Methods for treating respiratory diseases characterized by excess mucus secretion

Abstract

This invention provides a method for treating respiratory diseases characterized by excessive mucus secretion, comprising administering a gamma-secretase inhibitor (GSI) to a patient requiring such treatment, wherein the administration of the GSI effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. The method of this invention effectively treats respiratory diseases selected from: cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, respiratory infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infections. The method of this invention also includes a method for treating cystic fibrosis, wherein the GSI is administered to a patient who is receiving or requires a CFTR modulator, wherein mucus in the patient's lungs is reduced or mucus accumulation in the patient's lungs is inhibited.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202180051358.5, filed on August 19, 2021, entitled "Method for Treating Respiratory Diseases Characterized by Excessive Mucus Secretion".

[0002] Recognition of government rights

[0003] This invention was developed with government support under the GM098582 license granted by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0004] Cross-references to related applications

[0005] This application claims priority to U.S. Provisional Patent Application No. 63 / 068235, filed August 20, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0006] This application relates to a method for treating respiratory diseases characterized by excessive mucus secretion. Background Technology

[0007] Airway epithelial cells consist primarily of a mixture of multiciliated cells (MCCs) and mucus-secreting goblet cells exposed on the luminal surface, along with underlying basal (stem cell) cells. Each MCC has 200 to 300 active cilia that bend in a coordinated, directional manner to expel inhaled contaminants trapped in the mucus layer from the lungs (Tilley AE, Walters MS, Shaykhiev R, Crystal RG. Cilia dysfunction in lung disease. Annu RevPhysiol. 2015;77:379-406). Goblet cells secrete mucus to form a protective barrier for the respiratory epithelium; they can increase in activity and number to respond to harmful stimuli such as infection. Impaired airway clearance can lead to and / or exacerbate acute infections and chronic inflammatory conditions such as cystic fibrosis (CF), primary ciliary dyskinesis (PCD), chronic sinusitis (CRS), chronic obstructive pulmonary disease (COPD), and asthma (ibid.).

[0008] Cystic fibrosis (CF) is considered the most severe mucociliary clearance disorder (Bruscia EM, Bonfield TL. Innate and adaptive immunity in cystic fibrosis. Clin Chest Med. 2016;37(1):17–29). Mutations in the CF transmembrane transduction modulator (CFTR) lead to dehydration of the mucosal surface and abnormal accumulation of thick mucus, both of which impair airway clearance and become sites of infection for various microorganisms. These events result in severe chronic inflammation and a cycle of repeated injury and incomplete repair. These, in turn, lead to epithelial dysfunction, including structural and functional changes such as proliferation of mucinous secretory cells, reduced number of MCCs, abnormal tissue structure associated with scar formation, weakened barrier function, and decreased regenerative capacity (Adam D et al. Cystic fibrosis airway epithelium remodelling: involvement of inflammation. J Pathol. 2015;235(3):408-419). Airway damage is inevitable in patients with cystic fibrosis (CF), manifesting as bronchiectasis, chronic cough, dyspnea, sinusitis, persistent infections despite repeated antibiotic use, and oxygen dependence. Epithelial cell dysfunction in CF is considered a major factor in disease progression, and once medical options are exhausted, it will eventually lead to lung transplantation (Regamey N, Jeffery PK, Alton EW, Bush A, Davies JC. Airway remodeling and its relationship to inflammation in cystic fibrosis. Thorax. 2011;66(7):624-629).

[0009] The functional balance between mucus secretion from secretory cells and MCC-driven movement results in an efficient mucociliary clearance process, which is essential for respiratory health. MCCs are terminally differentiated, originating from basal or secretory cell types of airway epithelium, beginning in embryonic development and continuing as a regenerative process throughout life (Hogan BL et al. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell. 2014;15(2):123-138; Rock JR et al. Basal cells as stem cells of the mouse trachea and human airwayepithelium. Proc Natl Acad Sci US A. 2009;106(31):12771–12775). MCC differentiation begins with a Notch signaling event in which cells respond to activation of the Notch transmembrane protein to become secretory cells. Notch-unresponsive ligand-expressing cells are oriented to the MCC fate through an MCC-specific gene expression program that drives the final generation of hundreds of regulatory and structural components required for differentiation and motile cilia development (Choksi SP, Lauter G, Swoboda P, Roy S. Switching on cilia: transcriptional networks regulating ciliogenesis. Development. 2014;141(7):1427-1441). Robust mucosal cilia clearance requires the generation of cilia with the correct number, length, beating frequency, and waveform, and importantly, the correct orientation along the tissue axis. Furthermore, it has been demonstrated that inhibition of Notch signaling in differentiated epithelial cells, by inducing transdifferentiation of secretory cells to MCCs, shifts the cell composition from secretory to MCC cell fate (Lafkas et al., Nature 2015 Dec 3;528(7580):127-31).

[0010] Airway epithelial cells from patients with CF and other chronic inflammatory diseases have been shown to have sparse or absent MCCs, defective mucociliary clearance function, and decreased associated barrier function and regenerative capacity. In vitro and animal models have demonstrated that, by inhibiting Notch signaling, γ-secretase inhibitors can restore a healthy balance between secretory cells and MCCs by driving de novo MCC differentiation and by promoting the transdifferentiation of mature secretory cells into MCCs, thereby rescuing these cellular components, barrier function, and regenerative phenotypes (Vladar EK, Nayak JV, Milla CE, Axelrod JD. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation. JCI Insight. 2016;1(13);e88027). Furthermore, γ-secretase inhibitors exhibit relatively rapid transdifferentiation of mature secretory cells compared to relatively slow differentiation of new cells.

[0011] Recent advances in cystic fibrosis treatment have led to the development of a class of drugs called CFTR modulators. These drugs are examples of personalized medicine, designed to treat individuals carrying specific CFTR mutations. CFTR modulators can be divided into three main categories: enhancers, modifiers, and early stop codon inhibitors or read linkers. CFTR enhancers increase the probability of opening CFTR channels with gating or transduction mutations. CFTR modifiers aim to increase the amount of functional CFTR protein delivered to the cell surface. CFTR read linkers aim to “force” the reading of early stop codons, thereby producing more full-length CFTR protein (Derichs, N., Eur. Resp. Rev., 2013: 22: 127, 58-65). CFTR amplifiers are a class of CFTR modulators under development and testing that aim to increase the amount of CFTR protein produced by cells at the transcriptional level, potentially enhancing the function of CFTR-mutated patients with insufficient cell surface protein.

[0012] While CFTR modulators improve CFTR function in patients with corresponding CFTR mutations, they do not affect altered cellular composition, damage to epithelial cell structure, or corresponding epithelial dysfunction. In cystic fibrosis and other diseases characterized by excessive mucus secretion and / or inadequate mucociliary clearance, modified treatments are needed to restore MCC function and improve mucociliary clearance.

[0013] Due to the role of gamma-secretase in β-amyloid formation and plaque formation, gamma-secretase inhibitors (GSIs) have been extensively studied as agents for the treatment of Alzheimer's disease (Barten DM, Meredith JE, Zaczek R, Houston JG, Albright CF: Gamma-secretase inhibitors for Alzheimer's disease: balancing efficacy and toxicity. Drugs R D. 2006, 7: 87-97. Evin G, Sernee MF, Masters CL: Inhibition of gamma-secretase as a therapeutic intervention for Alzheimer's disease: prospects, limitations and strategies. CNS Drugs. 2006, 20: 351-372). Furthermore, the role of Notch signaling in human cancers has led to research on GSIs as a potential therapy for various tumor types (Shih I and Wang T, Notch Signaling, Gamma-Secretase Inhibitors, and Cancer Therapy, Cancer Res 2007, 67(5);1879-1882). The ability of GSIs to block Notch signaling has also prompted recommendations for the use of GSIs to treat respiratory diseases associated with epithelial cell dysfunction (EP 2932966 A1).

[0014] Gamma-secretase is a multi-unit transmembrane protease complex composed of four separate proteins. It is an aspartic protease that cleaves its substrate within the transmembrane region via a process called regulatory intramembrane proteolysis (RIP) (Kreft, AF, Martone, R, and Porte, A, Recent Advances in the Identification of gamma-secretase Inhibitors To Clinically Test the Ab Oligomer Hypothesis of Alzheimer's Disease, J. Med. Chem 2009, 52:6169-6188). Although there has been interest in gamma-secretase as a therapeutic target for several years, understanding its structure in detail and its structure-activity relationship has been challenging due to its complexity. However, significant progress has been made in elucidating certain structure-activity relationships (see Wolfe, MS, Gamma-Secretase Inhibition and Modulation for Alzheimer's Disease, Curr AlzheimerRes. 2008; 5(2): 158-164).

[0015] Based on the binding sites of GSIs to γ-secretase, GSIs can generally be classified into three types: (1) active site-bound GSIs, (2) substrate docking site-bound GSIs, and (3) alternative binding site GSIs. The latter type can be further subdivided into carboxamide-containing GSIs and arylsulfonamide-containing GSIs (Kreft et al., p. 6171).

[0016] Clinical trials of Alzheimer's disease have revealed toxicities believed to be associated with inhibition of gamma-secretase (David B. Henley, Karen L. Sundell, Gopalan Sethuraman, Sherie A. Dowsett & Patrick C. May (2014) Safety profile of semagacestat, a gamma-secretase inhibitor: IDENTITY trial findings, Current Medical Research and Opinion, 30:10, 2021-2032).

[0017] Other GSIs have been studied as potential cancer therapeutic agents, and they typically exhibit toxicity at high doses. Summary of the Invention

[0018] It has now been unexpectedly found that low doses of gamma-secretase inhibitors (GSIs) effectively reverse cellular abnormalities associated with respiratory diseases characterized by excessive mucus secretion, at doses that allow for therapeutic activity, and are expected to avoid or minimize previously associated harmful effects of such molecules. Furthermore, contrary to some popular notions, GSIs administered in combination with CFTR modulators have been found to effectively correct epithelial cell dysfunction in a cystic fibrosis cell-based model system (primary cells from patients), and indeed, the combination can be synergistic in improving CFTR ion channel function and epithelial cell correction.

[0019] Therefore, the present invention provides a method for treating respiratory diseases characterized by excessive mucus secretion, comprising administering GSI to a patient requiring such treatment, wherein the administration of a low dose of GSI effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. In some embodiments, the method of the present invention effectively treats respiratory diseases selected from: cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, respiratory infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infections.

[0020] In some implementations, GSI is selected from semagacestat, avagacestat, GS-1, DBZ, L-685458, BMS-906024, crenigascestat, MRK 560, nirogacestat, RO-4929097, MK-0752, itanapraced, LY-3056480, fosciclopirox, tarenflurbil, and begacestat.

[0021] In some embodiments, the GSI is selected from smashite, niroglustat, MK-0752, RO-492907, or keregastat. In some embodiments, the GSI is a carboxylamide-based GSI.

[0022] In some embodiments, a method for treating respiratory diseases characterized by excessive mucus secretion is provided, comprising systemic administration of semacid at a dose of about 0.1 mg to about 50 mg daily, wherein the administration of semacid effectively reduces mucus in the lungs of the patient or inhibits mucus accumulation in the lungs of the patient. In some embodiments, semacid is administered at a dose of about 0.5 mg to about 40 mg daily. In some embodiments, semacid is administered at a dose of about 0.5 mg to about 30 mg daily, or about 0.5 mg to about 20 mg daily, or about 0.5 mg to about 10 mg daily. For example, semacid can be administered at doses of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg daily. Preferably, semacid is administered orally.

[0023] In embodiments of the invention, a method for treating a respiratory disease characterized by excessive mucus secretion is provided, comprising systemically administering a therapeutically effective amount of semacid to a patient requiring such treatment, wherein the steady-state semacid plasma concentration in the patient after multiple doses comprises an AUC (area under the curve) of less than 2100 ng·hr / mL, such as less than 1220 ng·hr / mL, wherein systemic administration of semacid effectively reduces mucus in the lungs of such a patient or prevents mucus accumulation in the lungs of such a patient. In some embodiments, after multiple doses, the steady-state semacid plasma concentration in the patient comprises an AUC of less than 1500 ng·hr / mL, less than 1200 ng·hr / mL, or less than 900 ng·hr / mL, such as less than 1220 ng·hr / mL, less than 600 ng·hr / mL, or less than 250 ng·hr / mL.

[0024] In other embodiments of the invention, a method for treating cystic fibrosis is provided, comprising administering an effective amount of GSI to a patient taking a CFTR modulator, wherein mucus in the patient's lungs is reduced or mucus accumulation in the patient's lungs is inhibited. In some embodiments, the GSI is selected from smasitol, niroglustat, MK-0752, RO-492907, or cligastat. In some embodiments, the GSI is smasitol.

[0025] In some of these embodiments, the CFTR modulator is selected from CFTR enhancers, CFTR correctors, CFTR premature termination codon inhibitors, CFTR amplifiers, and combinations thereof. In some embodiments, the CFTR modulator is selected from ivacaftor, lumacaftor, tezacaftor, elexacaftor, and combinations thereof. Attached Figure Description

[0026] Figure 1 Dose-response data for smasitol in human primary nasal epithelial cells (HNECs) compared to untreated cells and DAPT-positive controls are shown. MCCs are labeled in green (acetylated tubulin), and cell junctions are labeled in red (ECAD). The percentage of MCCs increased from a baseline of 15.625 nM smasitol to a maximum of approximately 125 nM. Toxicity was observed at micromolar doses. Figure 1 The dose response of smasitol in HNEC is shown.

[0027] Figure 2 The ratio of MCC to total luminal cells in HNEC treated with DAPT and different doses of smectide is shown. Figure 2 The ratio of MCC to common lumen cells in HNEC treated with DAPT and various doses of smasitol is shown.

[0028] Figure 3 This paper illustrates a scoring method for the ratio of MCCs to non-ciliated cells in the airway epithelium in mice subjected to in vivo systemic treatment via intraperitoneal (IP) administration of smasitol. All cell nuclei (red; DAPI) and MCC cell fate (green; FoxJ1 and blue; acetylated tubulin) were labeled in airways from similarly sized lung slices. Cells were scored and the percentage of MCCs determined prior to unblinding treatment conditions. Alternatively, wild-type mice carrying FoxJ1::GFP were used to facilitate MCC scoring. Figure 3 Mouse epithelial cells were shown after 3 days of systemic (IP) administration.

[0029] Figure 4 The body weights of patients on days 9, 24, and 30 after daily systemic (IP) administration of smasitol and the load control are shown. Figure 4 Body weights on days 9, 24, and 30 after daily systemic (IP) administration of smasitol and the load-controlled regimen are shown.

[0030] Figure 5 The ratio of MCC to total cells on day 7 after three days of treatment with DAPT, low-dose and high-dose smasitol, and a load-controlled agent is shown. Figure 5The ratio of MCC to total cells on day 3 after treatment with DAPT, low-dose smectite, and high-dose smectite is shown.

[0031] Figure 6 The ratio of MCC to total cells is shown on day 31 after three weeks of treatment with smashite and a load control. Figure 6 The ratio of MCC to total cells is shown on day 31 after treatment with smashite.

[0032] Figure 7 The effects of GSI treatment on HNEC proliferation (pre-differentiation) and differentiation are shown. Figure 7 The effects of GSI treatment during proliferation and differentiation are shown.

[0033] Figure 8 It shows Figure 7 Quantification of the MCC of each total lumen cell in the data. Figure 8 It shows Figure 7 The treatment of each total lumen cell in the MCC.

[0034] Figure 9 The effect of the duration of GSI treatment on mature (ALI day 30) HNEC treated with DAPT and smasitronil for one week (ALI day 30 to day 37) or two weeks (ALI day 30 to day 44). Figure 9 The effect of GSI treatment time on mature primary human airway epithelial cells was demonstrated.

[0035] Figure 10 It shows Figure 9 Quantification of the MCC of each total lumen cell in the data. Figure 10 It shows Figure 9 The treatment of each total lumen cell in the MCC.

[0036] Figure 11 Results of HNEC treated with DAPT and smashite only during differentiation from the top or basal plane (day 0 to day 21 of ALI) are shown. Figure 11 The application of GSI treatment on the top and bottom surfaces is shown.

[0037] Figure 12 It shows Figure 11 Quantification of the MCC of each total lumen cell in the data. Figure 12 It shows Figure 11 The treatment of each total lumen cell in the MCC.

[0038] Figure 13The effects of smashite and DAPT treatments on a chronic airway inflammation epithelial culture model are illustrated. HNEC cultures were treated with IL-13 from day 7 to day 14 of ALI to induce inflammation. DAPT and smashite increased the percentage of MCCs in the control group (left). IL-13 treatment increased the percentage of mucin-positive secretory cells and decreased the percentage of MCCs. Subsequent DAPT and smashite treatments salvaged the cellular composition, increasing the percentage of MCCs and decreasing the percentage of mucin-positive secretory cells. Figure 13 GSI treatment in an IL-13 chronic inflammation model is shown.

[0039] Figure 14 It shows Figure 13 Quantification of the MCC of each total lumen cell in the data. Figure 14 It shows Figure 13 The ratio of MCC to total lumen cells in the treatment.

[0040] Figure 15 A representative using-chamber diagram of cultures treated with ETI, Smashite, or both is shown. Figure 15 Representative Uss chamber diagrams of epithelial cell cultures treated with the load control, the CFTR modulator combination Elexacaftor-tezacaftor-Ivacaftor (ETI), semasite (LY), or both (ETI+LY) are shown.

[0041] Figure 16 Representative plots of the Uss chamber short-circuit current (Isc) after treatment with smashite in wild-type and CF cells are shown. Figure 16 The diagram shows the representative figure of the Jus short-circuit current.

[0042] Figure 17 The Isc response of the Uss chamber after treatment with smasitol is shown in wild-type and CF cells. Two wild-type control samples (WT) and two CF patient samples (CF1: rare allele combination, CF2: F508 homozygote). CFTR current activity was assessed by CFTR inhibitor response, and CFTR current activity in wild-type control and CF samples was found to be as large as or greater than that in the load control. Values ​​were normalized to baseline current. Figure 17 The CFTRinh-172 Isc response is shown.

[0043] Figure 18 Simassitol demonstrated that in vitro treatment in combination with the CFTR modulator rumacotto reduced mucus production in human CF samples with different CFTR mutations. Figure 18 The study showed reduced mucus production in human CF samples treated with simassitol.

[0044] Figure 19 The effects of treatment with smashite, rumacator, and combinations thereof on human CF samples with different CFTR mutations are shown. In the presence of rumacator, smashite effectively increased the percentage of MCC, and the combination was more effective for some donors than the two drugs alone. Figure 19 CF samples after treatment with smasitax, rumacator, and their combinations are shown.

[0045] Figure 20 It shows Figure 19 Quantification of MCC per total lumen cell in healthy patients and CF donor 1. Figure 20 It shows Figure 19 The ratio of healthy and CF donor 1 treated MCCs to total lumen cells is shown.

[0046] Figure 21 The effects of treatment with smashite (LY45139) on primary healthy and cystic fibrotic airway epithelial cells were demonstrated only during the differentiation period (day 0 to day 21 of ALI). Figure 21 The effects of smasitol treatment during differentiation on primary healthy and cystic fibrotic airway epithelial cells were demonstrated.

[0047] Figure 22 SEM images of healthy and CF primary human airway epithelial cultures are shown, which demonstrate that the multiciliated cells formed under DAPT treatment are indistinguishable from those in untreated healthy cultures. Figure 22 SEM images of healthy and CF primary human airway epithelial cultures are shown.

[0048] Figure 23 The results of DAPT treatment in mature cystic fibrotic HNEC cultures are shown, demonstrating that GSI treatment induces the formation of additional multiciliated cells in mature cystic fibrotic cultures, while untreated cultures do not differentiate into any more multiciliated cells. Figure 23 This demonstrates the formation of additional multiciliated cells in mature cystic fibrosis cultures induced by GSI treatment.

[0049] Figure 24 Results of HNECs treated with DAPT and high and low concentrations of GSILY45139, PF-03084014, RO-4929097 and MK-0752 during differentiation period (ALI day 0 to day 21) are shown. Figure 24 Primary human airway epithelial cells treated with DAPT, high concentrations, and low concentrations of various GSIs during differentiation are shown.

[0050] Figure 25 It shows Figure 24The data shown represents the quantification of the MCC of each total lumen cell. Figure 25 It shows Figure 24 The ratio of MCC to total lumen cells shown in the treatment diagram.

[0051] Figure 26 Measurements of CFTR short-circuit current activity are shown in the Yus chamber of cultures from CF patients treated with LY45139, Elexcaftor / Tezacator / Ivacaftor (or "ETI"), or both. Figure 26 The CFTR activity of the Yus chamber was demonstrated in cultures from CF patients treated with LY45139, ETI, or both.

[0052] Figure 27 The measurements of CFTR current activity are shown in the Yus chamber of cultures from CF patients treated with MK04752, ETI, or both. Figure 27 The CFTR activity of the Yus chamber is demonstrated in cultures from CF patients treated with MK04752, ETI, or both.

[0053] Figure 28 Measurements of CFTR current activity are shown in the Yus chamber of cultures from CF patients treated with a reduced dose of MK04752, ETI, or the E component of Elexacaftor (an E component of the ETI modulator). Figure 28 The CFTR activity of the Yus chamber is demonstrated in cultures of CF patients treated with a combination of MK04752, ETI, or a reduced dose of ETI.

[0054] Figure 29 The results of GSI DAPT treatment on ionization in HNECs treated with DAPT only during differentiation (day 0 to day 21 of ALI) are shown. Figure 29 The study showed that GSI treatment had no effect on the formation of ionocytes.

[0055] Figure 30 The effects of different concentrations of GSI MK-0752 and Elexacaftor on ciliary wavy frequency (CBF) are shown. Figure 30 The effects of low doses of GSI MK-0752, Elexacaftor, and combinations thereof on ciliary beat frequency (CBF) are shown. Results are expressed as a relative increase in CBF (95% UCL) compared to untreated control cells.

[0056] Figure 31 The cilia beat frequency (CBF) and cilia length of HNE treated with DAPT are shown compared with the untreated control. Figure 31The ciliary beating frequency (CBF) and ciliary length of primary human epithelial cells treated with DAPT are shown.

[0057] Figure 32 The airway surface fluid (ASL) reabsorption characteristics of CF HNEC cultures treated with ETI, smashite, or a combination of both are shown. Figure 32 The reabsorption characteristics of airway surface fluid (ASL) in CF cultures treated with ETI, smashite, or both are shown.

[0058] Figure 33 Images obtained from high-speed video recordings of latex bead movement microscopy are shown, reflecting mucus transport on the surface of cell culture cilia. The cultures are HNECs from two CF donors (F508del homozygotes) treated with a carrier control, ETI, smashite (LY), or a combination of both treatments. Figure 33 Images of mucociliary transport on the surface of cell cultures treated with a carrier control, ETI, LY-45139, or both are shown.

[0059] Figure 34 It shows Figure 33 Calculated bead movement of the culture shown. Figure 34 It shows the use of Figure 33 The mucociliary transport in cultures treated with ETI, LY-45139, or both is shown. Detailed Implementation

[0060] definition

[0061] As used herein, "diseases characterized by excessive mucus secretion" refers to diseases in which at least one pathology is due to an amount of mucus present on the epithelial surface exceeding the amount present under normal conditions. This includes diseases in which excessive mucus is located in small airway pathways where it is not normally present, and can be attributed to excessive goblet cell production, mucous gland hypertrophy, reduced MCC, or other inadequate mucociliary clearance.

[0062] As used herein, "effective amount" or "therapeutic effective amount" means that the compounds of this invention effectively achieve the desired therapeutic outcome, such as reducing goblet cell production and / or increasing multiciliated cell production, thereby improving the amount of mucociliary clearance. In the context of this invention, the desired therapeutic outcome includes reducing mucus production in the patient's lungs or inhibiting mucus accumulation in the patient's lungs. While the dosages mentioned in this disclosure are guidelines, attending physicians may adjust the dosage according to the patient's specific needs, including, for example, the severity, size, and physical condition of the disease.

[0063] "Gamma secretase inhibitors" or "GSIs" refer to molecules that inhibit or regulate gamma secretase, thereby inhibiting Notch signaling. Examples include DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester), smasitol, celecoxib ((2R)-2-[(4-chlorophenyl)sulfonyl][[2-fluoro-4-(1,2,4-oxadiazol-3-yl)phenyl]methyl]amino]-5,5,5-trifluoropentanamide) (available at www.tocris.com), and DBZ (N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl) -6-oxo-5H-dibenzo[b,d]azapyro-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorophenylacetamide (available at www.tocris.com), L-685,458 ((5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucyl-L-phenylalanine amide (available at www.tocris.com), GS-1 (also known as L-685458) (CAS Registration No. 292632-98-5; WO0177144); BMS-906024 (bis(fluoroalkyl)-1,4-benzodiazepine; CAS Registration No. 1401066-79-2), Crizostat (also known as LY3039478) (Massard et al., "First-in-human study of LY3039478, a Notch signaling inhibitor in advanced or metastatic cancer," J Clin Oncol (2015) 33(15_suppl):2533), MRK 560 (N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide) (available at www.tocris.com), Niroglasta (also known as PF-03084014) ((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthyl-3-ylamino)-N-(1-(2-m-ethyl-1-(neopentylamino)propyl-2-yl)-1H-imidazol-4-yl)pentanamide; CAS Registry number 865773-15-5 (available commercially from www.adooq.com), RO-4929097 (RO-4929097 refers to 2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,diazaphen-7-yl)-N'-(2,2,3,3,3-pentafluoropropyl)-malondiamide). CAS Registry number 847925-91-1 (available from www.adooq.com).Purchased from .com), MK-0752 (CAS No. 471905-41-6 (www.medchemexpress.com)); Etalex (CAS No. 749269-83-8 (www.medchemexpress.com)); LY-3056480 (Samarajeewa, Anshula & Jacques, Bonnie & Dabdoub, Alain. (2019). Therapeutic Potential of Wnt and Notch Signaling and Epigenetic Regulation in Mammalian Sensory Hair Cell Regeneration. MolecularTherapy. 27. 10.1016 / j.ymthe.2019.03.017); fosciclopirox (provided as disodium heptahydrate) (Patel, MR et al., Safety, dose tolerance, pharmacokinetics, and pharmacodynamics of fosciclopirox (CPX-POM) in patients with advanced solid tumors. Journal of Clinical Oncology (2020) 38:6 suppl 518); Flurbiprofen (CAS No. 51543-40-9; (2R)-2-(3-fluoro-4-phenylphenyl)propionic acid; EVP-0962 (Rogers, K. et al., (2012). Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice. Molecular Neurodegeneration. 7. 61.10.1186 / 1750-1326-7-61.;NIC5-15;E-2212;GSI-1;NGP-555;PF-0664867);Begastrol (also known as GSI-953) (5-chloro-N-[(1S)-3,3,3-trifluoro-1-(hydroxymethyl)-2-(trifluoromethyl)propyl]-2-thiophene sulfonamide) (www.tocris.com);GSI-136 (5-chloro-N-[(2S)-3-ethyl-1-hydroxypentane-2-yl]thiophene-2-sulfonamide) (https: / / pubchem.ncbi.nlm.nih.gov / compound / gsi-136); and BMS-708163 (Gillman, KW et al., Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable Gamma-Secretase Inhibitor. ACS Med. Chem. Lett. (2010) 1(3)120-124). See also, Sekioka, R. et al., Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active gamma secretase modulators. Bioorg. & Med. Chem., (2020) 28(1): 115132. “Carboxamide-based GSIs” refers to GSIs having a carboxamide group and includes molecules formed by carboxamide substitution as well as known derivatives of carboxamide-based GSIs such as DAPT. Examples include DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethyl ethyl ester), smasitol, celecoxib ((2R)-2-[(4-chlorophenyl)sulfonyl][[2-fluoro-4-(1,2,4-oxadiazol-3-yl)phenyl]methyl]amino]-5,5,5-trifluoropentanamide) (available at www.tocris.com), DBZ (N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenzo[b,d]azapyro-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorophenylacetamide) (available at www.tocris.com), L-685,458 ((5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucyl-L-phenylalanine amide) (available at www.tocris.Purchased from .com), BMS-906024 (bis(fluoroalkyl)-1,4-benzodiazepine; CAS Registry No. 1401066-79-2), Crizostat (also known as LY3039478) (Massard et al., "First-in-human study of LY3039478, a Notch signaling inhibitor in advanced or metastatic cancer," JClin Oncol (2015) 33(15_suppl):2533), MRK 560 (N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide) (available at www.tocris.com), Niroglasta (also known as PF-03084014) ((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthyl-3-ylamino)-N-(1-(2-m-ethyl-1-(neopentylamino)propyl-2-yl)-1H-imidazol-4-yl)pentanamide; CAS CAS Registry No. 865773-15-5 (available at www.adooq.com) and RO-4929097 (RO-4929097 refers to 2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,diazaphen-7-yl)-N'-(2,-2,3,3,3-pentafluoropropyl)-malondiamide. CAS Registry No. 847925-91-1 (available at www.adooq.com) and BMS-708163 (Gillman, KW et al., Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable Gamma-Secretase Inhibitor. ACS Med. Chem. Lett. (2010) 1(3)120-124). See also Sekioka, R. et al., Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active gamma secretase modulators. Bioorg. & Med.Chem., (2020) 28(1): 115132. GSI includes any salt form, polymorph, hydrate, analogue, or prodrug that retains the inhibitory or regulatory activity of gamma secretase.

[0064] The terms “treatment,” “prevention,” “suppression,” and related terms include therapeutic treatment, preventative treatment, and treatment that reduces the risk of a subject developing the disease or risk factors. Treatment does not necessarily require a complete cure for the disease or condition; it includes reducing severity, decreasing symptoms, reducing other risk factors associated with the condition, and / or improving the disease's effects, such as slowing its progression.

[0065] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, and therefore, variations are naturally possible. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims.

[0066] Where numerical ranges are provided, it should be understood that, unless otherwise expressly stated herein, every intermediate value between the upper and lower limits of the range, accurate to one-tenth of the lower limit unit, and any other value or intermediate value within the specified range, is included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and also covered by this invention, subject to any specific exclusions within the ranges. When the range contains one or both of the limit values, the range excluding one or both of these contained limit values ​​is also included within the scope of this invention.

[0067] The numerical ranges provided in this document are preceded by the term "approximately". The term "approximately" is used in this document to provide textual support for the exact figures preceding it, as well as figures that are close to or approximate to the figures preceding the term. In determining whether a figure is close to or approximates a specifically listed figure, an unlisted figure that is close to or approximates a figure that is substantially equivalent to the specifically listed figure in its context.

[0068] 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in practicing or testing this invention, representative exemplary methods and materials are described hereafter.

[0069] All publications and patents referenced in this specification are incorporated herein by reference, as each individual publication or patent is specifically and individually indicated for inclusion by reference, and is incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publications. References to any publication are for disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to a prior art invention prior to that publication. Furthermore, the provided publication date may differ from the actual publication date, which may require independent confirmation.

[0070] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural indicators. It should be noted that claims may be drafted to exclude any arbitrary element. Therefore, such statements are intended to serve as a precondition for using exclusive terms such as “merely,” “only,” or using the restrictive term “negative” in conjunction with the elements of the claims.

[0071] Those skilled in the art will understand upon reading this disclosure that the individual embodiments described and illustrated herein have discrete components and features that can be readily separated from or combined with features of any other embodiments without departing from the scope or spirit of the invention.

[0072] Detailed description

[0073] γ-secretase inhibitors

[0074] Various GSIs have been developed as potential clinical candidates for Alzheimer's disease and cancer (see Kreft et al., 6171). DAPT was one of the earliest discovered GSIs. Modifications to DAPT have yielded clinical candidates.

[0075] Smashite, (2S)-2-hydroxy-3-methyl-N-[(1S)-1-methyl-2-oxo-2-[[(1S)-2,3,4,5-tetrahydro-3-methyl-2-oxo-1H-3-benzozazepine-1-yl]amino]ethyl]-butyramide, is a small molecule γ-secretase inhibitor originally developed for the treatment of Alzheimer's disease (see U.S. Patent No. 7,468,365). Smashite is known to exist in polymorphic forms, including a dihydrate and at least two anhydrous forms (ibid., see also U.S. Patent No. 8,299,059). See also Yi et al., DMD (2010) 38:554–565; http: / / doi:10.1124 / dmd.109.030841.

[0076] Niroglustat (also known as PF-03084014) is (S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthyl-3-ylamino)-N-(1-(2-m-ethyl-1-(neopentylamino)propyl-2-yl)-1H-imidazol-4-yl)pentanamide, a small molecule γ-secretase inhibitor developed for cancer indications. It is available in the form of hydrobromide (www.medchemexpress.com) and exists in solid form. (See U.S. Patent No. 10,590,087). See Wei P et al., Evaluation of selective gamma-secretase inhibitor PF-03084014 for its antitumor efficacy and gastrointestinal safety to guide optimal clinicaltrial design. Mol Cancer Ther. 2010 Jun;9(6):1618-28; and Kumar, S. et al., Clinical Activity of the gamma-secretase inhibitor PF-03084014 in adults with desmoid tumors (aggressive fibromatosis). J. Clin Oncol. (2017) May 10;35(14):1561-15. Seventeen patients were given an oral dose of 50 mg twice daily for 3 weeks as one cycle, for a total of 6 cycles (18 weeks).

[0077] MK-0752 is a small molecule gamma-secretase inhibitor under investigation for cancer indications. Phase I clinical data are detailed in Krop I et al., *Phase I pharmacologic and pharmacodynamic study of the gammasecretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors*. *J Clin Oncol.* 2012;30(19):2307-2313. In this study, among 103 patients treated with MK-0752, 21 received continuous once-daily doses of 450 mg and 600 mg; 17 received 450 mg and 600 mg doses on a 3-day intermittent dosing schedule over 7 days; and 65 received weekly doses of 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2400 mg, 3200 mg, and 4200 mg. The most common drug-related toxicities were diarrhea, nausea, vomiting, and fatigue. Toxicity findings depend on the dosing regimen, but once-weekly dosing is generally considered well-tolerated. See also Matthews et al., Journal of Chromatography B, 863 (2008) 36–45; https: / / doi:10.1016 / j.jchromb.2007.12.025.

[0078] RO-4929097 is a small molecule gamma secretase inhibitor being investigated for cancer indications. See, for example, Tolcher AW, Messersmith WA, Mikulski SM et al., Phase I study of RO4929097, agamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors. J Clin Oncol 2012; 30: 2348-2353; Wu et al., Journal of Chromatography B, 879 (2011) 1537–1543. In this study, patients received escalating doses of RO4929097 orally according to two dosing regimens: (A) three consecutive days per week for two weeks every three weeks; (B) seven consecutive days every three weeks; and (C) daily dosing. Toxicity included fatigue, thrombocytopenia, fever, rash, chills, and anorexia. The study concluded that RO4929097 was well tolerated at doses of 270 mg in regimen A and 135 mg in regimen B; however, the safety of regimen C was not adequately assessed.

[0079] Crenigasestat (also known as LY3039478) is a small molecule γ-secretase inhibitor being investigated for cancer indications. See Yuen E. et al., Evaluation of the effects of an oral notch inhibitor, crenigacestat (Ly3039478), on QT interval, and bioavailability studies conducted in healthy subjects. Cancer Chemother PHarmacol. 2019 Mar;83(3):483-492. In this study, healthy subjects were given a single oral dose of 25 mg, 50 mg, or 75 mg of crenigasestat, or an intravenous injection of 350 µg. 13 C 15 N 2 H. Clennigast.

[0080] In a phase III trial for Alzheimer's disease, smashite at daily doses of 100 mg and 140 mg did not improve cognitive status, and patients at the highest dose showed significant cognitive deterioration.

[0081] Simasit is also associated with a number of adverse events, including skin cancer and infection. Doody, RS et al., N EnglJ Med 369;4: 341-350 (July 25, 2013). An earlier phase I study reported placebo-like adverse events in subjects taking 5 mg, 20 mg, or 40 mg daily for 14 days, while 2 out of 7 subjects taking 50 mg daily reported potentially drug-related adverse events. Siemers E, Skinner M, Dean RA et al. Safety, tolerability, and changes in amyloid beta concentrations after administration of a gamma-secretase inhibitor in volunteers. Clin Neuropharmacol. 2005;28(3):126-132.

[0082] Given evidence that Notch signaling is dysregulated in many malignancies, GSI has been developed as a potential cancer treatment, as a monotherapy, or in combination with other drugs. See, for example, Takebe N, Nguyen D, Yang SX. Targeting notch signaling pathway in cancer: clinical development advances and challenges. Pharmacol Ther. 2014 Feb;141(2):140-9. doi: 10.1016 / j.pharmthera.2013.09.005.Epub 2013 Sep 27; Shao H, Huang Q, Liu ZJ. Targeting Notch signaling for cancer therapeutic intervention. Adv Pharmacol. 2012;65:191-234. doi: 10.1016 / B978-0-12-397927-8.00007-5.

[0083] It has been found that low-dose GSIs are effective in treating respiratory diseases characterized by excessive mucus secretion, and are effective at doses that allow for therapeutic activity, while avoiding or minimizing previously associated harmful effects with such molecules. As used herein, “low-dose” GSI refers to an effective dose for treating respiratory diseases characterized by excessive mucus secretion, and is a lower dose compared to the GSI doses appropriate for patients with neurodegenerative diseases, oncological diseases, or respiratory diseases not characterized by excessive mucus secretion. For example, low-dose means a dose that produces peak plasma levels in the submicromolar range. It should be understood that low-dose GSIs can be administered as a single daily dose, multiple daily doses (e.g., two or three times daily), intermittently, or weekly, depending on the dosage form (e.g., immediate-release or controlled-release) and the patient’s needs. Administration can be prolonged, intermittent, or limited in duration, and may be repeated if determined to some extent by the patient’s healthcare provider. For example, GSI can be administered daily for 1, 3, 5, 7, 10, 14, 18, 21, 24, 28, or 30 days, and then discontinued. In some embodiments, GSI is administered intermittently, such as every 3 days or weekly. It should be understood that the daily dosage mentioned herein can be achieved with a dosing regimen other than once-daily; for example, a weekly dose of 35 mg would correspond to a daily dose of 5 mg / day. Similarly, sustained-release formulations such as reservoir formulations or patch formulations are known in the art and can be used to provide doses equivalent to the daily doses described herein. The GSI dosing regimen may be repeated if necessary.

[0084] For example, when administered to human nasal epithelial cells in nanomolar ranges, each of the GSIs smasitol, niroglustat (PF-03084014), RO-4929097, and MK-0752 effectively blocked Notch signaling, drove differentiation into MCCs, and rescued conditions associated with excessive goblet cell mucus secretion. Therefore, relatively low systemic GSI levels can provide effective treatment for respiratory diseases associated with excessive mucus secretion while avoiding or minimizing the adverse events observed at higher doses.

[0085] Furthermore, contrary to some popular notions, GSIs administered in combination with CFTR modulators effectively corrected epithelial cell dysfunction in a cystic fibrosis cell-based model system (primary cells from patients), and the combination can indeed be synergistic in improving CFTR ion channel function and epithelial cell correction. For example, various GSIs have been shown not to interfere with CFTR ion channels and not to inhibit the effects of CFTR modulators on CFTR ion channels in cystic fibrosis airway epithelial cells. GSI treatment has been found to unexpectedly improve airway surface fluid (ASL) reabsorption in CF cells to the same extent as CFTR modulator drugs. Further evidence suggests that GSI treatment may be synergistic with CFTR modulator therapy, potentially reducing the dosage of one or both drugs and further reducing the potential toxicity of each drug.

[0086] GSIs can be administered in pharmaceutical dosage forms known in the art, including but not limited to oral solid doses, oral liquids, injections, transdermal patches, and inhalation. Dosage forms can be formulated with excipients and other compounds to facilitate administration to the recipient and maintain storage stability. See “Remington's Pharmaceutical Sciences” (Mack Publishing Co., Easton, PA). Oral pharmaceutical formulations include tablets, mini-tablets, pills, granules, capsules, gels, liquids, syrups, and suspensions. Preferably, GSIs are administered orally, typically via oral solid doses, although oral liquids may be suitable for certain populations who have difficulty taking tablets and capsules, such as children and elderly patients. Oral dosage forms can be immediate-release or controlled-release.

[0087] The tablet form of smasitax is known in the art (see U.S. Patent No. 8,299,059). Following oral administration, the half-life of smasitax has been reported to be approximately 2.5 hours. Therefore, in one embodiment of the invention, smasitax can be provided as an immediate-release formulation. Immediate-release smasitax can be provided as a single daily dose or divided into multiple daily doses, administered two, three, four, or more than four times daily. In another embodiment of the invention, smasitax is provided as a delayed-release formulation. Delayed-release formulations offer convenience to patients by reducing daily administration and can improve patient compliance. Furthermore, the controlled-release formulation of the present invention can be used to reduce serum peaks and troughs, thereby potentially reducing adverse events.

[0088] Oral controlled-release formulations are known in the art, including sustained-release, extended-release, delayed-release, and pulsatile-release formulations. See “Remington's Pharmaceutical Sciences” (Mack Publishing Co., Easton, PA). An active pharmaceutical ingredient can be formulated into a matrix formulation with one or more polymers, said polymers comprising hydrophilic or gelling agents, hydrophobic matrices, lipid or wax matrices, and biodegradable matrices, and enabling the slow release of the drug from the dosage form. The active ingredient can be formulated into beads, for example, having an inert sugar core, and coated with known excipients to delay or retard the release of the active ingredient by diffusion. Enteric coatings are known in the art for delaying the release of the active ingredient until the dosage form moves from the low pH environment of the stomach to the high pH environment of the small intestine, and can contain methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate, polyvinyl acetate phthalate (PVAP), shellac, sodium alginate, and cellulose acetate trimellitate.

[0089] In some implementations, GSI is selected from semagacestat, avagacestat, GS-1, DBZ, L-685458, BMS-906024, crenigascestat, MRK 560, nirogacestat, RO-4929097, MK-0752, etaralose, LY-3056480, fosciclopirox, tarenflurbil, and begacestat.

[0090] In some implementations, the GSI is selected from smashite, niroglustat, MK-0752, RO-492907, or keregastat. In one implementation, the GSI is smashite.

[0091] In some embodiments, a method for treating respiratory diseases characterized by excessive mucus secretion is provided, the method comprising administering smacide to a patient in need daily at a dose of about 0.1 mg to about 50 mg, wherein oral administration of smacide effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. Preferably, smacide is administered systemically at a daily dose of about 0.5 mg to about 40 mg, or about 0.5 mg to about 30 mg, and more preferably about 0.5 mg to about 20 mg, or about 0.5 mg to about 10 mg. For example, smacide can be administered daily at doses of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

[0092] In another embodiment, a method for treating a respiratory disease characterized by excessive mucus secretion is provided, the method comprising administering smashite systemically to a patient in need of the condition daily at a dose of about 5 µg / kg to about 1 mg / kg, preferably about 50 µg / kg to about 100 µg / kg.

[0093] In embodiments of the invention, a method for treating a respiratory disease characterized by excessive mucus secretion is provided, the method comprising systemically administering a therapeutically effective amount of semacid to a patient requiring such treatment, wherein after multiple doses, the patient's steady-state semacid plasma concentration comprises an AUC (area under the curve) of less than 1220 ng·hr / mL, wherein systemic administration of semacid effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. In some embodiments, after multiple doses, the patient's steady-state semacid plasma concentration comprises an AUC of less than 1220 ng·hr / mL, less than 600 ng·hr / mL, or less than 250 ng·hr / mL.

[0094] In some embodiments, a method is provided for treating respiratory diseases characterized by excessive mucus secretion, the method comprising systemically administering niroglustat at a dose of about 0.1 mg to about 50 mg daily, wherein administration of niroglustat effectively reduces mucus in the lungs of the patient or inhibits mucus accumulation in the lungs of the patient. In some embodiments, niroglustat is systemically administered at a dose of about 0.5 mg to about 40 mg, or about 0.5 mg to about 30 mg, or about 0.5 mg to about 20 mg daily.

[0095] In another embodiment, a method for treating a respiratory disease characterized by excessive mucus secretion is provided, the method comprising administering niroglustat systemically to a patient in need of the condition daily at a dose of about 8 µg / kg to about 0.9 mg / kg, preferably about 10 µg / kg to about 300 µg / kg.

[0096] In some embodiments, a method is provided for treating respiratory diseases characterized by excessive mucus secretion, the method comprising systemically administering RO-4929097 at a dose of about 0.1 mg to about 20 mg daily, wherein the administration of RO-4929097 effectively reduces mucus in the lungs of the patient or inhibits mucus accumulation in the lungs of the patient. In some embodiments, RO-4929097 is systemically administered at a dose of about 0.1 mg to about 10 mg, or about 0.5 mg to about 10 mg, or about 0.1 mg to about 5 mg daily.

[0097] In another embodiment, a method for treating a respiratory disease characterized by excessive mucus secretion is provided, the method comprising administering RO-4929097 systemically to a patient in need of the condition daily at a dose of about 5 µg / kg to about 0.4 mg / kg, preferably about 50 µg / kg to about 100 µg / kg.

[0098] In some embodiments, a method is provided for treating respiratory diseases characterized by excessive mucus secretion, the method comprising systemically administering MK-0752 at a dose of about 0.1 mg to about 40 mg daily, wherein the administration of MK-0752 effectively reduces mucus in the lungs of the patient or inhibits mucus accumulation in the lungs of the patient. In some embodiments, MK-0752 is administered at a dose of about 0.1 mg to about 30 mg, or about 0.1 mg to about 20 mg, and more preferably about 0.1 mg to about 10 mg daily.

[0099] In another embodiment, a method for treating a respiratory disease characterized by excessive mucus secretion is provided, the method comprising administering MK-0752 systemically to a patient in need of the treatment at a dose of about 2.5 µg / kg to about 0.6 mg / kg, preferably about 2.5 µg / kg to about 500 µg / kg, daily.

[0100] In some embodiments, the respiratory disease characterized by excessive mucus secretion is selected from cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, as well as respiratory infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infections. In some embodiments, the respiratory disease is cystic fibrosis. In other embodiments, the respiratory disease is chronic obstructive pulmonary disease.

[0101] In some embodiments, GSI is administered by inhalation. In a preferred embodiment, GSI is administered orally. In one embodiment, GSI is provided in an immediate-release solid oral dosage form. In another embodiment, GSI is provided in a controlled-release solid oral dosage form. In other embodiments, GSI is provided in a liquid dosage form. In still other embodiments, GSI is provided in an inhalation dosage form.

[0102] Combination therapy for cystic fibrosis

[0103] CFTR modulators have represented a significant advancement in the treatment of cystic fibrosis. However, they do not address the damage cystic fibrosis causes to the lung epithelium. Furthermore, CFTR modulators are limited to use in patients with specific CFTR mutations that can be treated with particular CFTR modulators.

[0104] The cell type most capable of expressing functional CFTR is not specified. It has been suggested that a rare cell type known as ionocytes may be the primary source of CFTR expression and thus activity (Plasschaert, LW et al., A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature (2018) 560: 377-381; https: / / doi.org / 10.1038 / s41586-018-0394-6). Furthermore, it has been suggested that Notch inhibition is expected to reduce the number of ionocytes and decrease CFTR activity (ibid., page 380). Other recently published data indicate that various cell types express different levels of CFTR (Carraro, G et al., Nat. Med. (2021) May;27(5):806-814. Doi: 10.1038 / s41591-021-01332-7. Epub 2021 May 5). Contrary to previously published claims that GSI interferes with CFTR activity, it has now been found that GSI treatment does not reduce CFTR activity. Instead, GSI treatment has been found to unexpectedly increase ciliary beat frequency (CBF) and mucus transport in CF cells to the same extent as CFTR modulator drugs. Further evidence suggests that GSI treatment may be synergistic with CFTR modulator therapy, potentially reducing the dosage of one or both drugs and further reducing the potential toxicity of each drug.

[0105] Therefore, the method of the present invention addresses the functional impairments in the airways and other epithelial cells of cystic fibrosis that lead to excessive mucus secretion (and often infection) by promoting MCC differentiation and reducing mucus-secreting cells, thereby improving mucociliary clearance. GSI administration improves epithelial function in cystic fibrosis, independent of CFTR mutations that cause the underlying disease. Therefore, in the treatment of cystic fibrosis, GSI can be administered alone or in combination with any CFTR modulator or a combination of CFTR modulators.

[0106] In some embodiments of the invention, a method for treating cystic fibrosis is provided, comprising administering a therapeutically effective amount of GSI and a CFTR modulator to a patient in need. The GSI may be administered before, after, or concurrently with the CFTR modulator. In some embodiments, the GSI is administered orally to a patient taking a CFTR modulator. The GSI may be provided in a single treatment course or intermittently in combination with a CFTR modulator dosing regimen.

[0107] For example, CFTR modifiers can be administered daily, and GSIs can be administered daily for 1, 3, 5, 7, 10, 14, 18, 21, 24, 28, or 30 days, and then discontinued. In some embodiments, GSIs are administered intermittently, such as every 3 days or weekly. It should be understood that the daily dosage mentioned herein can be achieved with a dosing regimen other than once-daily; for example, a weekly dose of 35 mg would correspond to a daily dose of 5 mg / day. Similarly, sustained-release formulations such as reservoir formulations or patch formulations are known in the art and can be used to provide doses equivalent to the daily doses described herein. The GSI dosing regimen can be repeated if necessary. In some embodiments, the GSI is selected from smasitol, niroglustat, MK-0752, RO-492907, or cligastat. In one embodiment, the GSI is smasitol.

[0108] CFTR modifiers that can be used in this invention include CFTR enhancers, correctors, premature stop codon inhibitors, amplifiers, and combinations thereof. Currently marketed CFTR modifiers include ivacaftor, rumacaftor, tizacator, and elexacaftor, and combinations thereof. Ivacaftor is marketed in tablet and granule form by KALYDEC (see U.S. Patent Nos. 7,495,103 and 8,754,224). Ivacaftor and tizacator are marketed by SYMDEKO (see U.S. Patent Nos. 7,745,789, 7,776,905, 8,623,905, and 10,239,867). Combinations of rumacaftor and ivacaftor are marketed by ORKAMBI (see U.S. Patent Nos. 8,507,534 and 10,597,384). Combinations of elexacaftor, ivacaftor, and tizacator (“ETI”) are marketed by TRIKAFTA. Other CFTR modifiers that can be used in this invention are under development (see, for example, U.S. Patent Nos. 10647717, 10604515; 10568867, 10428017, 10399940, 10259810, 10118916, 9895347, 10550106, 10548878, 10392378, 10494374, 10377762, 10450273, 989014, and 10258624).

[0109] In embodiments of the invention, a method for treating cystic fibrosis by administering therapeutically effective amounts of GSI and a CFTR modulator is provided. In another embodiment, a method for treating cystic fibrosis is provided, wherein GSI is administered systemically to a patient who is receiving or requires CFTR modulator administration. GSI may be administered concurrently with, before, or after the CFTR modulator. In some embodiments, GSI is delivered intermittently in combination with a CFTR modulator dosing regimen.

[0110] In one embodiment, a method is provided for treating cystic fibrosis in a patient who is receiving or needs to receive a CFTR modulator, comprising administering to the patient daily about 0.1 mg to about 50 mg of smacide, wherein oral administration of smacide effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. In some embodiments, smacide is administered systemically at a dose of about 0.5 mg to about 40 mg daily. In some embodiments, smacide is administered at a dose of about 0.5 mg to about 20 mg daily.

[0111] In one embodiment, a method for treating cystic fibrosis in a patient taking a CFTR modulator is provided, comprising administering smashite systemically to the patient in need daily at a dose of about 5 µg / kg to 1 mg / kg, preferably about 50 µg / kg to 100 µg / kg.

[0112] In some embodiments, semaciate is administered orally to a patient taking a CFTR modulator, wherein the steady-state semaciate plasma concentration after multiple doses includes an AUC (area under the curve) of less than 1220 ng·hr / mL, wherein systemic administration of semaciate effectively reduces or prevents mucus buildup in the patient's lungs. In some embodiments, the steady-state semaciate plasma concentration after multiple doses includes an AUC of less than 1220 ng·hr / mL, less than 600 ng·hr / mL, or less than 250 ng·hr / mL. The steady-state semaciate level can be determined approximately 1 week, approximately 2 weeks, or more than 2 weeks after administration of a therapeutically effective dose of semaciate.

[0113] In some embodiments, a method is provided for treating cystic fibrosis in patients who are receiving or require CFTR modulators, comprising administering niroglustat daily to the patient in need of it, from about 0.1 mg to about 50 mg, wherein oral administration of niroglustat effectively reduces or inhibits mucus accumulation in the patient's lungs. In some embodiments, niroglustat is administered systemically at a dose of about 0.5 mg to about 40 mg, or about 0.5 mg to about 30 mg, or about 0.5 mg to about 20 mg daily.

[0114] In another embodiment, a method for treating cystic fibrosis in patients taking CFTR modulators is provided, comprising administering niroglustat systemically daily to the patient in need of such treatment at a dose of about 8 µg / kg to 0.9 mg / kg, preferably about 10 µg / kg to 300 µg / kg.

[0115] In some embodiments, a method is provided for treating cystic fibrosis in patients taking CFTR modulators, comprising administering RO-4929097 daily to a patient in need of this treatment at a dose of about 0.1 mg to about 20 mg, wherein oral administration of RO-4929097 effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. In some embodiments, RO-4929097 is administered systemically at a dose of about 0.1 mg to about 10 mg, or about 0.5 mg to about 10 mg, or about 0.1 mg to about 5 mg daily.

[0116] In another embodiment, a method for treating cystic fibrosis in patients taking CFTR modulators is provided, comprising administering RO-4929097 systemically to the patient in need daily at a dose of about 5 µg / kg to 0.4 mg / kg, preferably about 50 µg / kg to 100 µg / kg.

[0117] In some embodiments, a method is provided for treating cystic fibrosis in patients taking CFTR modulators, comprising administering MK-0752 daily to a patient in need of such treatment at a dose of about 0.1 mg to about 40 mg, wherein oral administration of MK-0752 effectively reduces mucus in the patient's lungs or inhibits mucus accumulation in the patient's lungs. In some embodiments, MK-0752 is administered at a dose of about 0.1 mg to about 30 mg, or about 0.1 mg to about 20 mg, and more preferably about 0.1 mg to about 10 mg daily.

[0118] In another embodiment, a method for treating cystic fibrosis in patients taking CFTR modulators is provided, comprising administering MK-0752 systemically to the patient in need daily at a dose of about 2.5 µg / kg to 0.6 mg / kg, preferably about 2.5 µg / kg to 500 µg / kg.

[0119] Preferably, GSI is delivered orally. GSI may be delivered in the form of an immediate-release oral dosage form or a controlled-release oral dosage form.

[0120] Typically, the person being treated by the method of the present invention, such as that described above, is someone diagnosed with a respiratory disease characterized by excessive mucus secretion. In some cases, the respiratory disease characterized by excessive mucus secretion diagnosed in the subject is selected from cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, as well as respiratory infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infections. In some embodiments, the subject is someone diagnosed with cystic fibrosis. In other embodiments, the subject is someone diagnosed with chronic obstructive pulmonary disease.

[0121] The examples below are for illustrative purposes only and not for limitation.

[0122] Example

[0123] Example 1: Treatment of HNEC with GSI

[0124] Preparation of air-liquid interface (ALI) cultures was described in Vladar EK, Nayak JV, Milla CE, Axelrod JD et al., Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation. JCI Insight. 2016; 1(13); e88027. Human nasal epithelial cells (HNECs) were generated from human nasal epithelial brushes or tissue obtained from patients undergoing endoscopic sinus surgery at Stanford Hospital and cultured as described by Vladar et al.

[0125] Cultures were treated with different doses of semasite. DAPT (Abcam) (0.5 µg) was used as a positive control for GSI activity. On day 21 of ALI, cultures were labeled with anti-acetylated α-tubulin (green) and ECAD (red) antibodies to label cilia and epithelial junctions. Figure 1 The results shown demonstrate the dose-response of HNEC treated with smashite, and the nanomolar concentration of smashite demonstrates efficient conversion to MCC.

[0126] The total cell count was performed using ECAD, and the number of MCCs was counted using anti-acetylated α-tubulin (excluding immature MCCs that had been determined to be of a specific fate but had not yet formed cilia, as the focus was on "functional" MCCs). This ratio was defined as MCC number / total luminal cells. A representative image from five culture replicas from a single donor was used for the count. Figure 2 The results showed that both 1 µM DAPT and semasite at doses ranging from 500 nM to 31.25 nM significantly increased the ratio (p < 0.01, using ANOVA and post-hoc Dunnett multiple comparison test).

[0127] The effective dose of smashite is more than two orders of magnitude lower than the dose used in human Alzheimer's disease trials.

[0128] Example 2: In vivo mouse model

[0129] Smashite or its carrier was administered intraperitoneally (IP) to age-matched male and female Foxj1-GFP mice ranging from 10 to 40 weeks of age. In the first experiment, smashite was administered twice daily at doses of 0.1 mg / kg and 1 mg / kg for 3 days, and compared with administration of the carrier alone. Mice were sacrificed on day 7. Figure 3 A method for assessing airway cellular composition is illustrated. In PFA-fixed airways of approximately similar size, cell nuclei (red; DAPI) were scored as either MCC (green; GFP) or non-ciliated cells (no GFP). Acetylated tubulin (blue) labeled the cilia. Samples from the treatment group were blinded prior to scoring. Figure 5 The results showed that after 3 days of treatment with low- and high-dose systemic (IP) smasitol, the ratio of ciliated cells to non-ciliated cells increased.

[0130] In the second experiment, the carrier and smasitax were administered once daily for five consecutive days each week for three weeks, with a smasitax dose of 1 mg / kg. A significant toxicity observed in multiple GSI clinical trials, including a large Alzheimer's disease trial, is gastrointestinal toxicity. As a surrogate measure of GI toxicity, body weight was monitored throughout the experiment. Body weight was measured on days 1, 9, 24, and 30, and mice were sacrificed on day 31. No deaths or adverse reactions were observed in any group. Figure 4 The body weights on days 9, 24, and 30 are shown, and it is indicated that there were no significant differences between the treatment groups compared to the loading control. Figure 6 The results showed that after 3 weeks of systemic (IP) smashite, the ratio of ciliated cells to non-ciliated cells was significantly increased.

[0131] A dose-response trend was observed during the 3-day treatment, with the high dose reaching statistical significance. Figure 5The response to a high-dose treatment once daily for 3 weeks was very significant. Figure 6 ).

[0132] Example 3: The dependence of multiciliated cell formation in differentiated and mature airway epithelial cells on treatment time

[0133] Primary human airway epithelial cells were treated with DAPT and LY45139: I) only during proliferation (days 5 to 1 before ALI), ii) only during differentiation (days 0 to +21 of ALI), or iii) continuously throughout the culture period, followed by labeling with anti-acetylated α-tubulin (green) and ECAD (red) antibodies on day 21 of ALI. GSI treatment during multiciliated cell differentiation (differentiation only and continuous treatment) increased the number of MCC cells. Figure 7 The results indicate that GSI treatment during proliferation has no effect on differentiation, nor on subsequent differentiation or the overall epithelial structure. Figure 8 It shows Figure 7 The data shown is quantified (MCC per total lumen cell).

[0134] Mature (ALI day 30) primary human airway epithelial cells were treated with DAPT and smasidium for one week (ALI day 30 to 37) or two weeks (ALI day 30 to 44) and then labeled with anti-acetylated α-tubulin (green) and ECAD (red) antibodies. Figure 9 The results show that GSI treatment induced the formation of additional multiciliated cells in mature cultures, while untreated cultures did not differentiate into any more multiciliated cells. Figure 10 It shows Figure 9 The data shown is quantized.

[0135] Primary human airway epithelial cells were treated with DAPT and LY45139 only during differentiation from the apical or basal surface (days 0 to 21 of ALI), and then labeled with anti-acetylated α-tubulin (green) and ECAD (red) antibodies on day 21 of ALI. Figure 11 The results show that GSI treatment induces ciliated cell formation through top or base application. Top treatment eliminates the air-liquid interface, which leads to poor epithelial structure and multiciliated cell differentiation in untreated cultures; GSI treatment partially salvages this. Figure 12 It shows Figure 11 The data presented is quantified. This result suggests that both systemic and inhalational exposure may be effective in vivo.

[0136] The results showed that treatment during or after differentiation increased the ratio of MCC to total cells. Treatment during proliferation (pre-differentiation) had no significant effect, whether beneficial or detrimental.

[0137] Example 4: IL-13-induced chronic inflammation model

[0138] IL-13 was administered to ALI cultures to induce goblet cell proliferation, a useful model of chronic inflammation. ALI HNEC cultures were prepared according to Vladar et al. ALI cultures were treated with and without IL-13 administration from day 7 to day 14. DAPT (1 μm), smashite (125 nm), or a carrier control were administered from day 14 to day 21. Cultures immobilized with PFA were stained with Muc5AC (red; mucin-producing secretory cells), acetylated tubulin (green; MCC), and E-cadherin (blue showing cell boundaries).

[0139] Figure 13 The effects of semasite and DAPT treatments in a chronic inflammation model of ALI are illustrated. Inflammation was induced by treating HNEC cultures from CF patients with IL-13 from day 7 to day 14 of ALI. DAPT and semasite increased the percentage of MCCs in the control group (left). IL-13 treatment increased the percentage of mucin-positive secretory cells and decreased the percentage of MCCs. Subsequent DAPT or semasite treatment salvaged the cellular composition, increased the percentage of MCCs, and decreased the percentage of mucin-positive secretory cells.

[0140] Figure 14 It shows Figure 13 Quantification of the MCC of each total lumen cell in the data.

[0141] Example 5: A representative Uss chamber diagram

[0142] Cells grown at the gas-liquid interface were mounted on a fixed slider and inserted into a Uuse chamber for electrophysiological short-circuit current (Isc) measurements. Solutions were prepared in serous and mucosal baths to establish a chloride gradient between the two sides. After obtaining a stable baseline current recording, agonists were added in the following order: amiloride (10 µM) to block sodium channel activity, forscorin (10 µM) to stimulate CFTR, ivacaftor (10 µM) to enhance CFTR activity, and CFTRinh-172 (20 µM) to block CFTR current. The signal for each agonist was monitored until a steady current was observed before adding the next agonist. The δ-Isc response to CFTRinh-172 was used as the primary readout for CFTR-mediated chloride transport. Results are as follows: Figure 15 As shown.

[0143] Example 6: Electrophysiological assay of CFTR activity

[0144] To evaluate the effect of GSI (DAPT, simacita) on CFTR function in cultured epithelial cells, HNECs from CF patients and non-CF controls were collected by Vladar et al. and cultured at the gas-liquid interface until maturity (21 days). Cultures were then treated three times weekly for two weeks with DAPT, simacita, the CFTR modulator rumacator, or a carrier control added to basal medium. The short-circuit current (Isc) of the filter cartridge to the chloride gradient in the Uuse chamber was then assessed to evaluate CFTR activity. Figure 16 A representative figure is shown. Figure 17 CFTR channel activity was quantified by adding CFTR to two wild-type control cultures and two cultures from CF patients. inh Current suppression after 172 was evaluated (genotype: rare / rare = W1282X [class I] / I1234V [class II] and F508Δ / F508Δ). Note that in all cases, the CFTR current in cultures treated with smashite was equal to or greater than the CFTR current under control conditions.

[0145] Example 7: Mucus production in human CF cells

[0146] exist Figure 18 In this study, the PFA culture replicated in Example 6 was fixed and stained for Muc5AC (red; mucus) and acetylated tubulin (green; MCC). Note the mucus lines (red) in the wash-resistant, drug-loaded control CF cultures. Cultures treated with smasitronidazole revealed significantly less mucus without lines. This effect was observed in homozygous F508Δ and W1282X / I1234V (a rare regulator-responsive genotype) cells treated with the CFTR regulators rumacator and ivacaftor.

[0147] Example 8: Combination of Simassitol and CFTR modifier

[0148] To evaluate the effect of combined semacid and CFTR modulator therapy, wild-type control and CF(F508Δ / F508Δ) HNEC cultures were grown to maturity from day 0 to day 21 of ALI, with or without semacid. Prior to fixation, cultures from day 19 to day 21 of ALI were treated with or without rumacator (VX-809) and with Ivacaftor (VX-770) for 10 minutes. PFA-fixed membranes were then stained for acetylated tubulin (green; MCC) and E-cadherin (red). Results are as follows: Figure 19 As shown. Note that the ability of cultures treated with combination therapy to differentiate into MCCs is comparable to or better than that of cultures treated with smashite alone.

[0149] Figure 20It shows Figure 19 Quantitative data from healthy patients and CF donor 1. In some cases, such as this one, the increase in the smácitrate-induced MCC / total cell ratio was further increased by rumacator. Although this was not consistently observed in all quantified cultures and was not statistically significant, it can be concluded that no significant reduction in the smácitrate-induced MCC response was observed. CF patients who did not meet the Trikafta criteria responded similarly to smácitrate alone as those receiving the modifier (not shown).

[0150] Example 9: GSI treatment induces multiciliary cell formation in cystic fibrosis epithelium

[0151] Primary healthy and cystic fibrotic airway epithelial cells were treated with semasite (LY45139) only during differentiation (day 0 to day 21 of ALI), and then labeled with anti-acetylated α-tubulin (green) and ECAD (red) antibodies on day 21 of ALI. Figure 21 The results showed that LY45139 effectively increased the MCC / total cell ratio in cultures from CF patients. A healthy MCC / total cell ratio is expected to improve mucociliary clearance.

[0152] Example 10: GSI treatment induces structurally normal cilia in healthy and CF epithelial cells

[0153] Figure 22 SEM images of healthy and CF primary human airway epithelial cultures are shown, demonstrating that the multiciliated cells formed under DAPT treatment are indistinguishable from those in untreated healthy cultures.

[0154] Mature (ALI day 30) primary cystic fibrosis human airway epithelial cells were treated with DAPT for one week (ALI days 30 to 37) and then labeled with anti-acetylated α-tubulin (green) and ECAD (red) antibodies. Figure 23 The results show that GSI treatment induced the formation of additional multiciliated cells in mature cystic fibrosis cultures, while untreated cultures did not differentiate into any more multiciliated cells.

[0155] Example 11: The formation of multiciliated cells is induced by multiple GSIs

[0156] During differentiation (day 0 to day 21 of ALI), primary human airway epithelial cells were treated with DAPT and high and low concentrations of GSI LY45139, PF-03084014, RO-4929097, and MK-0752. On day 21 of ALI, cilia and epithelial junctions were labeled with anti-acetylated α-tubulin (green) and ECAD (red) antibodies. Results are as follows... Figure 24As shown, at high concentrations, GSI disrupted epithelial structure, but at low concentrations, all GSI induced the formation of multiciliated cells, similar to DAPT. Figure 25 It shows Figure 24 The data shown represents the quantification of the MCC of each total lumen cell.

[0157] Example 12: GSI treatment does not weaken CFTR current

[0158] CFTR activity in cultures from CF patients treated with GSI was directly measured, regardless of whether a known in vitro effective dose of Trikafta (Elexcaftor / Tezacator / Ivacaftor or “ETI”) was used. Veit, G. et al., JCI (2020) 10.1172 / jci.insight.139983. Measurements were performed in a Ussen chamber. LY45139( Figure 26 ) and MK04752 ( Figure 27 Neither GSI nor ETI-induced CFTR currents were attenuated. In some individual studies, combination therapy appeared to have a synergistic effect: GSI monotherapy was ineffective, but combination therapy produced a larger response than ETI monotherapy. This effect did not persist, but it increases the likelihood of observing a synergistic effect at lower ETI doses.

[0159] To further verify this, combinations of MK04752 with different doses of ETI were tested. Figure 28 The results showed that MK04752 appeared to enhance the suboptimal dose of ETI, indicating a synergistic effect.

[0160] Example 13: GSI treatment has no effect on the formation of ionocytes.

[0161] Due to previous assertions regarding CFTR expression, particular interest was generated in ionocytes; therefore, in addition to measuring current, the prevalence of ionocytes treated with GSI was analyzed. Primary healthy airway epithelial cells were treated with DAPT (ALI day 0 to day 21) only during differentiation, and on ALI day 21, they were labeled with anti-FOXI1 (green; ionocyte-specific marker) and acetylated α-tubulin (red) antibodies, and the nuclei were stained with DAPI (blue) to label the nuclei. Figure 29 The results showed that both untreated and DAPT-treated cultures contained a similar number of FOXI1-positive cell nuclei, indicating ionocytes. Therefore, previous claims that the number of ionocytes decreased with Notch inhibition appear to be incorrect.

[0162] Example 14: Effects of low concentrations of GSI MK-0752 and CFTR modifier Elexacaftor on ciliary beating frequency The role of (CBF)

[0163] As in previous experiments, primary nasal epithelial cells from two CF donors (F508del homozygous) were grown in filter cartridges until complete differentiation. The ability of lower doses of both MK-0752 and Elexacaftor to elicit a positive response in CBF, compared to the doses used in previous experiments, was assessed as evidence of a synergistic effect between the two drugs. During differentiation, cultures were treated with either a 125 nM load control or a combination of GSI MK-0752, or a triple modulator of 100 nM Elexacaftor, or a combination of both treatments. After cell maturation, the top surface was gently washed with PBS and then placed on an inverted microscope on a 37°C heated stage. High-speed video recording at 200x magnification was performed on the ciliary surface to estimate CBF in several regions. Figure 30 The bar graphs in the figure represent the mean CBF in Hz for each condition. Both treatments showed a significant enhancement of CBF (p < 0.001 for both compared to the control). Furthermore, the increase in CBF from the combination therapy was greater than that from either drug alone (p < 0.005 for all comparisons), demonstrating a synergistic effect, as the increase in CBF was significantly higher than the increase expected from simply adding either drug independently (36% vs. 28%, p = 0.049).

[0164] Figure 31 The results show the ciliary beating frequency (CBF) and ciliary length of primary human epithelial cells treated with DAPT compared to untreated controls. The results indicate that DAPT-treated multiciliated cells showed a moderate but significant increase in ciliary beating frequency, but no difference in ciliary length.

[0165] Example 15: GSI treatment reduced the airway surface fluid (ASL) reabsorption characteristics of CF, to a degree comparable to CFTR. The same regulator drug

[0166] As in previous experiments, primary nasal epithelial cells from two CF donors (F508del homozygous) were grown in the filter cartridges until complete differentiation. During differentiation, they were treated with a load control (blue), a triple combination of CFTR modulators (Elexcaftor / tizacato / Ivacaftor; orange), GSI smashite (LY-45139) (grey), or a combination of two treatments (yellow). Once mature, the top surface was gently washed with PBS, and then 30 µl of PBS was added to the surface. The filter cartridges were then weighed on a precision balance at 0, 12, and 24 hours after the addition of the liquid. The change in weight over time was used as a surrogate indicator of liquid reabsorption. Results are as follows: Figure 32As shown, control cells exhibited a typical ASL reabsorption pattern within 48 hours, while the reabsorption of treated cells was significantly reduced (p < 0.001 relative to control). Notably, there was no significant difference in the effect of all drug treatments on fluid reabsorption (p > 0.3 for all comparisons between drug treatments).

[0167] Example 16: Combined treatment with GSI and CFTR modulators significantly improved mucus transport.

[0168] Primary nasal epithelial cells from two CF donors (F508del homozygous) were grown to complete differentiation as in previous experiments under treatment with a load control, a triple combination of CFTR modulators (Elexcaftor / tizacato / Ivacaftor), GSI smasitol (LY-45139), or a combination of two, repeated twice. After maturation, a 20 µl suspension of 2 µm latex beads was added to the top surface, and the inserts were cut and placed under a microscope equipped with a high-speed video recorder. Images were then acquired at 1000 fps to track bead movement as a reflection of mucus transport on the ciliary surface and to estimate the distance traveled by individual beads. Figure 33 Representative images of each treatment are shown. Figure 34 The results showed that control cells exhibited almost no bead movement, reflecting poor mucus transport. Significantly increased motility was observed in the ETI or semacid treatment groups (p<0.01 for both treatment groups compared to the control group). A significant increase in transport was observed with the combination of ETI and semacid (p<0.05 compared to treatment alone). This can be seen as evidence of the strong enhancing effect of combination therapy on mucociliary transport and predicts substantial benefit for patients receiving CFTR modulators by increasing GSI treatment.

[0169] Notwithstanding the appended claims, this disclosure is also defined by the following provisions:

[0170] 1. A method for treating a respiratory disease characterized by excessive mucus secretion, comprising:

[0171] Administer a low dose of GSI to the patient who requires the treatment; and

[0172] The amount of mucus in the patient's lungs is reduced or the accumulation of mucus in the patient's lungs is inhibited.

[0173] 2. The method according to Clause 1, wherein the respiratory disease is selected from cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, and respiratory tract infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infections.

[0174] 3. The method described in accordance with Clause 1 or 2, wherein the GSI is selected from smashite, celecoxib, GS-1, DBZ, L-685458, BMS-906024, keregastat, MRK 560, niroglustat, RO-4929097, MK-0752, etanercept, LY-3056480, fosciclopirox, flurbiprofen, and begastat.

[0175] 4. The method according to Clause 3, wherein the GSI is selected from smashite, niroglustat, MK-0752, RO-492907 or keregastat.

[0176] 5. The method described in Clause 4, wherein GSI is Simashite.

[0177] 6. The method described in Clause 4, wherein GSI is MK-0752.

[0178] 7. The method described in Clause 4, wherein GSI is niroglustat.

[0179] 8. The method described in accordance with Clause 4, wherein GSI is RO-492907.

[0180] 9. The method described in Clause 4, wherein GSI is cregalstatin.

[0181] 10. The method according to Clause 2, wherein the administration of GSI is oral.

[0182] 11. The method described in Clause 2, wherein the respiratory disease is cystic fibrosis.

[0183] 12. The method described in Clause 2, wherein the respiratory disease is chronic obstructive pulmonary disease.

[0184] 13. The method according to Clause 5, wherein smashite is administered orally in doses of about 0.1 mg to about 50 mg daily.

[0185] 14. The method according to Clause 13, wherein approximately 0.5 mg to approximately 40 mg of smashite is administered daily.

[0186] 15. The method of claim 14, wherein approximately 0.5 mg to approximately 30 mg of smashite is administered daily.

[0187] 16. The method of claim 15, wherein approximately 0.5 mg to approximately 20 mg of smashite is administered daily.

[0188] 17. The method according to Clause 7, wherein niroglustat is administered orally in doses of about 8 μg to about 0.9 mg daily.

[0189] 18. The method of procedure according to Clause 17, wherein approximately 10 μg to approximately 300 μg of niroglustat is administered daily.

[0190] 19. The method according to Clause 8, wherein RO-492907 is administered orally in doses of about 0.1 mg to about 20 mg daily.

[0191] 20. The method according to Clause 19, wherein RO-492907 is administered daily from about 0.1 mg to about 10 mg.

[0192] 21. The method according to Clause 20, wherein about 0.1 mg to about 5 mg of RO-492907 is administered daily.

[0193] 22. The method according to Clause 6, wherein MK-0752 is administered orally in doses of about 0.1 mg to about 40 mg daily.

[0194] 23. The method of claim 22, wherein approximately 0.1 mg to approximately 20 mg of MK-0752 is administered daily.

[0195] 24. The method of claim 23, wherein approximately 0.1 mg to approximately 10 mg of MK-0752 is administered daily.

[0196] 25. A method for treating a respiratory disease characterized by excessive mucus secretion, comprising:

[0197] A therapeutically effective dose of semacid is administered systemically to a patient requiring the treatment, wherein, approximately 1 week, approximately 2 weeks, or more after administration of the therapeutically effective dose, the patient's steady-state semacid plasma concentration has an AUC of less than 1220 ng•hr / mL.

[0198] The application of smasithide effectively reduces or inhibits the accumulation of mucus in the patient's lungs.

[0199] 26. The method described in Clause 25, wherein the respiratory disease is selected from cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, obliterative bronchiolitis, idiopathic pulmonary fibrosis and other fibrotic lung disorders, and respiratory infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infections.

[0200] 27. The method according to Clause 26, wherein after multiple doses, the patient’s steady-state smasitol plasma concentration includes an AUC of less than 600 ng•hr / mL.

[0201] 28. The method according to Clause 27, wherein after multiple doses, the patient’s steady-state smasitol plasma concentration comprises an AUC of less than 250 ng•hr / mL.

[0202] 29. The method described in Clause 26, wherein the respiratory disease is cystic fibrosis.

[0203] 30. The method described in Clause 26, wherein the respiratory disease is chronic obstructive pulmonary disease.

[0204] 31. A method for treating cystic fibrosis, comprising:

[0205] Administer an effective amount of GSI to a patient who is currently receiving or requires one or more CFTR modulators.

[0206] Furthermore, after GSI administration, the amount of mucus in the patient's lungs decreased or the accumulation of mucus in the patient's lungs was inhibited.

[0207] 32. The method described in Clause 31, wherein GSI is selected from smash, celecoxib, GS-1, DBZ, L-685458, BMS-906024, cregalstat, MRK 560, niroglustat, RO-4929097, MK-0752, etanercept, LY-3056480, fosciclopirox, flurbiprofen, and begastat.

[0208] 33. The method described in accordance with Clause 32, wherein the GSI is selected from smashite, niroglustat, MK-0752, RO-492907 or keregastat.

[0209] 34. The method described in Clause 33, wherein GSI is Simsit.

[0210] 35. The method described in accordance with Clause 33, wherein GSI is MK-0752.

[0211] 36. The method described in accordance with Clause 33, wherein GSI is niroglustat.

[0212] 37. The method described in accordance with Clause 33, wherein the GSI is RO-492907.

[0213] 38. The method described in accordance with Clause 33, wherein GSI is cregalstatin.

[0214] 39. The method according to Clause 31, wherein the administration of GSI is oral.

[0215] 40. The method according to Clause 31, wherein the CFTR modifier is a CFTR enhancer.

[0216] 41. The method according to Clause 31, wherein the CFTR modifier is a CFTR corrector.

[0217] 42. The method according to Clause 31, wherein the CFTR modifier is a CFTR amplifier.

[0218] 43. The method according to Clause 31, wherein the CFTR modifier is selected from ivacaftor, rumacaftor, tizacato, elexacaftor, and combinations thereof.

[0219] 44. The method according to Clause 31, wherein GSI is administered daily.

[0220] 45. The method described in Clause 44, wherein GSI is applied for a maximum of thirty days, and then discontinued.

[0221] 46. ​​The method described in Clause 31, wherein GSI is applied weekly.

[0222] 47. A method for treating cystic fibrosis, comprising:

[0223] Administer an effective dose of GSI to patients taking CFTR modulators;

[0224] GSI is selected from Simasit, Celexib, GS-1, DBZ, L-685458, BMS-906024, Crizostat, MRK 560, Niroglustat, RO-4929097, MK-0752, Etalex, LY-3056480, Fosciclopirox, Flurbiprofen, and Begastidine;

[0225] The CFTR modifiers are selected from ivacaftor, rumacator, tizacator, elexacaftor, and combinations thereof; and

[0226] After GSI administration, the amount of mucus in the patient's lungs decreased or the accumulation of mucus in the patient's lungs was inhibited.

[0227] 48. The method described in accordance with Clause 47, wherein the GSI is selected from smashite, niroglustat, MK-0752, RO-492907 or keregastat.

[0228] Although the invention has been described in some detail by way of illustration and example for clarity, those skilled in the art will be able to make certain changes and modifications to it without departing from the spirit or scope of the appended claims, based on the teachings of the invention.

Claims

1. The use of low-dose GSI in the preparation of medicaments for treating respiratory diseases characterized by excessive mucus secretion, wherein: A low dose of GSI is administered to the patient who requires the treatment. and After GSI administration, the amount of mucus in the patient's lungs was reduced or the accumulation of mucus in the patient's lungs was substantially improved or suppressed.

2. The use according to claim 1, wherein the low dose is an effective amount for treating respiratory diseases characterized by excessive mucus secretion, and is a lower dose compared to the GSI dose suitable for administration to patients with neurodegenerative diseases, neoplastic diseases, or respiratory diseases not characterized by excessive mucus secretion.

3. The use according to claim 2, wherein the low dose of GSI produces peak plasma levels in the submicromolar range.

4. The use according to claim 1, wherein the respiratory disease is selected from cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, obliterative bronchiolitis, idiopathic pulmonary fibrosis and other fibrotic lung disorders, and respiratory tract infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infection.

5. The use according to any one of claims 1 to 4, wherein GSI is selected from smashite, celecoxib, GS-1, DBZ, L-685458, BMS-906024, cregalstat, MRK 560, niroglustat, RO-4929097, MK-0752, etanercept, LY-3056480, fosciclopirox, flurbiprofen, and begasstat.

6. The use according to claim 5, wherein the GSI is selected from smashite, nirogastrostat, MK-0752, RO-492907 or keregastat.

7. The use according to claim 6, wherein GSI is Simsit.

8. The use according to claim 6, wherein GSI is MK-0752.

9. The use according to claim 6, wherein GSI is niroglustat.

10. The use according to claim 6, wherein GSI is RO-492907.

11. The use according to claim 6, wherein GSI is cregalactostat.

12. The use according to any one of claims 1 to 4, wherein GSI is administered orally.

13. The use according to any one of claims 1 to 4, wherein the respiratory disease is cystic fibrosis or chronic obstructive pulmonary disease.

14. The use of smashite in the preparation of medicaments for treating respiratory diseases characterized by excessive mucus secretion, wherein: A therapeutically effective amount of semasite was systemically administered to patients requiring the treatment, wherein, after multiple doses, the steady-state plasma concentration of semasite in the patients had an AUC of less than 1220 ng•hr / mL. The application of smasithide effectively reduces or inhibits the accumulation of mucus in the patient's lungs.

15. The use according to claim 14, wherein the respiratory disease is selected from cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, obliterative bronchiolitis, idiopathic pulmonary fibrosis and other fibrotic lung disorders, and respiratory tract infections, including exacerbations of chronic respiratory disorders and mucus accumulation in response to acute infection.

16. The use according to claim 15, wherein the respiratory disease is cystic fibrosis or chronic obstructive pulmonary disease.

17. The use according to claim 14, 15 or 16, wherein smashite is administered in doses from 0.1 mg to 50 mg daily.

18. The use according to claim 17, wherein smashite is administered daily at a dose of 0.5 mg to 40 mg.

19. The use according to claim 18, wherein smashite is administered daily in doses of 0.5 mg to 40 mg.

20. The use according to claim 19, wherein smashite is administered daily at a dose of 0.5 mg to 30 mg.

21. The use according to claim 20, wherein smashite is administered daily at a dose of 0.5 mg to 20 mg.

22. The use of GSI in the preparation of medicaments for the treatment of cystic fibrosis, wherein: An effective dose of GSI is administered to patients who are currently receiving or require CFTR modulators. The amount of mucus in the patient's lungs is reduced or the accumulation of mucus in the patient's lungs is inhibited.

23. The use according to claim 22, wherein GSI is selected from smashite, celecoxib, GS-1, DBZ, L-685458, BMS-906024, cregalstat, MRK 560, niroglustat, RO-4929097, MK-0752, etanercept, LY-3056480, fosciclopirox, flurbiprofen, and begasstat.

24. The use according to claim 23, wherein GSI is selected from smashite, niroglustat, MK-0752, RO-492907 or keregastat.

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