembrane conductance regulator), which is important for fluid secretion in the airways. CF airways appear normal at birth, but their airway surface liquid (ASL) has a reduced ability to kill bacteria (1), and mucus clearance is slowed (2). Chronic pulmonary infection is a major driver of human lung function decline (3, 4). However, even when chronic infections in transgenic ferrets with CF (“CF ferrets”) are prevented with antibiotics, the mucus obstruction phenotype with bronchial obstruction and inflammation still persists (5).

[0004] Therefore, improving mucociliary clearance (MCC) in the airways is an important therapeutic goal in CF. Improvements in mucus clearance can be achieved with inhaled therapies using recombinant human DNAse (Pulmozyme) (6, 7), hypertonic saline (8, 9), or powdered mannitol (10), sometimes sufficient to show clinical efficacy. For the majority of the CF population, the most effective improvements in mucus clearance are provided by small - molecule modulators that can partially restore the function of CFTR with specific mutations (11, 12). For patients in whom the mutations cannot be treated with existing modulators, or for patients in whom lung function still declines despite the use of modulators, additional improvements in mucus clearance may have therapeutic effects. MCC is a function of the volume and composition of airway surface liquid (ASL), the rheological properties of the secreted mucus, and ciliary beat frequency (CBF) (13). Summary of the Invention

[0005] The present disclosure provides methods and compositions for treating cystic fibrosis in an individual in need thereof.

[0006] The present disclosure provides a method of treating an individual suffering from a mucosal obstructive disorder, the method comprising: administering to the individual a combination of a β - adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to treat the mucosal obstructive disorder of the individual.

[0007] Also provided is a method of increasing the rate of submucosal gland secretion in an individual, the method comprising: administering to the individual a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to increase the rate of submucosal gland secretion in the individual.

[0008] The present disclosure also provides a method of inhibiting cholinergic agonist-induced airway smooth muscle contraction in an individual, the method comprising: administering to the individual a β-adrenergic agonist or an adenylate cyclase activator, wherein the administration of the β-adrenergic agonist or the adenylate cyclase activator is performed before or simultaneously with the administration of the cholinergic agonist to the individual to inhibit airway smooth muscle contraction.

[0009] Also provided are compositions and systems for practicing the subject methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The present invention can be better understood by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, as a matter of convention, the various features in the drawings are not drawn to scale. Instead, for clarity, the dimensions of the various features are arbitrarily enlarged or reduced. The accompanying drawings include the following figures.

[0011] Figure 1. Synergistic mucus clearance in CF ferrets and WT pigs tracheas. (A) Time course of MCC velocity (MCCV, measured value of MCC velocity) of CF ferrets in response to the direct adenylate cyclase activator - 10 μM forskolin (blue open circles, Fsk, n = 4), the cholinergic agonist - 0.3 μM carbachol (red open squares, Carb, n = 4) or the combination (filled symbols). The CF ferret genotypes were five CFTRG551D, one CFTRΔF / ΔF and one CFTRG551D / KO. (B) Summary data as box plots. Bs: basal / unstimulated MCCV, sum: arithmetic sum of the MCCV measurements of the agonists used alone, SR: synergistic response, MCCV measured for the combined agonists. SR was 5.7-fold the sum, representing synergy (P = 0.006, n = 3 - 7). (C) MCCV of 2 - 5-day-old piglets: same protocol and symbols as for CF ferrets. (D) Summary data. SR was 3.9-fold the sum, (P = 3.8E-05, n = 4 - 8). (E) Response of pigs to the β-adrenergic agonist - 10 μM formoterol / Fmt (blue circles) rather than forskolin, same other protocols and symbols. (F) Summary data. SR was 3.4-fold the sum, (P = 0.005, n = 3 - 7).

[0012] Figure 2. Enhancement of cAMP by activation of adenylate cyclase inhibits carbachol-induced muscle tension and airway narrowing. (A, B) To measure muscle tension, one end of an isolated WT ferret tracheal muscle bundle was fixed in a Sylgard-lined dish containing KRB solution, and the other end was attached by a 26-gauge wire to a previously calibrated strain gauge. Tension responses to increasing doses of carbachol are shown in the absence (A) and presence (B) of 10 μM forskolin. (C–F) To measure airway narrowing, tracheal ring slices (∼2 mm) from WT pigs and WT or CF ferrets were treated with carbachol alone or in the presence of forskolin (F or Fsk) or formoterol (Fmt); their lumens were imaged over time, and the area was measured as a determination of muscle contraction. After a baseline period (black open squares), carbachol (0.3 μM, red open squares) was added, or carbachol was added in the presence of 10 μM forskolin or formoterol (red solid circles). Mean responses at 10-min intervals were as follows: (C) WT piglet tracheas (n = 3–7) plus forskolin. (D) WT piglet tracheas plus formoterol (n = 5). (E) WT ferret tracheas (2 tracheas, 5 experiments). (F) CF ferrets (n = 2, one CFTRΔF / ΔF and one CFTRΔF / G551D).

[0013] Figure 3. Combinatorial agonists synergistically increase glandular mucus secretion. Using the same notation as in Fig. 1, the mean secretion rates of 15 WT pigs (A), 12 WT ferrets (D), and 2 CF ferrets (G) are summarized as box plots. Each agonist increased secretion above baseline, and the rate of the combinatorial agonist (SR, synergistic response) was significantly greater than the arithmetic sum of their individual responses (sum). (B) Mean secretion rates of individual WT pigs to 10 μM forskolin alone and 10 μM forskolin in combination with 0.3 μM carbachol (60 glands, 8 pigs). (C) Same as B, except for carbachol alone and the combination of carbachol and forskolin (60–61 glands, 7 pigs). (E, F) WT adult ferret data, conditions the same as for pigs (26–29 glands, 7 ferrets). (H, I) CF ferret data, generated under the same conditions as for pigs and WT ferrets. One ferret was run under each condition, and the secretion rate was measured in 7–14 glands. The time course of the mean response was plotted at 10-min intervals.

[0014] Figure 4. Combinatorial agonists inhibit sodium absorption and stimulate anion secretion by surface epithelium. (A) Cartoon of two electrogenic ion transport pathways across the apical epithelium of the airway: increased anion secretion raises surface fluid, and Na+ absorption reduces surface fluid. These two pathways have opposite effects on fluid depth but have an additive effect on short-circuit current (Isc) because of their opposite valence and transport directions. (B) Raw tracing of Isc across porcine tracheal epithelium using Chart 4 software. After achieving a stable, unstimulated Isc (here, >2 h after fixation), 10 μM forskolin, 0.3 μM carbachol, 10 μM benzamil, 20 μM benzopyrimidopyrazinedione (BPO-27), and 200 μM niflumic acid were added sequentially at the times indicated. (C, D) Porcine tracheal mucosa: mean ΔIsc plots over time in response to (C) forskolin alone, then forskolin + carbachol and (D) agonists added in the reverse order. (E&F) Ferret tracheal mucosa: mean ΔIsc plots drawn with the same protocol as for pigs. Porcine traces (C, D) are based on 10 - 12 experiments where tissue was from 6 to 7 pigs. Ferret traces (E, F) are from 7 experiments where tissue was from 5 ferrets.

[0015] Figure 5 . Agonists stimulate ciliary beat frequency and produce an additive effect. Ciliary beat frequency was measured in human nasal mucosa from 4 subjects. The CBF (in Hz) of unstimulated tissue in Krebs buffer (KRB) at 37 °C was 10.46 ± 0.95. Each agonist alone produced a small increase in CBF, but this did not reach significance in this small sample: carbachol: 11.04 ± 1.3 (5.3%) and forskolin 12.06 ± 1.22 (9.8%). When comparing the difference between CBF (Δ) and unstimulated CBF (KRB), the combinatorial agonists significantly increased CBF to 13.31 ± 0.77 (27.2%, n = 4, P < 0.05), but not compared to the arithmetic sum of the ΔCBF of the combinatorial agonists: 2.85 ± 0.76 (synergistic paradigm) vs 2.19 ± 0.66 (arithmetic sum) (n = 4, P = 0.47). (Note that in this experiment, any effect of the agonists on the ASL was diluted because the ciliated tissue was submerged in Krebs solution during the study.)

[0016] Figure 6. Summary figure linking increased ASL generation to increased mucus clearance. (A) Relationship between ΔIsc, ASL depth, and MCCV. MCCV (redrawn from Fig. 1C) is shown on the main graph; inset shows ΔIsc (from Fig. 4C) with time points aligned to the MCCV graph. Brown dashed line represents inferred ASL depth change in the absence of MCC. (B) Cartoon of major ion fluxes across the tracheal surface epithelium under four conditions: baseline, β-adrenergic (β-Adn), carbachol (CCh), and β-Adn + CCh. Each panel shows inferred anion secretion status, Na+ absorption, and resulting ASL depth change. Inferred changes in ASL depth. In our experiments, the major change was an increase in mucus clearance rate, which would tend to offset the increase in ASL depth. Since carbachol inhibits Na+ (and fluid) absorption and stimulates anion (and fluid) secretion, the combined agonists have opposite effects on Isc but at least an additive increase in ASL depth. (C) Summary figure of component processes leading to a synergistic increase in MCCV in ex vivo tracheas of WT ferrets, WT pigs, and CF ferrets. The end result is a significant increase in MCCV.

[0017] Figure 7. Individual MCCV responses of 7 CF ferrets to the synergistic paradigm. (A–G) Protocol and genotype are shown on each time-MCC velocity plot. Note that a reduced y-axis scale is used in (E) and (G) to display the synergistic MCCV. Also note that the mean MCCV to 0.3 μM carbachol at T10–30 in (E) is less than 5% of those in (A) and (C).

[0018] Figure 8 Depicts synergistic MCC generated by sequential agonists with methacholine and formoterol.

[0019] Figure 9 Depicts the protective effect of formoterol on methacholine-induced muscle contraction.

[0020] Figure 10 Depicts simultaneous treatment with formoterol and methacholine that induces a synergistic response.

[0021] Figure 11 Depicts that simultaneous treatment with formoterol and methacholine does not induce muscle contraction.

[0022] Figure 12Depicts the mean squared displacement of particles for ASL transport in cystic fibrosis and healthy, mature human nasal epithelial cell cultures in response to treatment with DMSO control, forskolin, carbachol, forskolin + carbachol (SP), or the CFTR modulator combination elexacaftor (3 μM) - tezacaftor (3 μM) - ivacaftor (10 μM) (ETI).

[0023] Figure 13 Depicts the effective diffusion rate of mucus transport in cystic fibrosis and healthy, mature human nasal epithelial cell cultures in response to treatment with DMSO control, forskolin, carbachol, forskolin + carbachol (SP), or the combination elexacaftor (3 μM) - tezacaftor (3 μM) - ivacaftor (10 μM) (ETI).

[0024] Figure 14 Depicts the effects of formoterol (Fmt) and Fmt + methacholine (MCh) on TMV in a sheep model of cystic fibrosis.

[0025] Figure 15 Depicts the effects of formoterol (Fmt) and Fmt + methacholine (MCh) on whole lung clearance in a sheep CF model.

[0026] Figure 16 Depicts the effects of salbutamol + methacholine on single - ascending - dose tolerance in healthy volunteers.

[0027] Figure 17 Depicts the effects of formoterol + methacholine on single - ascending - dose tolerance in healthy volunteers.

[0028] Figure 18 Depicts the effects of formoterol + methacholine on single - ascending - dose tolerance in CF patients.

[0029] Figure 19 Depicts sputum production (in grams) in CF patients in a single - ascending - dose tolerance study.

[0030] Figure 20 Depicts the percentage of solids in sputum produced by CF patients in a single - ascending - dose tolerance study.

[0031] Figure 21 . ENaC inhibition increases baseline MCCV but has no additive effect on dual - agonist stimulation. Time course of MCCV in WT neonatal piglet tracheas (n = 4 each) in response to 10 μM benzamil (Bz, red open squares) or dual agonists in the presence (solid squares) and absence (open blue triangles) of benzamil.

[0032] Figure 22 The co - agonist increases HCO3− secretion. Compared with the baseline condition, the co - agonist significantly increases the HCO3− secretion rate (p = 2E - 05, n = 13, from tracheas of 8 pigs).

[0033] Figure 23 The co - agonist increases the ASL height in ex - vivo WT porcine tracheas. A. Changes in the mean ASL height of DMSO (open black squares, n = 2 pigs) or drug (solid orange squares, n = 4 pigs). B. Summary of the rates of increase in ASL height in baseline (Bs), formoterol, and co - agonist. Detailed implementation manners

[0034] Definitions

[0035] Before describing the exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the specification.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994) and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide the general meaning of many of the terms used herein. Nevertheless, for clarity and ease of reference, certain terms are defined below.

[0037] Certain ranges of numerical values given herein are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number preceding it as well as a number that is close to or approximately the number preceding it. In determining whether a number is close to or approximates a specifically recited number, a number that is close to or approximates the recited number may be a number that provides a substantial equivalent to the specifically recited number in the context presented.

[0038] It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "β-adrenergic agonist" refers to one or more β-adrenergic agonists, i.e., a single β-adrenergic agonist and multiple β-adrenergic agonists. It should also be noted that claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a basis for antecedent recitation of claim elements using exclusive terms such as "solely", "only", etc., or using "negative" limitations.

[0039] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides applicable to the described embodiments.

[0040] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0041] As used herein, the term "naturally occurring" when applied to a nucleic acid, protein, cell, or organism refers to a nucleic acid, protein, cell, or organism found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and has not been deliberately modified by humans in the laboratory is naturally occurring.

[0042] As used herein, the term "exogenous" when applied to a nucleic acid or protein refers to a nucleic acid or protein that does not normally or naturally occur in a given bacterium, organism, or cell and / or is not produced by a given bacterium, organism, or cell. As used herein, the term "endogenous nucleic acid" refers to a nucleic acid that normally occurs in a given bacterium, organism, or cell and / or is produced by a given bacterium, organism, or cell in nature. "Endogenous nucleic acid" is also referred to as "native nucleic acid" or nucleic acid "native" to a given bacterium, organism, or cell. As used herein, the term "endogenous polypeptide" refers to a polypeptide that normally occurs in a given bacterium, organism, or cell and / or is produced by a given bacterium, organism, or cell in nature.

[0043] As used herein, "recombinant" refers to a particular nucleic acid or protein that is the product of various combinations of cloning, restriction, and / or ligation steps, and that results in a construct having a structural coding or non-coding sequence that is distinguishable from the endogenous nucleic acids found in natural systems. Typically, the DNA sequences encoding the structural coding sequences can be assembled from cDNA fragments and short oligonucleotide linkers or a series of synthetic oligonucleotides to provide a synthetic nucleic acid capable of being expressed from a recombinant transcription unit contained in a cellular or cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns, which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences can also be used to form recombinant genes or transcription units. Untranslated DNA sequences can be present at the 5' or 3' of the open reading frame, where such sequences do not interfere with the operation or expression of the coding region and can in fact regulate the production of the desired product by various mechanisms.

[0044] Thus, for example, the term "recombinant" nucleic acid or "recombinant" protein refers to a nucleic acid or protein that is not naturally occurring, e.g., is artificially combined from two originally separated sequence segments by human intervention. Such artificial combination is typically achieved by chemical synthesis methods or by manually manipulating the separated nucleic acid segments (e.g., by genetic engineering techniques). This typically involves replacing codons with redundant codons encoding the same or conserved amino acids, and usually introducing or removing sequence recognition sites. Alternatively, it is performed to join nucleic acid segments having desired functions together to produce a desired functional combination. Such artificial combination is typically achieved by chemical synthesis methods or by manually manipulating the separated nucleic acid segments (e.g., by genetic engineering techniques).

[0045] As used herein, the term "sample" refers to a material or mixture of materials that contains one or more components of interest, typically (but not necessarily) in fluid form, i.e., in aqueous form. Samples can be obtained from a variety of sources, such as food, environmental materials, biological samples, or solids, such as tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, skin, external sections of the respiratory, intestinal, and urogenital tracts, tears, saliva, milk, blood cells, tumors, organs, and samples of in vitro cell culture components (including but not limited to conditioned media produced by cells growing in cell culture, putative virus-infected cells, recombinant cells, and cell components). In certain embodiments of the method, the sample includes cells. In some cases of the method, the cells are in vitro. In some cases of the method, the cells are in vivo.

[0046] The term "biological sample" encompasses clinical or non-clinical samples and also includes tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, etc. "Biological sample" includes samples obtained from a patient's sample cells, e.g., a sample containing polynucleotides and / or polypeptides obtained from a patient's sample cells (e.g., a cell lysate or other cell extract containing polynucleotides and / or polypeptides); and a sample containing a patient's sample cells. A biological sample containing a patient's sample cells may also include normal, non-diseased cells. Biological samples can be from plants or animals. Biological samples can also be from any species. In certain embodiments of the method, the biological sample includes cells. In some cases of the method, the cells are in vitro. In some cases of the method, the cells are in vivo.

[0047] The term "antibody" encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies, and also: hybrid (chimeric) antibody molecules (see, e.g., Winter et al. (1991) Nature 349:293-299; and U.S. Patent No. 4,816,567); F(ab′)2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, e.g., Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies (see, e.g., Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and UK Patent Publication No. GB 2,276,169, published September 21, 1994); and any functional fragment obtained from such molecules, wherein such fragment retains the specific binding properties of the parental antibody molecule.

[0048] A "single-chain antibody", "single-chain variable fragment", or "scFv" has a variable heavy chain domain (VH) and a variable light chain domain (VL) of an antibody linked together by a flexible peptide linker. The peptide linker typically has a length of 10 - 25 amino acids. The single-chain antibody retains the antigen-binding properties of the native full-length antibody, but is smaller than the native intact antibody or Fab fragment due to the lack of the Fc domain.

[0049] As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V HH ), and is known to those skilled in the art. They are derived from heavy-chain only antibodies, found in Camelidae (Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). Immunoglobulins lacking light polypeptide chains are found in the "Camelidae" family. "Camelidae" includes Old World Camelidae (Bactrian camel and dromedary) and New World Camelidae (e.g., llama (Llama paccos), guanaco (Llama glama), vicuña (Llama guanicoe), and alpaca (Llama vicugna)). The single variable domain heavy-chain antibody is referred to herein as a nanobody or V HH antibody. Nanobodies are smaller than human antibodies. Nanobodies are typically 12 - 15 kDa, human antibodies are typically 150 - 160 kDa, Fab fragments are approximately 50 kDa, and single-chain variable fragments are approximately 25 kDa. Nanobodies have certain advantages over conventional antibodies, including smaller size, easier engineering, higher chemical and thermal stability, better solubility, deeper tissue penetration, the ability to bind small cavities and inaccessible target protein epitopes, the ability to be produced in microbial cells (i.e., lower production costs relative to animal immunization), etc. Specific nanobodies have been successfully generated using yeast surface display, as shown by McMahon et al. (2018) Nature Structural Molecular Biology 25(3):289 - 296, which is hereby specifically incorporated by reference.

[0050] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present disclosure that is effective to achieve a desired therapeutic outcome, e.g., to improve the symptoms of an individual suffering from cystic fibrosis and / or reduce disease severity, improve mucociliary clearance, increase submucosal gland secretion, or inhibit cholinergic agonist-induced muscle contraction. In the context of the present invention, desired therapeutic outcomes include clearing mucus from the lungs of such patients or inhibiting mucus accumulation in the lungs of such patients. Although the dosages mentioned in the present disclosure are guidelines, the attending physician may adjust the dosage according to the specific needs of the patient, including, for example, the severity of the disease, body size, and physical condition.

[0051] "Treatment", "treating", "preventing", "inhibiting", and the corresponding terms include therapeutic treatment, prophylactic treatment, and treatment to reduce the risk of a subject developing a disorder or risk factor. Treatment does not require complete cure of the disorder or condition and includes reducing severity, alleviating symptoms, reducing other risk factors associated with the condition, and / or improving the effects of the disease, e.g., slowing the progression of the disease.

[0052] Cystic fibrosis is a genetic disorder that disrupts anion transport in exocrine glands and "wet" epithelia, primarily affecting the gastrointestinal and respiratory systems. It results in chronic lung disease, exocrine pancreatic insufficiency, hepatobiliary disease, and abnormally high sweat electrolytes. Diagnosis is by sweat testing or identification of 2 cystic fibrosis-causing gene variants in patients with positive newborn screening test results or characteristic clinical features. Treatment is supported by aggressive multidisciplinary care and small molecule correctors and potentiators for cystic fibrosis transmembrane conductance regulator (CTFR) protein defects.

[0053] Approximately 3% of white people carry the autosomal recessive trait for cystic fibrosis, with a lower carrier rate in non-whites. The responsible gene is located on the long arm of chromosome 7. It encodes a membrane-associated protein called cystic fibrosis transmembrane conductance regulator (CFTR). The most common gene variant, F508del, occurs in approximately 85% of CF alleles; over 2000 less common CFTR variants have been identified.

[0054] CFTR is a cyclic adenosine monophosphate (cAMP)-regulated anion channel. It conducts chloride and bicarbonate ions and affects the transport of other ions (notably sodium ions) across epithelial membranes. There may be many other functions. The disease only manifests in homozygotes. Heterozygotes show subtle abnormalities in epithelial electrolyte transport and are clinically unaffected, but have a slightly increased risk of many CF-related conditions (Miller, Proc Natl Acad Sci USA, 2020, 117, 1621).

[0055] CFTR mutations have been classified into six classes according to how they affect the function or processing of the CFTR protein. Patients with class I, II, or III mutations are considered to have a more severe genotype, resulting in little or no CFTR function, while patients with one or two class IV, V, or VI mutations are considered to have a milder genotype, resulting in residual CFTR function. However, there is no strict relationship between a specific mutation and disease manifestation, so clinical testing (i.e., organ function) rather than genotyping is a better guide for prognosis. CFTR mutations can involve frameshifts (deletions or insertions in the DNA sequence that change the way the sequence is read) or nonsense (stop) mutations.

[0056] Fifty percent of patients who are not diagnosed by newborn screening present with pulmonary manifestations, usually starting in infancy. Lung disease is the result of mucus obstructing the airways. This mucus obstruction promotes recurrent or chronic infections, typically presenting as coughing, sputum production, and wheezing. Cough is the most troublesome symptom, often accompanied by phlegm, retching, vomiting, and sleep disturbances. As the disease progresses, intercostal retractions, use of accessory respiratory muscles, barrel chest deformity, clubbing of the fingers, cyanosis, and decreased exercise tolerance occur. Upper airway involvement includes nasal polyps and chronic or recurrent sinusitis.

[0057] β-adrenergic agonists are drugs that relax the airway muscles, causing the airways to dilate, thus making breathing easier. They are a class of sympathomimetic drugs, each acting on β-adrenergic receptors. In general, pure β-adrenergic agonists have the opposite function to β-blockers: β-adrenergic receptor agonist ligands mimic the action of adrenaline and noradrenaline signaling in the heart and lungs, as well as in smooth muscle tissue; adrenaline shows a higher affinity. β 1 、β 2 and β 3 activation activates adenylate cyclase. This in turn leads to the activation of the secondary messenger cyclic adenosine monophosphate (cAMP); then cAMP activates protein kinase A (PKA), which phosphorylates target proteins, ultimately inducing smooth muscle relaxation and cardiac tissue contraction.

[0058] Cholinergic agonists are a class of agents that act on the neurotransmitter acetylcholine, which is the main neurotransmitter within the parasympathetic nervous system (PNS). Cholinergic agonists stimulate cholinergic receptors, including nicotinic and muscarinic receptors. There are two main classes of cholinergic drugs: direct-acting and indirect-acting. Direct-acting cholinergic agonists act by directly binding to and activating muscarinic receptors. Examples of direct-acting cholinergic drugs include choline esters (acetylcholine, methacholine, carbachol, bethanechol) and alkaloids (muscarine, pilocarpine, cevimeline). Indirect-acting cholinergic drugs increase the availability of acetylcholine at cholinergic receptors. These include reversible drugs (physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine) and irreversible drugs (echothiophate, parathion, malathion, diazinon, sarin, soman). Detailed Description

[0060] Before describing the various embodiments, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and thus may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the teachings will be limited only by the appended claims.

[0061] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. While the teachings are described in connection with various embodiments, the teachings are not limited to these embodiments. Instead, the teachings cover various alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Some exemplary methods and materials are now described, but any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the teachings.

[0063] Any reference to a publication is for its disclosure prior to the filing date and should not be construed as an admission that the claimed invention is not entitled to antedate such publication by virtue of a prior invention. In addition, the provided publication dates may be different from the actual publication dates that can be independently verified.

[0064] As will be apparent to those skilled in the art upon reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of the several other embodiments without departing from the scope or spirit of the present teachings. Any of the recited methods can be performed in the order of the recited events or in any other order that is logically possible.

[0065] All patents and publications cited herein, including all sequences disclosed in such patents and publications, are hereby expressly incorporated by reference.

[0066] Where a numerical range is provided, it is to be understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range and any other stated value or intervening value in the stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specific exclusions recited in the stated range. When the stated range includes one or both of the limits, ranges excluding either one or both of those included limits are also included in the invention.

[0067] It should be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. The invention expressly encompasses all combinations of embodiments related to the invention and is hereby disclosed as if each such combination were separately and expressly disclosed. In addition, the invention also expressly encompasses all sub-combinations of the various embodiments and their elements and is hereby disclosed as if each such sub-combination were separately and expressly disclosed.

[0068] In further describing aspects of the invention, methods for treating an individual with cystic fibrosis are first described in more detail. Next, methods for increasing submucosal gland secretion are described. Next, methods for inhibiting cholinergic agonist-induced muscle contraction are described. Finally, compositions for practicing the methods disclosed herein are reviewed. Methods for treating mucosal obstructive disorders in an individual

[0069] The present disclosure provides a method for treating a mucosal obstructive disorder in an individual, the method comprising: administering to the individual a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to treat the mucosal obstructive disorder in the individual.

[0070] The methods disclosed herein can be used to treat a variety of mucosal obstructive disorders to directly treat the disorder or alleviate symptoms associated with the disorder. Mucosal obstructive disorders that can be treated with these methods include, but are not limited to, cystic fibrosis, primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, chronic bronchitis, non-CF bronchiectasis, and the like.

[0071] When the mucosal obstructive disorder is cystic fibrosis, the individual can be any individual predicted to have or diagnosed with cystic fibrosis. The diagnosis can be based on specific genetic variations in the CFTR gene that result in a non-functional CTFR protein or a CFTR with reduced functionality. The diagnosis can also be based on symptoms or a collection of symptoms of clinical features. Genetic variations associated with cystic fibrosis include, but are not limited to, G85E, R117H, 621+1G→T, 711+1G→T, 1078delT, R334W, R347P, A455E, ΔI507, ΔF508, 1717-1G-A, G542X, S549N, G551D, R553X, R560T, 1898+1G→A, 2184delA, 2789+5G→A, R1162X, 3659delC, 3849+10kbC, W1282X, N1303K, and the like. The individual can have genetic variations other than those described above. Individuals that can be treated with the method for cystic fibrosis or other mucosal obstructive disorders are typically mammals. Non-limiting examples of mammals that can be treated with the method include, but are not limited to, pigs, ferrets, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, and the like. In some embodiments, the individual is a human.

[0072] The β-adrenergic agonists of the present disclosure can be any β-adrenergic agonist that activates β-adrenergic receptors. The β-adrenergic agonists can target any β-adrenergic receptor that is considered useful, including β 1 -adrenergic receptor, β 2 -adrenergic receptor, or β 3 -adrenergic receptor. In some embodiments, the β-adrenergic agonist is a β 2 -adrenergic agonist that targets the β 2 -adrenergic receptor. A variety of different β 2β-adrenergic agonists are used to practice the methods disclosed herein, including but not limited to bitolterol, fenoterol, isoprenaline, isoproterenol, levalbuterol, levalbuterol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, ibuterol, mabuterol, zilpaterol, etc. In some embodiments, the β 2 -adrenergic agonist is formoterol. In some embodiments, an adenylate cyclase activator (such as forskolin) is used instead of the β 2 -adrenergic agonist. Adenylate cyclase activators include forskolin and corforsin.

[0073] The cholinergic agonists of the present disclosure are any molecules that mimic the activity of the neurotransmitter acetylcholine and act on muscarinic receptors. A range of different cholinergic agonists can be utilized in the methods practiced herein. Cholinergic agonists can be direct-acting or indirect-acting. Non-limiting examples of direct-acting cholinergic agonists are acetylcholine, methacholine, carbachol, bethanechol, muscarine, pilocarpine, and cevimeline. Non-limiting examples of indirect-acting cholinergic agonists are physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine, ecothiopate, parathion, malathion, diazinon, sarin, and soman. In some embodiments, the direct-acting cholinergic agonist is methacholine. In some embodiments, the direct-acting cholinergic agonist is carbachol.

[0074] The combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist can be administered at a specific time and / or in a specific order in order to treat cystic fibrosis or other mucosal obstructive disorders in an individual. Thus, the β-adrenergic agonist or adenylate cyclase activator and the cholinergic agonist can be administered sequentially or simultaneously. In some embodiments, the β-adrenergic agonist or adenylate cyclase activator is administered and then the cholinergic agonist is administered after a delay. In some embodiments, the cholinergic agonist is administered and then the β-adrenergic agonist or adenylate cyclase activator is administered after a delay. The delay can be a range of different time periods. For example, the delay can be 5 min or longer, 10 min or longer, 15 min or longer, 20 min or longer, 25 min or longer, 30 min or longer, 35 min or longer, 40 min or longer, 45 min or longer, 50 min or longer, 55 min or longer, 60 min or longer, or greater than about 60 min.

[0075] In some embodiments, a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist is administered simultaneously. When a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist is administered simultaneously, or when a β-adrenergic agonist or an adenylate cyclase activator is administered prior to a cholinergic agonist, certain benefits can be conferred. For example, administering a β-adrenergic agonist or an adenylate cyclase activator simultaneously or prior to cholinergic administration can inhibit cholinergic agonist-induced airway smooth muscle contraction. Prior to the present disclosure, it was well established that a β-adrenergic agonist should not be administered prior to a cholinergic agonist. It has been unexpectedly found that administering a β-adrenergic agonist prior to a cholinergic agonist can inhibit cholinergic agonist-induced airway smooth muscle contraction and airway narrowing. In some embodiments, administration of a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist results in a synergistic increase in mucus transport relative to mucus transport using either agonist alone.

[0076] The method involves administering locally or systemically a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist. When the combination is administered locally, the combination can be administered directly to the trachea or lungs, or to a site near the trachea or lungs. When the combination is administered locally, the combination can be administered using an oral or nasal inhaler. When the combination is administered systemically, the combination can be administered in a convenient systemic form, such as in the form of a pill or oral tablet.

[0077] The combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist can be administered as a single daily dose, multiple daily doses (e.g., 2 or 3 doses per day), intermittently, or weekly, and the dosing regimen depends on the dosage form (e.g., immediate release or controlled release) and individual needs. Administration can be for an extended period of time, intermittently, or for a limited time, and can be repeated if a qualified professional determines and to a determined extent. For example, the combination can be provided daily for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, or greater than 30 days, and then stopped. In some embodiments, the combination is administered intermittently, such as every 2 days, or 3 days, or weekly.

[0078] In some embodiments, the method further comprises administering one or more cystic fibrosis transmembrane conductance regulator (CFTR) modulators. It has been found that the combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist, when co-administered with a CFTR modulator, can be effective in increasing mucus clearance. When a CFTR modulator is co-administered with the combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist, any CFTR modulator that can effectively treat cystic fibrosis can be used. CFTR modulators that can be used in the present disclosure include, but are not limited to, elexacaftor, tezacaftor, ivacaftor, lumacaftor, voxelotor, deuterated ivacaftor, etc. In one embodiment, the one or more CFTR modulators are elexacaftor, tezacaftor, and ivacaftor. In some embodiments, co-administration of the combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist with one or more CFTR modulators results in a synergistic increase in mucus transport relative to the combination or the one or more CFTR modulators alone.

[0079] In some embodiments, the combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist can be co-administered with other drugs in addition to one or more CFTR modulators. Other drugs include any drug that directly treats cystic fibrosis or other mucosal obstructive disorders or their symptoms, such as substances that improve mucus clearance. For example, other drugs include, but are not limited to, recombinant human DNase, such as Pulmozyme, hypertonic saline, powdered mannitol, etc., and carriers designed to transfect airway cells with agents designed to enhance defective CFTR proteins by providing reagents (such as cDNA or mRNA).

[0080] The treatments disclosed herein can have many different effects on individuals suffering from cystic fibrosis or other mucosal obstructive disorders. For example, administration can increase the mucociliary clearance rate, increase the rate of submucosal gland secretion in the airway, and inhibit cholinergic agonist-induced airway smooth muscle contraction. Administration can also alleviate symptoms associated with cystic fibrosis or other mucosal obstructive disorders, including but not limited to recurrent or chronic infections, cough, sputum production, wheezing, abdominal distension, constipation, etc. Methods for increasing the rate of submucosal gland secretion in the airway

[0081] The present disclosure provides a method for increasing the rate of submucosal gland secretion in the airway of an individual, the method comprising: administering to the individual a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to increase the rate of submucosal gland secretion in the airway of the individual.

[0082] The β-adrenergic agonists of the present disclosure can be any β-adrenergic agonist that activates β-adrenergic receptors. In embodiments, the β-adrenergic agonist is a β 2 -adrenergic agonist that targets the β 2 -adrenergic receptor. A variety of different β 2 -adrenergic agonists can be used to practice the methods disclosed herein, including but not limited to bitolterol, fenoterol, isoproterenol, isoprenaline, levalbuterol, levosalbutamol, metaproterenol, pirbuterol, procaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, etc. In some embodiments, the β 2 -adrenergic agonist is formoterol. In some embodiments, an adenylate cyclase activator (such as forskolin) is used in place of the β 2 -adrenergic agonist.

[0083] The cholinergic agonists of the present disclosure are any molecules that mimic the activity of the neurotransmitter acetylcholine and act on muscarinic receptors. Cholinergic agonists can be direct-acting or indirect-acting. Non-limiting examples of direct-acting cholinergic agonists are acetylcholine, methacholine, carbachol, bethanechol, muscarine, pilocarpine, and cevimeline. Non-limiting examples of indirect-acting cholinergic agonists are physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine, ecothiopate, parathion, malathion, diazinon, sarin, and soman. In some embodiments, the cholinergic agonist is methacholine. In some embodiments, the cholinergic agonist is carbachol.

[0084] The combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist can be administered at a specific time and / or in a specific order in order to treat a subject with cystic fibrosis. In some embodiments, a β-adrenergic agonist or an adenylate cyclase activator is administered and then a cholinergic agonist is administered after a delay. In some embodiments, the combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist is administered simultaneously.

[0085] The methods disclosed herein increase the rate of submucosal gland secretion in the airway. The rate of submucosal gland secretion can be increased by a series of different values. For example, administration of a combination of an α-adrenergic agonist and a cholinergic agonist can increase the rate of submucosal gland secretion by at least about 0.5 nL / min, at least about 1.0 nL / min, at least about 1.5 nL / min, at least about 2.0 nL / min, at least about 3.0 nL / min, at least about 4.0 nL / min, at least about 5.0 nL / min, at least about 6.0 nL / min, at least about 7.0 nL / min, at least about 8.0 nL / min, at least about 9.0 nL / min, at least about 10.0 nL / min, or greater than about 10.0 nL / min.

[0086] The rate of submucosal gland secretion can be increased in different individuals. Individuals who can benefit from such methods include individuals with reduced mucus production, individuals who produce overly viscous mucus, or individuals with chronic airway inflammation. Individuals who can respond to these methods are typically mammals. Non-limiting examples of mammals that can be treated using this method include, but are not limited to, pigs, ferrets, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, and the like. In some embodiments, the individual is a human. In some embodiments, the individual is a pig. In some embodiments, the individual is a ferret.

[0087] In some embodiments, the individual has a genetic variation in CFTR. Genetic variations in CFTR include, but are not limited to, G85E, R117H, 621+1G→T, 711+1G→T, 1078delT, R334W, R347P, A455E, ΔI507, ΔF508, 1717-1G-A, G542X, S549N, G551D, R553X, R560T, 1898+1G→A, 2184delA, 2789+5G→A, R1162X, 3659delC, 3849+10kbC, W1282X, N1303K, etc. The individual can have genetic variations other than those described above.

[0088] Methods for inhibiting cholinergic agonist-induced airway smooth muscle contraction

[0089] The present disclosure provides a method for inhibiting cholinergic agonist-induced airway smooth muscle contraction in an individual, the method comprising administering to the individual a β-adrenergic agonist or an adenylate cyclase activator, wherein the administration of the β-adrenergic agonist or the adenylate cyclase activator is performed before or simultaneously with the administration of the cholinergic agonist to the individual to inhibit airway smooth muscle contraction.

[0090] The β-adrenergic agonist of the present disclosure can be any β-adrenergic agonist that activates the β-adrenergic receptor. In an embodiment, the β-adrenergic agonist targets β2 β - adrenergic receptor 2 -adrenergic agonist. A variety of different β 2 -adrenergic agonists can be used to practice the methods disclosed herein, including but not limited to bitolterol, fenoterol, isoproterenol, isoprenaline, levosalbutamol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, salbutamol, terbutaline, arformoterol, bambuterol, clenbuterol, formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, etc. In some embodiments, the β 2 -adrenergic agonist is formoterol. In some embodiments, an adenylate cyclase activator (such as forskolin) is used in place of the β 2 -adrenergic agonist.

[0091] The cholinergic agonists disclosed herein are any molecules that mimic the activity of the neurotransmitter acetylcholine and act on muscarinic receptors. Cholinergic agonists can be direct-acting or indirect-acting. Non-limiting examples of direct-acting cholinergic agonists are acetylcholine, methacholine, carbachol, bethanechol, muscarine, pilocarpine, and cevimeline. Non-limiting examples of indirect-acting cholinergic agonists are physostigmine, neostigmine, pyridostigmine, edrophonium, rivastigmine, donepezil, galantamine, ecothiopate, parathion, malathion, diazinon, sarin, and soman. In some embodiments, the cholinergic agonist is methacholine. In some embodiments, the cholinergic agonist is carbachol.

[0092] The methods disclosed herein inhibit cholinergic agonist-induced airway smooth muscle contraction. Cholinergic agonist-induced airway smooth muscle contraction can be inhibited by a series of different amounts. For example, administration of a β-adrenergic agonist or an adenylate cyclase activator can inhibit cholinergic agonist-induced muscle contraction by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0093] Cholinergic agonist-induced airway smooth muscle contractions can be inhibited in a series of different individuals. Individuals who can obtain specific benefits from such methods are those who have taken or regularly take cholinergic agonist-based drugs. Cholinergic agonist-based drugs are used to treat a variety of different conditions, including but not limited to myasthenia gravis, xerostomia, urinary retention, neurogenic bladder, ophthalmic surgery assistance, glaucoma, dementia, acute colonic pseudo-obstruction, anticholinergic drug overdose, Sjogren's syndrome, etc. Individuals who can respond to these methods are usually mammals. Non-limiting examples of mammals that can be treated using this method include but are not limited to pigs, weasels, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc. In some embodiments, the individual is a human. In some embodiments, the individual is a pig. In some embodiments, the individual is a weasel.

[0094] In some embodiments, the individual has a genetic variation of CFTR. Genetic variations of CFTR include but are not limited to G85E, R117H, 621+1G→T, 711+1G→T, 1078delT, R334W, R347P, A455E, ΔI507, ΔF508, 1717-1G-A, G542X, S549N, G551D, R553X, R560T, 1898+1G→A, 2184delA, 2789+5G→A, R1162X, 3659delC, 3849+10kbC, W1282X, N1303K, etc. The individual may have genetic variations other than those described above.

[0095] Composition

[0096] The present disclosure also describes compositions for practicing this method. Generally speaking, the subject compositions may further have a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist as described above, in addition to the pharmaceutical excipients as described above. The composition may be contained in a device. In some embodiments, the device is an inhaler.

[0097] In some embodiments, the composition may further comprise one or more CFTR modulators. The CFTR modulators of the present disclosure have been described in more detail above. In some embodiments, the composition may further comprise other drugs in addition to one or more CFTR modulators. Other drugs include any drugs that directly treat cystic fibrosis or other mucosal obstructive conditions or their symptoms, such as substances that improve mucus clearance. For example, other drugs include but are not limited to recombinant human DNAse, such as Pulmozyme, hypertonic saline, powdered mannitol, etc., and carriers designed to transfect airway cells with agents designed to enhance the defective CFTR protein by providing reagents (such as cDNA or mRNA).

[0098] In some embodiments, the composition is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers, with strengths ranging from 5 mM to 100 mM. In some embodiments, the aqueous buffer includes a reagent that provides an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars, such as mannitol, glucose, sucrose, etc. In some embodiments, the aqueous buffer further includes a nonionic surfactant, such as polysorbate 20 or 80. Optionally, the composition may also include a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, etc. In many cases, the formulation is stored at about 4°C. The pharmaceutical composition can also be lyophilized, in which case they typically include cryoprotectants, such as sucrose, trehalose, lactose, maltose, mannitol, etc. The lyophilized formulation can be stored for an extended period of time, even at ambient temperature.

[0099] Each active agent can be provided in unit doses of about 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg, 500 mg, 750 mg, or more.

[0100] The composition can be administered in unit dosage forms and can be prepared by any method known in the art. Such methods include combining a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist with a pharmaceutically acceptable excipient or diluent, which constitutes one or more accessory components. The pharmaceutically acceptable excipient is selected according to the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable", i.e., compatible with the other components in the formulation and not harmful to the subject. The carrier can be solid or liquid, and the type is generally selected according to the type of administration used.

[0101] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar, and bulk powders. Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions, and / or suspensions, and solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid carriers can contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickening agents, and solubilizing agents. Preferred carriers are edible oils, such as corn oil or rapeseed oil. Polyethylene glycol, such as PEG, is also a good carrier.

[0102] The combination of a β - adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist can be administered via pharmaceutical dosage forms known in the art, including but not limited to oral solid dosage forms, oral liquids, injections, transdermal patches, and inhalants. Oral dosage forms are formulated for systemic delivery, while inhalant dosage forms are formulated for local delivery. Systemic formulations are designed to be digested in the stomach and intestines, while inhalant delivery is designed to deliver locally and directly to the affected tissues, including the trachea and lungs. Dosage forms can be formulated with excipients and other compounds to facilitate administration to a subject and maintain shelf life. See "Remington’s Pharmaceutical Sciences" (Mack Publishing Co., Easton, PA). Oral pharmaceutical preparations include tablets, tablets, pills, granules, capsules, gels, liquids, syrups, and suspensions. The combination can be administered orally, typically via oral solid dosage forms, although oral liquids may be an ideal option for certain populations who have difficulty taking tablets and capsules, such as pediatric and elderly patients. Oral dosage forms can be immediate - release or controlled - release.

[0103] In one embodiment of the present invention, the combination of a β - adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist can be provided as an immediate - release formulation. The immediate - release of the combination can be provided as a single daily dose or divided into multiple daily doses, which can be administered 2, 3, 4 times or more per day. In another embodiment of the present invention, the combination is provided as an extended - release formulation. Extended - release formulations can provide convenience to patients by reducing the number of daily administrations and can improve patient compliance. In addition, the controlled - release formulations of the present invention can be used to reduce serum peak - to - trough levels, thereby reducing adverse events.

[0104] Oral controlled - release formulations are known in the art and include sustained - release, extended - release, delayed - release, and pulsatile - release formulations. See "Remington’s Pharmaceutical Sciences" (Mack Publishing Co., Easton, PA). The active agent can be formulated into a matrix formulation, where one or more polymers slow the release of the drug from the dosage form, including hydrophilic agents or gelling agents, hydrophobic matrices, lipid or wax matrices, and biodegradable matrices. The active agent can be formulated in the form of beads, for example, having an inert sugar core and coated with known excipients to delay or slow the release of the active agent by diffusion. Enteric coatings are known in the art for delaying the release of the active agent until the dosage form passes from the low - pH environment of the stomach into the higher - pH environment of the small intestine, and enteric coatings can include copolymers of methyl acrylate - methacrylic acid, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate (PVAP), shellac, sodium alginate, and cellulose acetate trimellitate.

[0105] The compositions of the present disclosure can be included within a device. The device can be any device that permits delivery of the composition to an affected tissue (such as the trachea or lungs). The device includes, but is not limited to, nebulizers, oral inhalers, nasal inhalers, and the like. When the device is an inhaler, the inhaler can disperse a dry powder or a liquid in the form of an aerosol spray. The aerosol spray is typically provided by a pressurized package, using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (such as gelatin) for inhalers can be formulated to contain a powder mixture of the compound and a suitable powder matrix (such as lactose or starch). For example, preparations for inhaled administration can be prepared according to the teachings of U.S. Patent No. 7,812,120 B2 to Quay et al.

[0106] Kit

[0107] Kits for practicing certain methods described herein are also provided. In certain embodiments, the kit contains a β-adrenergic agonist and a cholinergic agonist, such as described above. In a given kit, the active agent can be packaged separately or present in a formulation, e.g., where the active agents are delivered to the subject simultaneously. As needed, the active agents can be present in the same or separate containers.

[0108] In certain embodiments, the kit will further include instructions for practicing the subject methods or means for obtaining the subject methods (e.g., a website URL that directs the user to a web page providing the instructions), where the instructions can be printed on a substrate, where the substrate can be one or more of the following: a package insert, the packaging, a reagent container, and the like. Another form of these instructions is a computer-readable medium having information recorded thereon, such as a floppy disk, a compact disc (CD), a portable flash drive, a USB storage, a DVD, a Blu-ray disc, etc.). Another form of these instructions is a website address that can be used via the Internet to access information on a remote site.

[0109] Examples

[0110] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art on how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; i.m., intramuscular; i.p., intraperitoneal; s.c., subcutaneous; and so on.

[0111] Example 1

[0112] The MCCV of CF ferrets and WT pigs increases synergistically. The'synergy paradigm' is defined as sequential exposure to cAMP or Ca 2+ elevating agonists for 30 min and combined agonists for at least 30 min. 10 μM forskolin, isoproterenol, or formoterol was used as the cAMP agonist, while 0.3 μM carbachol was used as the Ca 2+ elevating agonist (all in the basolateral). In previous studies, these combinations resulted in an increase in MCCV in ferret trachea that was much greater than the predicted additive effect of the two agonists and close to the maximum value (16).

[0113] To determine whether a synergistic increase in MCCV could be generated in the tracheas of CF ferrets, tracheas from 7 transgenic adult CF ferrets of mixed genotype were tested (see "Methods" section). The 7 CF ferret tracheas were divided into two groups for testing, with 4 first treated with forskolin and 3 first treated with carbachol, followed by the combined agonist. MCCV was then measured for 90 - 150 min (all values in mm / min) and plotted as MCCV vs. time and agonist in Fig. 1A. In the absence of stimulation, MCCV declined to near zero within the first 30 min (Fig. 1A, see legend for details). Forskolin did not cause an increase in MCCV, while carbachol caused only a small increase. However, when the agonists were combined in either order, they caused a sustained and substantial increase in MCCV to approximately 20 mm / min. Mean data for the last 20 min of each basal and single-drug treatment period and the 10 - 80 min period after addition of the combined agonist are shown as box plots in Fig. 1B. MCCV values were: unstimulated: 1.6 ± 1.09 (n = 3); 10 μM forskolin: 0.18 ± 0.09 (n = 4); 0.3 μM carbachol: 3.29 ± 2.08 (n = 3), "sum" is the arithmetic sum of MCCV caused by the two agonists used alone: 3.5 ± 2.07; SR, synergistic response to the combined agonist: 19.95 ± 4.12, (P = 0.006 SR vs sum).

[0114] Encouraged by these results, we began to wonder whether synergy could be observed in different species. MCCV was measured in the tracheas of WT piglets aged 2 - 5 days (see "Methods" section for details). The MCC velocity in the unstimulated state was less than 1 mm / min (mean T10 - 30 MCCV, 0.93 ± 0.39, n = 8 piglet tracheas), similar to that in WT ferrets (16). The 8 piglet tracheas were divided into two groups for testing, with 4 first treated with forskolin and the other 4 first treated with carbachol, followed by the combined agonist. Carbachol or forskolin alone caused only a slight increase in MCCV, but the combined agonist caused a sustained and substantial increase in MCCV to 12 - 17 mm / min regardless of the addition order (Fig. 1C). Mean data for the last 20 min of each basal and single agonist treatment period and the last 50 min of the synergistic paradigm period are shown as box plots in Fig. 1D. Mean MCCV values were: 0.3 μM carbachol: 0.91 ± 0.63 to 1.12 ± 0.82 (P = 0.32, Carb vs Bs / basal, 4 piglets); 10 μM forskolin: 0.95 ± 0.65 to 2.46 ± 0.68 (P = 0.13, n = 4); sum of individual responses (sum): 3.58 ± 1.06, synergistic paradigm (SR): 13.92 ± 0.94.

[0115] Box plots clearly show that for both CF ferrets and WT pigs, for the combined agonists, the increase in MCCV of the combined agonists is significantly greater than the arithmetic sum of the responses. Thus, MCCV synergy exists in at least two species and persists at least in part after loss of CFTR function. The MCCV of CF ferrets under synergistic conditions is 5.7-fold faster than the arithmetic sum of the responses to the agonists alone, but the actually important value lies in the comparison with WT ferrets. The values for CF ferrets are compared with those previously obtained from WT ferrets (16). For forskolin alone, the MCCV values for WT and CF ferrets are 6.75 ± 0.84 (n = 28) and 0.18 ± 0.09 (n = 4), respectively. For carbachol alone, the MCCV values for WT and CF ferrets are 8.24 ± 0.82 (n = 12) and 3.29 ± 2.08 (n = 3), respectively. For the synergistic response of the combined agonists, the MCCV values for WT and CF are 36.24 ± 0.9 (n = 40) and 19.95 ± 4.12 (n = 7), respectively. Thus, compared with WT ferrets, the response of CF ferrets to forskolin is approximately 0%, to carbachol is approximately 40%, and to synergy is approximately 55%.

[0116] These experiments used forskolin to elevate cAMP. To evaluate clinically accessible β-adrenergic drugs, the response of MCCV to the β2-adrenergic receptor agonist formoterol at 10 μM was measured instead of forskolin. A similar synergistic increase in MCCV (in mm / min) was observed in porcine trachea: baseline, 0.3 ± 0.1 (n = 12); 10 μM formoterol, 2.4 ± 0.9 (n = 5), 0.3 μM carbachol, 1.3 ± 0.8 (n = 3); combined agonists were 10.9 ± 0.8 (n = 7 piglet tracheas) (Figs. 1E,F).

[0117] The combined agonists do not induce airway smooth muscle contraction or airway narrowing. The cAMP and Ca that increase MCCV 2+ The elevated agonists also affect airway smooth muscle. When used alone, they have opposite effects: Ca 2+Elevating agonists causes muscle contraction, while cAMP-elevating agonists cause muscle relaxation. For therapeutic use, combined agonists may produce unwanted bronchoconstriction, which is a safety concern. To determine which effect predominates, the responses of airway smooth muscle to carbachol ± 10 mM forskolin or formoterol were measured using two different methods: measuring muscle tension and luminal area. The tension of ferret tracheal muscle bundles was measured against increasing concentrations of carbachol ± 10 mM forskolin. Forskolin abolished the increase in tension for 0.3 and 0.6 mM carbachol and greatly reduced the response to higher doses of carbachol (Figures 2A,B). The luminal area imaged in thin sections of porcine or ferret tracheal rings decreased continuously by 20 - 40% upon exposure to 0.3 mM carbachol, but when forskolin or formoterol was used prior to carbachol, it only caused a transient decrease of 5% or less( Figure 2C-F ). Importantly, a protective effect was also observed in CF ferrets (Figure 2F).

[0118] The rate of mucus clearance reflects the transportability of mucus and the ciliary beat frequency. Transportability depends mainly on the hydration / concentration (17) and pH (or bicarbonate content) (18, 19) of mucus. The main source of upper airway fluid is the submucosal gland, and agonists used to stimulate MCCV also stimulate submucosal gland secretion (20 - 29). The surface epithelium that secretes and absorbs electrolytes / fluid also alters the depth and composition of the ASL. In fact, this is the main method of controlling the ASL in airways lacking submucosal glands (30). In previous studies by us and others (26, 31 - 33), evidence was found for cholinergic inhibition of Na+ absorption, which tends to increase the fluidity and transportability of mucus. Finally, CBF was increased by raising Ca 2+ (34) or cAMP (35). The following experiments sought evidence to support or challenge the possible contribution of these mechanisms to the synergistic increase in MCCV.

[0119] Synergistic glandular mucus secretion in WT pigs, WT ferrets, and CF ferrets. The synergistic increase in MCCV depends in part on an increase in submucosal gland mucus secretion. This hypothesis stems from the evidence that [Ca 2+ i -elevating agonists and [cAMP] i -elevating agonists synergistically increase the rate of mucus secretion from submucosal glands in humans (22), pigs (24), and ferrets (21). However, different specific concentrations of agonists were used in these experiments. To determine whether the same protocol used here produced a synergistic increase in submucosal gland secretion, the rate of mucus secretion from individual tracheal glands in WT pigs and ferrets and CF ferrets was measured by time-lapse optical imaging (28), while stimulating them with the same concentrations of agonists and exposure durations used in the MCCV studies. All secretion rates were reported in nanoliters / min / gland.​

[0120] In WT pig tracheal glands, the mean unstimulated secretion rate was nl / min / gland (0.21 ± 0.06, 121 glands, 8 pigs, Figure 3A). Each agonist alone significantly increased the basal rate, and combinations in any order further increased the basal rate. The rate of the combined agonists was significantly greater than the arithmetic sum of their individual responses: sum = 1.26 ± 0.19, 7 pigs vs combined agonists = 2.86 ± 0.25 (2.3-fold greater, P < 0.01, 8 pigs). Figure 3B shows data for an individual pig with forskolin first, and Figure 3C shows data for an individual pig with carbachol first.

[0121] WT ferrets gave similar results ( Figure 3D-F ). In ferret tracheal glands, the mean unstimulated secretion rate was approximately zero (0.003 ± 0.001, 67 glands, 7 ferrets, Figure 3D). Forskolin (0.26 ± 0.07, 37 glands, 7 ferrets P < 0.05) and carbachol (0.94 ± 0.28, 30 glands, 7 ferrets P < 0.05) significantly increased this rate. The secretion rate of the combined agonists was significantly greater than the arithmetic sum of their individual responses (Figure 3D): total arithmetic sum = 1.27 ± 0.23, while the combined agonists were 2.46 ± 0.39 (1.9-fold greater, P < 0.05, 55 - 67 glands, 5 - 7 ferrets). Figure 3E shows data for an individual WT control ferret with forskolin first, and Figure 3F shows data for an individual WT control ferret with carbachol first.

[0122] Importantly, CF ferrets (CFTRKO / KO) also exhibited synergistic glandular secretion, although there was no response to forskolin alone. Only two CF ferrets could be tested ( Figure 3G-I ). One CF ferret was first stimulated with 10 mM forskolin and the other with 0.3 mM carbachol, both using the synergistic paradigm. The unstimulated secretion rate was approximately zero, as in WT ferrets. Forskolin alone failed to stimulate secretion as expected (0.01, 7 glands), carbachol alone increased the mean secretion rate to 0.45 ± 0.16, while the combined agonists increased the mean secretion rate to 1.23 ± 0.35, 7 glands, and 1.31 ± 0.19, 7 glands. Synergy was observed in both addition orders when the agonists were combined. The mean secretion rate for the combined agonists in the two ferrets was 1.27 ± 0.15, 2.8-fold the arithmetic sum of the two agonists alone, and approximately half of the WT ferret response of 2.46 ± 0.39 (16).

[0123] In summary, the mucus secretion rates of two species, including CF ferrets, increased to values exceeding the sum of the agents used alone, providing indirect evidence that glandular secretion rates contribute to MCCV in our system.

[0124] The combined agonists stimulate anion secretion from the epithelial surface and inhibit Na + absorption. The surface epithelium also modifies the ASL. Figure 4A is a cartoon of the major ion fluxes that control ASL depth: increased anion-mediated fluid secretion and decreased Na + -mediated fluid absorption reduce ASL depth. It is hypothesized that the combined agonists increase ASL depth by stimulating secretion and inhibiting absorption, thereby increasing MCCV (see also Figure 6). Figure 4B shows an example of our best Isc trace, which was obtained from porcine tracheal mucosa stimulated with forskolin followed by carbachol. Forskolin caused a sustained increase in Isc with no measurable change in conductance. When 0.3 μM carbachol was subsequently added, it induced an instantaneous increase in Isc (anion secretion), followed by a slow decline in Isc and conductance, such that after approximately 30 min, the conductance decreased to 84% of its pre-forskolin and immediately post-forskolin values. The ENaC inhibitor benzamil (Bz) did not cause a further change in Isc or conductance, indicating that carbachol completely inhibited ENaC-dependent Na + absorption. At this time, the epithelial cells were secreting anions, as indicated by the sharp decline in Isc and conductance produced by the two anion channel inhibitors BPO-27 and niflumic acid. Without balanced absorption, the ASL depth is expected to increase unless MCCV increases (dashed gold line in Figure 6A). Our evidence indicates that MCCV did increase.

[0125] Figures 4C-F show summary plots of ΔIsc as a function of time and stimulation. Each subplot shows the response of WT pigs (Figures 4C, D) and WT ferrets (Figures 4E, F) to 10 μM forskolin or 0.3 μM carbachol for the first 30 min, followed by the response to the combined agonists for the next 30 min. Forskolin increased ΔIsc in both species as expected, but when carbachol was added, ΔIsc slowly decreased (Figures 4C, E). Our interpretation of the Isc in the forskolin + carbachol condition is that forskolin increases Isc mainly by stimulating anion secretion, while carbachol greatly reduces Isc by inhibiting Na + absorption. Inhibiting Na + absorption increases the net fluid accumulation at the surface. When carbachol was added first, ΔIsc either decreased directly or decreased after a brief increase (Figures 4D, F). In both cases, the subsequent increase in ΔIsc for carbachol + forskolin was less than that for forskolin alone because their effects on Isc were opposite but additive on ASL depth.

[0126] Agonists stimulate human ciliary beat frequency (CBF). CBF is known to increase in response to an elevation of 2+ i (34) or [[cAMP]] i (35). It was tested whether CBF might increase synergistically with a combination of agonists. The CBF (in Hz) of unstimulated human nasal cells in KRB (Krebs buffer solution) was 6.79 ± 1.69 at 25 °C and 10.46 ± 0.95 at 37 °C (4 subjects, P = 0.01) (see "Methods" section). As Figure 5 shown, neither agonist significantly increased CBF, but when combined, their additive effect produced a 27.2% increase at 37 °C, reaching 13.31 ± 0.77 Hz. This was a significant increase compared to unstimulated CBF (n = 4, P < 0.05), but not compared to the arithmetic sum of the ΔCBF of the two agonists: combined agonists: 2.85 ± 0.76, arithmetic sum: 2.19 ± 0.66 (n = 4, P = 0.47). Thus, while the increase in CBF leads to an increase in MCCV, they are unlikely to explain the synergistic increase in MCCV seen with the combined agonists (see "Discussion" section).

[0127] Discussion

[0128] Main findings. There were six main findings. (1) The combined agonists synergistically increased the MCCV of CF transgenic ferrets to 19.95 ± 4.12 mm / min, which is approximately 55% of the MCCV of WT ferrets tested under similar conditions. (2) Pigs also showed a synergistic increase in MCCV, so this effect is not species-specific. (3) The combined agonists produced little airway narrowing in WT ferrets, WT pigs, and CF transgenic ferrets. As for the mechanism: (4) there was a synergistic increase in glandular mucus secretion in pigs, ferrets, and CF ferrets; (5) there was an increase in anion secretion from the surface epithelium; (6) there was a decrease in Na + absorption from the surface epithelium; and (7) there was an increase in CBF. The synergistic increase in MCCV was several-fold higher than that of the individual agonists or their combined response, and was close to the maximum reported in vivo. In anesthetized ferrets, the in vivo basal MCCV was 18.2 ± 1.0 mm / min and increased to 32.0 ± 3.8 mm / min under maximum anticholinesterase treatment (36). In anesthetized pigs, the in vivo mean basal MCCV was approximately 7 mm / min and the mean maximum MCCV was approximately 12 mm / min (37). If synergism also occurs in vivo, it should help to move mucus in some obstructive airway diseases.

[0129] ​The most notable result was the synergistic increase in MCCV in CF ferrets. This is therapeutically relevant and mechanistically interesting because in CF ferret trachea, forskolin alone does not increase MCCV (Figure 1A) or stimulate glandular mucus secretion (Figure 3H). (Using different synergy paradigms, human submucosal gland secretion is lost in the airways of subjects with CF (22)).

[0130] Thus, the combined agonist used in this study must activate an anion secretion pathway independent of CFTR that is resistant to forskolin alone (see below).

[0131] Strategies to increase mucus clearance. Strategies to increase mucus clearance are a mainstay of cystic fibrosis therapy but have limited effectiveness (6, 8 - 10). Pulmozyme (recombinant human DNase), hypertonic saline, and mannitol all improve mucus clearance in CF, while inhaled bicarbonate or tromethamine improves CF sputum rheology (38).

[0132] Well before the advent of Pulmozyme or hypertonic saline therapy, numerous studies had demonstrated that β - adrenergic (cAMP) agonists increase MCC (13, 39). In fact, β - adrenergic agonists (viewed as bronchodilators) are now widely used to treat obstructive diseases. However, the doses required to stimulate an increase in MCC are higher than those that reliably produce bronchodilation (39), and thus it is unclear to what extent currently used doses increase MCC. Unlike β - adrenergic drugs, cholinergic (Ca 2+ ) drugs cause bronchoconstriction, which is the basis of the methacholine challenge test (40), although cholinergic stimulation has been reported to increase human mucus transport (41). Cholinergic drugs also stimulate mucus secretion, and mucus overproduction is generally thought to contribute to mucus - obstructive diseases (42, 43). Thus, it is not surprising that no one has previously advocated the use of inhaled drugs that stimulate mucus secretion and cause bronchoconstriction for therapy. In fact, anticholinergic drugs are used to treat COPD, with modest efficacy, apparently due mainly to increased bronchodilation (44). Thus, we found it unexpected that the combination of forskolin (or the β - adrenergic drug formoterol) and a low - dose cholinergic drug significantly increased MCCV.

[0133] Our hypothesis is that the combinatorial agonists increase MCCV mainly because they increase ASL volume through three processes: synergistically increasing glandular mucus secretion, increasing fluid secretion, and decreasing surface epithelial absorption (Figure 6). The combinatorial agonists produced only a modest additional increase in CBF measured in Krebs solution. The greater increase in CBF may depend on the increase in ASL volume, which occurred in the MCCV experiments but not in the CBF experiments. Using micro-optical coherence tomography (μOCT) to visualize transport in the intact trachea, an increase in CBF in response to cholinergic stimulation was observed (45, 46). Importantly, all of this occurred without airway narrowing.

[0134] The concept that MCCV is faster if ASL depth increases is supported by studies in patients with pseudohypoaldosteronism (PHA), in which loss-of-function mutations in ENaC subunits abolish Na + absorption at the airway surface, which more than doubles the volume of ASL and results in a four-fold increase in the clearance of inhaled tracer from the lungs at 0 - 20 min (47). Previously, it has been shown that agonist-induced MCCV increases by approximately two-fold in ferrets when ENaC is inhibited (16). In these experiments (16), stimulation with forskolin or carbachol in the presence of ENaC inhibition increased MCCV to values similar to those seen with the combinatorial agonists, providing additional evidence that synergistic MCCV arises in part from ENaC inhibition. The idea that increased ASL provides faster clearance is also the logical basis for the increased clearance using β-agonists (39) and hypertonic saline (8, 9). In vitro porcine trachea studies also support this view, where stimulating secretion increases MCCV, blocking secretion slows MCCV, and blocking absorption increases MCCV in the trachea after secretion has been blocked (20).

[0135] Potential molecular and cellular mechanisms. The molecular and cellular mechanisms of synergistic MCCV by β-adrenergic and cholinergic agonists were not explored in this study, but given our evidence that ENaC inhibition contributes, previous studies on the molecular mechanisms of ENaC inhibition are relevant. A common theme is the role of elevated [Ca 2+ i (48 - 50), which can be achieved by a variety of agonists, including ATP, UTP, histamine, thapsigargin, and bradykinin (51). Cholinergic agonists increase [Ca 2+ i ; other mechanisms include increasing extracellular antiproteases (27, 52); while other ENaC inhibitors (25, 53) act by stimulating secretion from airway glands and surface epithelium.

[0136] ​​A synergistic increase in MCCV and glandular secretion was observed in CF ferrets. Therefore, mechanisms bypassing CFTR must be partly involved. It has been shown that intracellular crosstalk between the cAMP and Ca 2+ signaling pathways via inositol 1,4,5-trisphosphate receptor-binding protein and the release of IP3 (IRBIT) can mediate synergy in salivary glands and pancreatic ducts (54). The synergistic secretion of lacrimal glands in response to cAMP and cholinergic agonists is partly due to the inhibition of p44 / p42 mitogen-activated protein kinase (MAPK) by cAMP agonists (55). A previous study (50) showed that in isolated serous cells of human nasal cavities and WT&CFTR− / − pig tracheal glands, the synergistic fluid secretion provided by cAMP+Ca 2+ agonists might be induced by a cAMP-dependent Ca 2+ release mechanism and Ca 2+ agonists. However, another study on HEK293 cells (49) showed that cAMP agonists (such as parathyroid hormone or isoproterenol) alone did not increase [Ca 2+ i , but when combined with carbachol, cAMP agonists strengthened carbachol-induced Ca 2+ release by revealing non-continuous Ca 2+ pools in the endoplasmic reticulum. The differences in previous reports might partly result from using different cell or organ preparations and partly from using different measurement parameters, such as [Ca 2+ i versus [HCO 3 - i ([pH] i ). Our earlier studies (22, 24) showed that there are CFTR-dependent and non-CFTR-dependent pathways in synergistic glandular mucus secretion, depending on the doses of β-adrenergic and cholinergic agonists.

[0137] Potential therapeutic relevance for mucus obstructive diseases. Procedures to enhance mucociliary clearance are needed in people with mucus obstructive airway diseases, including a large number of people with CF (11, 56). Since β-agonists and methacholine are routinely used (the latter for testing airway hyperactivity), there is little to prevent their combined testing, unless this seems counterintuitive. Our in vitro data show that this combination can effectively accelerate mucus clearance without causing airway narrowing, even in CF animals (Figure 2), whose airway muscles have increased sensitivity to cholinergic agonists (57). Our findings on CF airways are consistent with an earlier study that found that in CF children, a substantial reduction in bronchoconstriction was observed when a β-adrenergic agonist was administered before methacholine (58).​​

[0138] Whether this combination is safe for individuals with airway hyperactivity remains to be seen. If the results are indeed worthy of further testing in CF patients, it will be important to initiate the use of a healthier airway as early as possible, as the observed trend of β-agonist improvement of MCC is that a healthier airway shows more benefits than a diseased airway (39).

[0139] Materials and Methods

[0140] Airway tissue procurement. CF ferret tissue. Seven transgenic CF ferret tracheas (five CFTR G551D / G551D , one CFTR ΔF508 / ΔF508 , one CFTR G551D / KO ) were used for MCC assays. These ferrets were maintained on the CFTR modulator VX770. Administration was stopped at least 3 weeks prior to euthanasia; no residual drug effects were anticipated or observed (zero response to forskolin). Two CFTRKO / KO ferret tracheas were used for tracheal single gland mucus secretion rate assays. Two CF ferret tracheas (one CFTR ΔF508 / ΔF508 and one CFTRG 551D / ΔF508 ) ferret tracheas were used for tracheal smooth muscle contraction assays. All isolated CF ferret tracheal trimmings (2 - 3 cm in length) were placed immediately in DMEM medium after euthanasia and shipped by overnight priority courier from the University of Iowa.

[0141] Porcine tissue. Neonatal WT piglet tracheas (2 - 5 days old) were obtained directly from the swine farm at the University of California, Davis or the laboratory of David Stoltz at the University of Iowa by overnight priority courier. Postmortem (<1 hour) tracheas from adult Yorkshire pigs (30 - 50 kg) and 5 - 12 - month - old European ferrets were from animal facilities in Stanford and Gilroy / California. All methods using animal tracheas were conducted in accordance with the relevant guidelines and regulations of Stanford University, and the animal protocol was approved (Stanford IACUC protocol number: 10,048). Porcine tracheas were transported in DMEM cell culture medium, and other animal tissues were transported to the laboratory in cold PhysioSol TM solution (Hospira, IL / USA) and then transferred to ice - cold Krebs Ringer bicarbonate (KRB) buffer, which was aerated with 95% O 2 and 5% CO 2 and stored at 4°C until use. The KRB buffer contained (in mM): 115 NaCl, 25 NaHCO3, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, and 1.0 μM indomethacin, and was adjusted to pH 7.2 and approximately 290 mOsm at room temperature.

[0142] Human tissues. During endoscopic sinus surgery at Yonsei University Hospital, human nasal mucosal tissues were obtained from nasal biopsies. All methods using human tissues were performed in accordance with the relevant guidelines and regulations of Yonsei University in Seoul, Korea. All experimental protocols were approved by Yonsei University, and informed consent was obtained from all participants prior to the study (Yonsei University - IRB protocol number: 4 - 2016 - 1153).

[0143] MCCV measurement. Details are described in previous reports (16, 59, 60). Briefly, each full - length ferret or piglet trachea or CF tracheal preparation was cut along the mid - dorsal line and mounted mucosa - side up onto a Sylgard elastomer platform. For pig experiments, tracheas from neonatal piglets were used because the tracheas of adult pigs (the source of our submucosal gland experiments) that had undergone acute experiments had unstable MCC velocities due to epithelial damage caused by intubation. The prepared tracheas were placed into a sealed humidified chamber that was continuously bubbled with gas (95% / 5% - O 2 / CO 2 ), and the serosal surface was soaked with KRB buffer ± drug. During an initial 30 - min stabilization period, the tissue was submerged in the bath and the temperature was gradually increased to 37°C. Then, the excess apical solution was drained, and the tissue was incubated for an additional 10 min before starting baseline measurements of MCCV. Drugs were added by replacing the bath with a pre - warmed bath + drug. Unless otherwise stated, MCCV data were reported as a single number and were based on the mean MCCV during the last 20 min of the treatment period.

[0144] Electrophysiology. Intact ferret tracheal preparations (approx. 0.5×1.0 cm 2 ) or pig mucosal preparations cut from cartilage were mounted in an EasyMount Ussing chamber (Physiologic Instruments, CA, USA) with an exposed surface area of 0.45 cm 2 , soaked in KRB buffer at 37°C, and ventilated with 95% O 2 / 5% CO 2 . The transepithelial short - circuit current (Isc) was obtained and displayed using a VCC - 600 voltage clamp (Physiologic Instruments, CA / USA) and PowerLab Chart4 software (V.4.1.2, https: / / adinstruments.com, ADInstruments, CO / USA). The total tissue conductivity was calculated by applying Ohm's law to the Isc deflection caused by a 1 - mV pulse applied to the tissue every 20 s during the experiment. Unless otherwise stated, the reported mean response was for the last 20 min of each measurement period.

[0145] Optical measurement of glandular mucus secretion rate. Details are given in a previous report (28). Intact ferret tracheal trimmings (approx. 1.5 cm 2 ) or porcine tracheal mucosa cut from the underlying cartilage in cold Krebs Ringer bicarbonate buffer were mounted, mucosa side up, in a 35 mm Petri dish lined with flexible silicone to immerse the glands in KRB buffer while keeping the surface dry and covered with water-saturated mineral oil. The appearance of "mucus bubbles" within the oil layer was observed by oblique illumination and digital images were captured using a macro lens of a Nikon digital camera. Images were analyzed either by direct measurement or using ImageJ software (V.1.50i, https: / / imagej.nih.gov / ij / , NIH, MD / USA). Rates of the indicated drugs were calculated over 5 min intervals based on the mean sustained T10-30 secretion rate of either 10 mM forskolin or 0.3 mM carbachol or T5-30 of the combined agonists to include the rapidly coalescing bubbles induced by the combined agonists.

[0146] Ciliary beat frequency measurement. Ciliary beat frequency was measured using human nasal mucosa in the laboratory where ferret and porcine tracheal mucosa are not easily accessible. Human nasal mucosa from endoscopic nasal biopsies was further dissected under the microscope and placed in a chamber where temperature and pH could be controlled. The perfused Krebs bicarbonate buffer was maintained at 37 °C and pH 7.4. Cilia were visualized using a differential interference contrast (DIC) optical system with a Zeiss microscope (Munich, Germany) equipped with 40× or 60× objective lenses. Images were viewed in real time and automatically captured at 2,000 fps using a high frame rate digital camera (optiMOS and NIS-Elements microscope imaging software (Nikon, Japan)) and then converted to TIFF images. Images were acquired for 10 s under each condition and the experiment was conducted in the following order: (1) unstimulated CBF at room temperature; (2) unstimulated at 37 °C; (3) CBF at 37 °C with 0.3 μM carbachol; (4) wash for 10 min; (5) CBF with 10 μM forskolin; and (6) CBF with 0.3 μM carbachol added to forskolin. Each condition was maintained for at least 10 min. Note that this paradigm is different from the synergistic paradigm used to measure MCCV and glandular mucus secretion rate in that the exposure time to agonists was ≥10 min instead of 30 min and it omitted the condition of adding forskolin in addition to carbachol. All recordings under each condition were made in three different regions of the epithelium and the analyzed CBF for each experiment was averaged. To analyze the captured images and calculate CBF, in-house coding was performed using MATLAB software (MA, USA).

[0147] Airway smooth muscle contraction measurement. Two methods were used to measure tracheal smooth muscle contraction. One method was to use thin-sectioned tracheal rings to measure airway narrowing. Tracheal ring preparations of approximately 2 mm from piglets or ferrets were immersed and firmly fixed on Sylgard-lined Petri dishes containing KRB solution at 37 °C and pH 7.4. Digital images of the tracheal rings contracting in response to agonists at 1-10 min intervals were recorded using a Nikon digital camera, and the luminal surface area of the tracheal rings was calculated using ImageJ (NIH, MD / USA). The other method was to use a force sensor. One end of an isolated ferret tracheal muscle bundle was fixed in a Sylgard-lined Petri dish containing KRB solution, and the other end was attached via a 26-gauge wire to a previously calibrated strain gauge (400A series force sensor system, Cambridge Technology, MA / USA). Tension responses to increasing doses of carbachol ± 10 μM forskolin were obtained and displayed using PowerLab Chart4 software (ADInstruments, CO / USA).

[0148] Reagents. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO / USA), Calbiochem (Billerica, MA / USA), and Alomone Labs (Jerusalem, Israel). BPO-27 was a generous gift from Alan Verkman at the University of California, San Francisco. Forskolin, benzamil, BPO-27, niflumic acid, formoterol fumarate were dissolved in dimethyl sulfoxide (DMSO), carbachol was dissolved in sterile double-distilled water, and indomethacin was dissolved in absolute ethanol. Solutions were freshly prepared or stored as aliquots of the stock concentration at -20 °C. All chemicals were diluted in KRB solution at a ratio of 1:1000 (except for indomethacin, which was diluted 1:10,000), and then used at the indicated concentrations.

[0149] Statistics. Unless otherwise stated, data are expressed as mean ± S.E.M. To compare the means of different treatment groups, student paired and unpaired t-tests or Mann-Whitney U tests were used.

[0150] Example 2

[0151] Effect of sequential and simultaneous administration of formoterol and methacholine on mucociliary clearance rate

[0152] To determine whether methacholine and formoterol can also cause a synergistic increase in MCCV, piglet tracheas were treated first with 0.3 μM methacholine and then with 10 μM formoterol, or first with 10 μM formoterol and then with 0.3 μM methacholine. MCCV was then measured for 90 - 120 min (all values are in mm / min) and plotted in Figure 8 as a graph of MCCV vs. time and agonist. In the absence of stimulation, MCCV decreased to near zero within the first 30 min ( Figure 8 ). Formoterol did not cause an increase in MCCV, and methacholine caused only a small increase. However, when the agonists were combined in either order, they caused a large, sustained increase in MCCV to approximately 12 mm / min. This synergistic increase in MCCV was observed when methacholine and formoterol were administered simultaneously ( Figure 10 )

[0153] Effects of sequential and simultaneous administration of formoterol and methacholine on airway smooth muscle contraction

[0154] To determine whether formoterol can also inhibit cholinergic agonist-induced airway smooth muscle contraction, the response of airway smooth muscle to methacholine (0.3 or 0.6 μM) ± 10 mM formoterol was measured by measuring the luminal area. The luminal area imaged in thin-sectioned pig tracheal rings showed a continuous 20 - 40% decrease upon exposure to 0.3 or 0.6 mM methacholine, but when formoterol was administered prior to methacholine, it caused only a transient 5 - 10% or less decrease ( Figure 9 ). Protection against methacholine-induced airway smooth muscle contraction was also observed when methacholine and formoterol were administered simultaneously ( Figure 11 ).

[0155] Effects on mucus transport at the epithelial level in an in vitro cell culture model

[0156] Human nasal epithelial cells (HNEC) were obtained from patients with CF (homozygous for F508del) and healthy WT controls according to established standard operating procedures. The cells were then isolated, seeded onto collagen-coated 0.4 μM pore size polyester membrane inserts (Corning Inc.), and expanded in Pneumacult Ex-Plus medium (StemCell Technologies), which was added to both the basal and apical chambers. The apical medium was then removed to create an air-liquid interface (ALI), and the basal medium was replaced with Pneumacult ALI medium (StemCell Technologies). Once the HNEC ALI cultures were mature (evidenced by the presence of active cilia), the CF cells were treated with DMSO control or the elexacaftor (3 μM)-tezacaftor (3 μM)-ivacaftor (10 μM) (ETI) combination for 48 hours. After 48 hours, the cells were treated with DMSO control, forskolin, carbachol, or forskolin + carbachol (SP), and the inserts were cut from their supports and placed on a well slide filled with medium such that only the basal side was exposed to the medium, and 20 μL of a 0.1% 2 μM fluorescent polystyrene bead suspension (Thermo Scientific R0200) was added to the apical surface.

[0157] The slides were then placed on a custom system that included a heated stage at 37 °C and imaged from above using a digital microscope (Keyence Inc., Elmwood Pk, NJ) equipped with a high-speed camera. Images were acquired at a frame rate of 1000 fps and exported to Image J to analyze particle movement using the MTrackJ plugin (v.1.5.1). Wild-type cells were used as a reference control. The distance traveled by each particle was then estimated using the extracted frame-by-frame coordinates. The coordinates and time lag between frames were used to define the time scale (τ) following the multi-particle transport (MPT) method, and the mean squared displacement (MSD) was then determined as the squared displacement of the particles for all possible time lags. The effective diffusivity (D eff ) was then calculated based on the extracted MSD and time scale. The MSD and D eff data generated by all tracked particles were analyzed by Loess smooth regression to produce an ensemble plot of MSD and D D eff versus the time scale to facilitate comparison of treatment conditions ( Figure 12 and 13 ).

[0158] Example 3

[0159] The combination agonist significantly improves MCC in sheep with induced CF airway disease when administered by inhalation.

[0160] In order to determine whether the MCCV synergistic increase observed in the above-mentioned in vitro and in vivo experiments can be produced in vivo in CF animal models, adult sheep were studied. In this established CF model (Abraham, W. Pulm Pharm Ther 2008; Kim et al.; Am J Respir Crit Care Med 2020, 201: 313-324), a CF-like condition was produced in the airways of adult sheep by aerosolization of CFTRinh172 and subsequent human neutrophil elastase. As a large animal model, it provides the opportunity to evaluate the impact on the tracheobronchial tree with similar characteristics to CF humans, as well as the opportunity to almost directly convert inhalation administration considerations. In brief, adult sheep were placed in a special body sling, maintained in an upright position, and under local anesthesia, the animals were intubated nasally with a standard endotracheal (ET) tube. Tracheal mucus velocity (TMV), the in vivo equivalent of MCCV, was measured by fluoroscopic technology. Five to seven radiopaque Teflon / bismuth trioxide disks (1 mm diameter, 0.8 mm thickness, 1.8 mg weight) were insufflated into the mid-trachea of ​​the animals. After the disks were delivered into the trachea, the head axis velocity of each disk was recorded on videotape by a portable image intensifier synchronized with the fluoroscopy instrument. The average of all disk velocities at a given time point was calculated, and new disks were insufflated at subsequent time points. After establishing baseline values, animals received 10 mg CFTRinh172 by nebulization, and TMV measurements were repeated every hour for the next 2 hours. Animals then received 1190 mU human neutrophil elastase (hNE) by nebulization, and TMV measurements were repeated every hour for the next 2 hours. This resulted in a severe and persistent impairment of TMV, which is typical of CF. Once CF conditions were established, animals received vehicle control (n=4), 20 μg formoterol (n=2), or a combination of 20 μg formoterol plus 12 μg methacholine (n=2) by inhalation. Methacholine alone was not tested because it would cause severe bronchospasm and endanger the life of the animals. TMV was measured hourly for 12 and 24 hours after administration of the test agent by inhalation. Figure 14As shown, after inhibiting TMV with CFTRinh172 + hNE, compared with the control group, formoterol alone had little effect on TMV, while the co - agonist increased TMV to approximately 80% of the pre - inhibition baseline. Importantly, the effect lasted for 24 hours after a single dose administration. Since the TMV study only evaluated MCC in the trachea and our ultimate goal was to address pulmonary mucus accumulation, the TMV study was supplemented with a whole - lung clearance study. To achieve this, the same CF sheep model was used, but whole - lung MCC was evaluated by radionuclide clearance techniques. For this purpose, after administering CFTRinh172 and hNE, 99mTc - sulfur colloid was administered by inhalation, followed by inhalation of either a vehicle control or a combination of 20 μg formoterol and 12 μg methacholine. Then, the pulmonary radio - tracer activity was monitored using a gamma camera over a 2 - hour period, as described previously (Abraham, Pulm Pharmacol Ther 2008, 21, 743 - 75). As Figure 15 shown, under control conditions (n = 2), during the 2 - hour observation period, the radio - tracer was retained and there was an artificial increase in the signal, while in contrast, the radio - tracer activity in the co - treated animals (n = 2) decreased by 11%. Notably, no adverse reactions were observed in the sheep exposed to the co - combination.

[0161] Healthy human volunteers and CF patients tolerated the combination agonist administered by inhalation in a single dose well. Encouraged by the experimental results, it was desired to evaluate whether humans could tolerate the combination of a β - adrenergic agonist and a cholinergic agonist delivered by inhalation, as this would lay the foundation for the full development of a treatment for clinical use. A series of 3 dose - escalation, single - administration studies were conducted in human volunteers and CF patients. In these studies, we used the available clinical - grade drugs. In the first study, it was desired to test the effect of the short - acting β - adrenergic drug salbutamol and its combination with methacholine (0, 1, 3, or 12 μg) in a dosing cohort of 3 healthy volunteers. Overall, all tested doses were well - tolerated, no adverse reactions were observed, and the predefined safety endpoint was met, that is, no individual experienced a decrease in forced expiratory volume in 1 second (FEV1, a standard measure of airway obstruction) of 20 points or more ( Figure 16 ). After this study, a similar study was conducted, but this time using the long - acting β - adrenergic drug formoterol, with which most of our experimental work was carried out. This study was similar to the first study, with volunteers divided into 3 cohorts and formoterol and 0, 1, 3, or 12 μg of methacholine administered by inhalation. Again, we observed no signs of intolerance to any of the tested doses, and the study met the predefined safety endpoint, which was set more stringently this time, that is, a decrease in FEV1 of 10 points or more ( Figure 17)。With these encouraging results, a study was conducted in CF patients similar to the healthy volunteer study with formoterol and methacholine, but the only difference was that the dosing cohort size was increased to 6 subjects. As in the previous study, no adverse reactions were observed at any test dose, and the study also reached its safety endpoint, i.e., no decrease in FEV1 > 10 points ( Figure 18 )). As an exploratory endpoint in the CF study, sputum production in the subjects was evaluated to reflect the effect on pulmonary mucus clearance. Notably, at the highest dose of methacholine, sputum production (in g, Figure 19 ) increased more, although this did not reach statistical significance, probably due to the small sample size in each dosing cohort (p = 0.056). However, in the group receiving methacholine, the sputum solid content increased significantly (p = 0.024; Figure 20 ), which we interpret as reflecting the movement of denser material, a characteristic of retained secretions in the CF lung.

[0162] Example 4

[0163] Further evidence indicates that inhibition of ENaC-mediated transport is part of the synergistic increase in MCCV.

[0164] It has previously been shown that in the presence of ENaC inhibition, stimulation of ferret trachea with forskolin or carbachol increases MCCV values similar to those obtained with the combined agonist, suggesting that ENaC inhibition may be involved in the synergistic MCC induced by the combined agonist. It is hypothesized that the synergistic MCC induced by β-adrenergic and cholinergic agonists involves ENaC inhibition. Consistent with this hypothesis, the MCCV results (in mm / min) of 10 μM formoterol + 0.3 μM methacholine were comparable in the presence (10 μM benzamil) or absence (0.1% DMSO as control treatment) of ENaC inhibition: 14.0 ± 1.6 in the presence of benzamil and 13.9 ± 1.6 in the absence of benzamil (P = 0.97, n = 4 piglets). As Figure 21 shown, ENaC inhibition significantly increased the baseline MCCV, almost 6-fold: 3.0 ± 0.7 in the presence of benzamil and 0.5 ± 0.7 in the absence of benzamil (P = 0.02, 4 piglets).

[0165] ASL pH can affect mucus viscosity and mucociliary clearance.

[0166] Generally, when pH decreases, the viscoelasticity of mucus increases and clearance slows. It was desired to evaluate whether the combined agonist increases the secretion of HCO 3 - in the airway. The secretion of HCO was evaluated using porcine tracheal mucosa and pH stat3 - Production was found such that 10 μM formoterol + 0.3 μM methacholine increased HCO 3 - secretion rate by 68% (units μM / cm2 / hr): 0.76 ± 0.11 at baseline, and 1.24 ± 0.14 ( Figure 22 ). HCO 3 - secretion was increased by co - agonist stimulation. Compared to the baseline condition, the co - agonist significantly increased the HCO 3 - secretion rate (p = 2E - 05, n = 13, from 8 porcine tracheas).

[0167] The co - agonist increased the ASL height in ex vivo WT porcine tracheas.

[0168] The ASL height in porcine tracheal preparations was determined using synchrotron - based phase - contrast imaging (PCI). The synchrotron beamline generates sufficiently parallel spatially coherent X - rays to increase the contrast between the airway lumen and the ASL layer through PCI. When X - rays pass through the intact tracheal preparation, the refractive index difference between the ASL and the airway lumen causes a phase shift of the X - rays, resulting in a unique interference pattern displayed as a change in X - ray intensity on a charge - coupled device (CCD) detector. After instilling agarose beads as a barometer, ASL height measurements were obtained every 5 min: T15 - baseline, T30 - 10 μM formoterol alone, and then T30 - 0.3 μM methacholine + 10 μM formoterol. As Figure 23 shown in A, both the β - adrenergic drug alone and the combined co - agonist (n = 4 porcine tracheas) increased the ASL height, while the control pigs treated with vehicle (DMSO treatment) (n = 2 porcine tracheas) failed to increase the ASL height during the same measurement period. The average rate of increase in ASL height during the co - drug period was significantly greater (P < 0.005, n = 4 pigs) than the average rate during formoterol (units μM / min): baseline (T5 - T15), 0.5; 10 μM formoterol (T30 - T50), 1.7 ± 0.1; and co - agonist (T65 - T85), 3.6 ± 0.4 ( Figure 23 B).

[0169] Although, for the purposes of clear understanding, the foregoing invention has been described in some detail by way of illustration and example, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0170] Accordingly, the foregoing merely illustrates the principles of the present invention. It is to be understood that those skilled in the art will be able to conceive of various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. In addition, all of the examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention and specific examples thereof are intended to cover both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed regardless of structure that perform the same function. Further, nothing disclosed herein is intended to be dedicated to the public, whether or not such disclosure is explicitly recited in the claims.

[0171] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked for such limitations in the claims only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of a recited limitation in the claims; if such exact phrases are not used in a limitation of the claims, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

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[0233] Notwithstanding the appended claims, the disclosure herein is also described by the following items:

[0234] 1. A method of treating a mucosal obstructive disorder in an individual, the method comprising:

[0235] administering to the individual a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to treat the mucosal obstructive disorder in the individual.

[0236] 2. The method of item 1, wherein the mucosal obstructive disorder is selected from cystic fibrosis, primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, and chronic bronchitis.

[0237] 3. The method of item 1, wherein the β-adrenergic agonist is a β 2 -adrenergic agonist.

[0238] 4. The method of item 3, wherein the β 2 -adrenergic agonist is selected from formoterol, salbutamol, isoprenaline, pirbuterol, levosalbutamol, clenbuterol, salmeterol, indacaterol, and vilanterol.

[0239] 5. The method of item 1, wherein the adenylate cyclase activator is forskolin or corforsin.

[0240] 6. The method of any one of the preceding items, wherein the cholinergic agonist is a direct-acting cholinergic agonist.

[0241] 7. The method of item 6, wherein the direct-acting cholinergic agonist is selected from methacholine, acetylcholine, carbachol, pilocarpine, and bethanechol.

[0242] 8. The method of any one of the preceding items, wherein the β-adrenergic agonist and the cholinergic agonist are administered sequentially.

[0243] 9. The method of item 8, wherein the β-adrenergic agonist is administered before the cholinergic agonist.

[0244] 10. The method of any one of items 1-9, wherein the β-adrenergic agonist and the cholinergic agonist are administered simultaneously.

[0245] 11. The method of any one of the preceding items, wherein the β-adrenergic agonist and the cholinergic agonist are administered systemically.

[0246] 12. The method according to any one of items 1-10, wherein the β-adrenergic agonist and the cholinergic agonist are administered locally.

[0247] 13. The method according to any one of the preceding items, wherein the administration does not cause airway smooth muscle contraction.

[0248] 14. The method according to any one of the preceding items, further comprising administering one or more cystic fibrosis transmembrane conductance regulator (CFTR) modulators.

[0249] 15. The method according to item 14, wherein the one or more CFTR modulators are elexacaftor, tezacaftor, deuteracaftor, vanzacaftor, and ivacaftor.

[0250] 16. The method according to any one of the preceding items, wherein the administration results in a synergistic increase in mucus transport relative to mucus transport using any one of the agonists alone.

[0251] 17. The method according to any one of the preceding items, wherein the individual is human.

[0252] 18. A method of increasing the rate of submucosal gland secretion in the airways of an individual, the method comprising:

[0253] administering to the individual a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to increase the rate of submucosal gland secretion in the airways of the individual.

[0254] 19. The method according to item 18, wherein the β-adrenergic agonist is a β 2 -adrenergic agonist.

[0255] 20. The method according to item 19, wherein the β2-adrenergic agonist is selected from formoterol, salbutamol, isoprenaline, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol.

[0256] 21. The method according to item 18, wherein the adenylate cyclase activator is forskolin or corforsin.

[0257] 22. The method according to any one of items 18-21, wherein the cholinergic agonist is a direct-acting cholinergic agonist.

[0258] 23. The method according to item 22, wherein the direct-acting cholinergic agonist is selected from methacholine, acetylcholine, carbachol, pilocarpine, and bethanechol.

[0259] 24. The method according to any one of items 18 - 23, wherein the β - adrenergic agonist and the cholinergic agonist are administered sequentially.

[0260] 25. The method according to any one of items 18 - 23, wherein the β - adrenergic agonist and the cholinergic agonist are administered simultaneously.

[0261] 26. The method according to any one of items 18 - 25, wherein the β - adrenergic agonist and the cholinergic agonist are administered systemically.

[0262] 27. The method according to any one of items 18 - 26, wherein the administration results in a synergistic increase in the submucosal gland secretion rate as compared to the submucosal gland secretion rate when using either agonist alone.

[0263] 28. The method according to any one of items 18 - 27, wherein the individual is a mammal.

[0264] 29. The method according to any one of items 18 - 27, wherein the individual is a pig.

[0265] 30. The method according to any one of items 18 - 27, wherein the individual is a weasel.

[0266] 31. The method according to any one of items 18 - 27, wherein the individual is a human.

[0267] 32. The method according to any one of items 18 - 31, wherein the individual has a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the mutation results in a non - functional CTFR protein, a CTFR protein with reduced functionality, or no CFTR protein.

[0268] 33. A method for inhibiting cholinergic agonist - induced airway smooth muscle contraction in an individual, the method comprising:

[0269] administering a β - adrenergic agonist or an adenylate cyclase activator to the individual, wherein the administration of the β - adrenergic agonist or the adenylate cyclase activator is performed before or simultaneously with the administration of the cholinergic agonist to the individual to inhibit airway smooth muscle contraction.

[0270] 34. The method according to item 33, wherein the β - adrenergic agonist is a β 2 - adrenergic agonist.

[0271] 35. The method according to item 34, wherein the β2 - adrenergic agonist is selected from formoterol, salbutamol, isoprenaline, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol.

[0272] 36. The method as described in item 35, wherein the adenylate cyclase activator is forskolin or calforsin.

[0273] 37. The method as described in any one of items 34 - 36, wherein the cholinergic agonist is a direct-acting cholinergic agonist.

[0274] 38. The method as described in item 37, wherein the direct-acting cholinergic agonist is selected from methacholine, acetylcholine, carbachol, pilocarpine, and bethanechol.

[0275] 39. The method as described in any one of items 34 - 38, wherein the β-adrenergic agonist is administered systemically.

[0276] 40. The method as described in any one of items 34 - 38, wherein the β-adrenergic agonist is administered locally.

[0277] 41. The method as described in any one of items 34 - 40, wherein the individual is a mammal.

[0278] 42. The method as described in any one of items 34 - 40, wherein the individual is a pig.

[0279] 43. The method as described in any one of items 34 - 40, wherein the individual is a ferret.

[0280] 44. The method as described in any one of items 34 - 40, wherein the individual is a human.

[0281] 45. The method as described in any one of items 34 - 44, wherein the individual has a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the mutation results in a non-functional CTFR protein or a CTFR protein with reduced functionality.

[0282] 46. A pharmaceutical composition comprising:

[0283] A β-adrenergic agonist or an adenylate cyclase activator;

[0284] A cholinergic agonist; and

[0285] A pharmaceutical excipient.

[0286] 47. The method as described in item 46, wherein the β-adrenergic agonist is a β 2 -adrenergic agonist.

[0287] 48. The method according to item 47, wherein the β2-adrenergic agonist is selected from formoterol, salbutamol, isoprenaline, pirbuterol, levosalbutamol, clenbuterol, salmeterol, indacaterol, and vilanterol.

[0288] 49. The method according to item 46, wherein the adenylate cyclase activator is forskolin or corforsin.

[0289] 50. The method according to any one of items 46-49, wherein the cholinergic agonist is a direct-acting cholinergic agonist.

[0290] 51. The method according to item 50, wherein the direct-acting cholinergic agonist is selected from methacholine, acetylcholine, carbachol, pilocarpine, and bethanechol.

[0291] 52. The composition according to any one of items 46-51, wherein the composition is formulated for systemic administration.

[0292] 53. The composition according to any one of items 46-52, wherein the composition is formulated for topical administration.

[0293] 54. The composition according to any one of items 46-53, wherein the composition is a solid composition.

[0294] 55. The composition according to any one of items 46-53, wherein the composition is a liquid composition.

[0295] 56. A device comprising the composition according to any one of items 46-55.

[0296] 57. The device according to item 50, wherein the device is a mouth inhaler.

[0297] 58. The device according to item 50, wherein the device is a nasal inhaler.

[0298] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art should understand that various other omissions, additions, and modifications can be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0299] Those skilled in the art should understand that, generally speaking, the terms used herein, especially the terms used in the appended claims (e.g., the subject matter of the appended claims), are usually intended to be "open" terms (e.g., the term "comprising" should be interpreted as "comprising but not limited to", the term "having" should be interpreted as "having at least", the term "including" should be interpreted as "including but not limited to", etc.). Those skilled in the art should also understand that if the intention is to introduce a specific number of recited claims, such intention will be explicitly recited in the claims, and in the absence of such recitation, there is no such intention. For example, for the sake of understanding, the following appended claims may contain the use of introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that introducing a claim recitation by the indefinite article "a" or "an" will limit any particular claim containing such introduced claim recitation to an embodiment containing only one such recitation, even when the same claim includes an introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the case of using a definite article to introduce a claim recitation. Additionally, even if the specific number of the introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted as meaning at least the recited number (e.g., the mere recitation "two recitations" without other modifiers means at least two recitations, or two or more recitations). Moreover, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally speaking, such a construction is intended to be understood by those skilled in the art as to the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include but not be limited to the following systems: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, in those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally speaking, such a construction is intended to be understood by those skilled in the art as to the meaning of the convention (e.g., "a system having at least one of A, B, or C" will include but not be limited to the following systems: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art should also understand that in fact any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood as contemplating the possibility of including one of the terms, any one of the terms, or both terms. For example, the phrase "A or B" will be understood as including the possibilities of "A" or "B" or "A and B".

[0300] In addition, where features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0301] As will be understood by those skilled in the art, for any and all purposes, such as to provide a written description, all ranges disclosed herein also cover any and all possible subranges and combinations of subranges thereof. Any listed range can be readily deemed to be sufficiently described and enables the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all such language as “up to,” “at least,” “greater than,” “less than,” and the like include the recited number and refer to ranges that can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0302] Although the foregoing invention has been described in some detail for purposes of clarity of understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0303] Accordingly, the foregoing merely illustrates the principles of the invention. It is to be understood that those skilled in the art will be able to devise various arrangements, which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Moreover, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to further the art and are to be construed as not being limited to such specifically recited examples and conditions. Additionally, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof are intended to cover both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public, whether or not such disclosure is explicitly recited in the claims.

[0304] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked for such a limitation in a claim only if the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of a recited limitation in the claim; if such exact phrases are not used in a limitation of the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

Claims

1. A method of treating a mucosal obstructive disorder in an individual, the method comprising: administering to the individual a combination of a β-adrenergic agonist or an adenylate cyclase activator and a cholinergic agonist to treat the mucosal obstructive disorder in the individual.

2. The method according to claim 1, wherein the mucosal obstructive disorder is selected from cystic fibrosis, primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, chronic bronchitis, and non-CF bronchiectasis.

3. The method according to claim 1, wherein the β-adrenergic agonist is a β 2 -adrenergic agonist.

4. The method according to claim 3, wherein the β2-adrenergic agonist is selected from formoterol, salbutamol, isoprenaline, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, and vilanterol.

5. The method according to claim 1, wherein the adenylate cyclase activator is forskolin or corforsin.

6. The method according to any one of the preceding claims, wherein the cholinergic agonist is a direct-acting cholinergic agonist.

7. The method according to claim 6, wherein the direct-acting cholinergic agonist is selected from methacholine, acetylcholine, carbachol, pilocarpine, and bethanechol.

8. The method according to any one of the preceding claims, wherein the β-adrenergic agonist and the cholinergic agonist are administered sequentially.

9. The method according to claim 8, wherein the β-adrenergic agonist is administered before the cholinergic agonist.

10. The method according to any one of claims 1-9, wherein the β-adrenergic agonist and the cholinergic agonist are administered simultaneously.

11. The method according to any one of the preceding claims, wherein the β-adrenergic agonist and the cholinergic agonist are administered systemically.

12. The method according to any one of claims 1-10, wherein the β-adrenergic agonist and the cholinergic agonist are administered topically.

13. The method according to any one of the preceding claims, wherein the administration does not result in airway smooth muscle contraction.

14. The method according to any one of the preceding claims, further comprising administering one or more cystic fibrosis transmembrane conductance regulator (CFTR) modulators.

15. The method according to claim 14, wherein the one or more CFTR modulators are elexacaftor, tezacaftor, and ivacaftor.

16. The method according to any one of the preceding claims, wherein, relative to mucus transport using either agonist alone, the administration results in a synergistic increase in mucus transport.

17. The method according to any one of the preceding claims, wherein the individual is human.

18. A pharmaceutical composition comprising: a β-adrenergic agonist or an adenylate cyclase activator; a cholinergic agonist; and a pharmaceutical excipient.

19. The composition according to claim 18, wherein the β-adrenergic agonist or the adenylate cyclase activator is selected from formoterol, salbutamol, isoprenaline, pirbuterol, levalbuterol, clenbuterol, salmeterol, indacaterol, vilanterol, forskolin, and corforsin.

20. The composition according to claim 19, wherein the direct-acting cholinergic agonist is selected from methacholine, acetylcholine, bethanechol, pilocarpine, and carbachol.

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