Vanzacaftor enantiomer for BK potentiation in airway diseases

Abstract

Described herein are methods of treating cystic fibrosis, asthma, COPD, and other airway related diseases in a patient by administering a therapeutically effective amount of a specific enantiomer, R-vanzacaftor or a pharmaceutically acceptable salt thereof, to the patient.

Description

25KU017M-0262724-PCTVANZACAFTOR ENANTIOMER FOR BK POTENTIATION IN AIRWAY DISEASESCROSS-REFRENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 738,609, filed December 24, 2024, entitled VANZACAFTOR ENANTIOMER FOR BK POTENTIATION IN AIRWAY DISEASES, incorporated by reference in its entirety herein.SEQUENCE LISTING

[0002] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled "SequenceListing_62724.xml," created on December 19, 2025, as 38,796 bytes in size, the contents of which are incorporated herein.TECHNICAL FIELD

[0003] This disclosure is directed to vanzacaftor compounds, therapeutic composition comprising the vanzacaftor compounds, and methods of treating airway conditions using the same.BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] In patients with CF, mutations in CFTR (cystic fibrosis transmembrane conductance regulator) endogenously expressed in respiratory epithelia lead to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to enhanced mucus accumulation in the lung and accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, result in death. In addition, the majority of males with cystic fibrosis are infertile, and fertility is reduced among females with cystic fibrosis.

[0006] Sequence analysis of the CFTR gene has revealed a variety of disease-causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 2000 mutations in the CF25KU017M-0262724-PCTgene have been identified; currently, the CFTR2 database contains information on only 322 of these identified mutations, with sufficient evidence to define 281 mutations as disease-causing. The most prevalent disease-causing mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence, and is commonly referred to as the F508del mutation. This mutation occurs in approximately 70% of the cases of cystic fibrosis and is associated with severe disease.

[0007] The deletion of residue 508 in CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit the endoplasmic reticulum (ER) and traffic to the plasma membrane. As a result, the number of CFTR channels for anion transport present in the membrane is far less than observed in cells expressing wild-type CFTR, i.e., CFTR having no mutations. In addition to impaired trafficking, the mutation results in defective channel gating. Together, the reduced number of channels in the membrane and the defective gating lead to reduced anion and fluid transport across epithelia. (Quinton, P. M. (1990) FASEB J. 4: 2709-2727). The channels that are defective because of the F508del mutation are still functional, albeit less functional than wild-type CFTR channels. (Dalemans et al. (1991) Nature Lond. 354: 526-528; Pasyk and Foskett (1995) J. Cell. Biochem. 270: 12347-50). In addition to F508del, other disease-causing mutations in CFTR that result in defective trafficking, synthesis, and / or channel gating could be up- or down-regulated to alter anion secretion and modify disease progression and / or severity.

[0008] CFTR is a cAMP / ATP-mediated anion channel that is expressed in a variety of cell types, including absorptive and secretory epithelia cells, where it regulates anion flux across the membrane, as well as the activity of other ion channels and proteins. In epithelial cells, normal functioning of CFTR is critical for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. CFTR is composed of approximately 1480 amino acids that encode a protein which is made up of a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are linked by a large, polar, regulatory (R)-domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.

[0009] Chloride transport takes place by the coordinated activity of ENaC and CFTR present on the apical membrane and the Na+— K-ATPase pump and CE channels expressed on the basolateral surface of the cell. Secondary active transport of chloride from the luminal side leads to the accumulation of intracellular chloride, which can then passively leave the cell via CE channels, resulting in a vectorial transport. Arrangement of Na / 2C1 / !< co-transporter, Na+— K+-ATPase pump and the basolateral membrane K+channels on the basolateral surface25KU017M-0262724-PCTand CFTR on the luminal side coordinate the secretion of chloride via CFTR on the luminal side. However, apically expressed BK channels provide an important driver for Cl" exit via the so called apical loop current discussed below. (Manzanares D. et al. (2011) J. Biol. Chem.286(22):.19830-9) (Cook, D. I. and Young, J. A. (1989) J. Membr. Biol. 110(2): 139-46.) Because water is probably never actively transported itself, its flow across epithelia depends on tiny transepithelial osmotic gradients generated by the bulk flow of sodium and chloride. BK (big potassium) channels (aka Slol, Maxi-K) are large conductance calcium-activated potassium channels. BK channels are voltage-gated potassium channels that conduct large amounts of potassium ions (K+) across the cell membrane, hence their name, big potassium. These channels can be activated (opened) by either electrical means, or by increasing Ca2+concentrations in the cell. BK channels help regulate physiological processes, such as circadian behavioral rhythms and neuronal excitability. BK channels are also involved in many processes in the body, as it is a ubiquitous channel.

[0010] A number of CFTR modulators have recently been identified. These modulators can be characterized as, for example, potentiators, correctors, potentiator enhancers / co-potentiators, amplifiers, readthrough agents, and nucleic acid therapies. CFTR modulators that increase the channel gating activity of mutant and wild-type CFTR at the epithelial cell surface are known as potentiators. Correctors improve faulty protein processing and resulting trafficking to the epithelial surface. Ghelani and Schneider-Futschik (2020) ACS Pharmacol. Transl. Sci. 3:4-10. There are three CFTR correctors approved by the U.S. FDA for treatment of cystic fibrosis. However, monotherapy with some CFTR correctors has not been found to be effective enough and as a result combination therapy with a potentiator is needed to enhance CFTR activity. There is currently only one CFTR potentiator that is approved for the treatment of cystic fibrosis. Thus, although the treatment of cystic fibrosis has been transformed by these new small molecule CFTR modulators, new and better modulators are needed to prevent disease progression, reduce the severity of the cystic fibrosis and other CFTR-mediated diseases, and to treat the more severe forms of these diseases.

[0011] Modulators of CFTR have made large differences for more than 80% of people with cystic fibrosis (pwCF) by partially correcting the function of certain disease-causing CFTR variants (Middleton, P.G. et al. (2019) N. Engl. J. Med. 381(19): 1809-19; Keating, D. et al. (2018) N. Engl. J. Med. 379(17): 1612-20). However, pwCF with minimal function CFTR variants (no response to modulators, no baseline function) have limited therapeutic options. Alternative ion channels to CFTR could serve as therapeutic targets in CF and other airway diseases. BK channels play important roles throughout the body due to their large conductance25KU017M-0262724-PCTand dual sensitivity to membrane voltage (V) and intracellular calcium (Ca2+). BK channels consist of four a subunits (Slot) encoded by the KCNMA1 gene, plus a variable number of auxiliary subunits. Slol contains the pore and Ca2+- and V-sensor domains. Channels are formed from Slol alone in some tissues but more often are formed with auxiliary subunits (e.g. pi-4, yl-4) that tune the channel’s function to tissue-specific roles. For example, the yl subunit (LRRC26) shifts the V-dependence of activation by more than -150 mV, which is essential to allow BK channels to activate in non-excitable secretory epithelial cells in the airway (Manzanares, D. et al. (2015) J. Biol. Chem. 290(42):25710-6; Kim, M.D. et al. (2020) Am. J. Respir. Crit. Care Med. 201(3): 313-24). The P4 subunit relevant to large airway secretory cells enhances Ca2+-sensitivity and slows activation (Brenner, R. (2000) J. Biol. Chem. 275(9): 6453-61; Contreras, G. F. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109(46): 18991-6; Yan, J. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109(20):7917-22).

[0012] BK channels provide a driving force for chloride secretion. BK channel function is critical for adequate airway surface liquid (ASL) volume as BK inhibition, by knockdown of the pore-forming subunit or the y-regulatory subunit LRRC26 (critical for BK function in non-excitable cells), causes significant ASL volume loss, even in the presence of functional CFTR and TMEM16A. Apical K+secretion is a driving force for CL exit, forming the apical loop current. (Cook, D. I. and Young, J. A. (1989) J. Membr. Biol. 110(2): 139-46.) Using the model of Cook and Young, it was demonstrated that apical CL secretion increases 5- to 30-fold when apical BK channels are activated (Manzanares D. et al. (2011) J. Biol. Chem. 286(22):.19830-9). Importantly, even small increases in BK activity can lead to large changes in CL secretion (FIG. 1, A-B). In simulations, CL secretion changes with increasing apical K+conductance. Thus, enhancing BK function can be an important strategy for improving mucociliary clearance in the airways of pwCF.

[0013] BK potentiators or activators exist, but none for clinical use (Al-Khazali, H.M. et al. (2024) J. Headache Pain. 25(1): 102). Clinical CFTR modulators, e.g., elexacaftor and vanzacaftor, potentiate BK (Clancy, J. P. et al. (2019) J. Cyst. Fibros. 18(l):22-34). In vitro studies have shown that elexacaftor is a weak potentiator, active in the presence of LRRC26 (Kolski-Andreaco A et al. (2024) J. Clin. Invest. 134(16)). In another study related to clinical use, pwCF were provided elexacaftor, tezacaftor, and / or ivacaftor for a month, no matter their CFTR variants (Burgel, P. R. et al. (2023) Eur. Respir. J. 2023). In FIG 2, selected clinical results of pwCF with two minimal function CFTR variants showed that 9 out of 21 have meaningful FEV1 increases and up to 11% predicted FEV1. As expected with minimal function CFTR variants, sweat chloride did not change. These data support the hypothesis that25KU017M-0262724-PCTan ion channel other than CFTR has been variably potentiated by elexacaftor, likely BK ion channel. In the case of nonfunctioning CFTR (such as in minimal function variants), the alternative Cl" channel TMEM16A or AN01 is co-expressed in secretory cells in large and small airways (FIG. 3, A-F).

[0014] Airway inflammation is invariably present in pwCF off modulators. TGF- 1 is linked to worse pulmonary outcomes (Chen, G. et al. (2019) J. Clin. Invest. 129(10):4433-50) with levels inversely related to pediatric lung function (Harris, W. T. et al. (2011) Pediatr. Pulmonol.46(7): 688-95). TGF-pi decreases BK activity by downregulating LRRC26 (Manzanares, D. et al. (2015) J. Biol. Chem. 290(42): 25710-6; Kim, M. D. et al. (2020) Am. J. Respir. Crit. Care Med. 201(3):313-24). Mechanisms by which TGF-PI -induced BK dysfunction can be targeted to restore apical loop currents, are insufficiently explored. Anti-inflammatory therapies can counter TGF-PI effects in CF airway epithelia in vitro (Kim, M. D. et al. (2022) J. Clin. Invest. 132(11); Kim, M. D. et al. (2020) Am. J. Respir. Crit. Care Med. 201(3):313-24) and in pwCF, however better strategies are needed to recover BK in TGF-pi -dominated inflammation.SUMMARY

[0015] The present disclosure concerns the use of a particular vanzacaftor enantiomer, R-vanzacaftor, as a potent BK potentiator. In particular, the data demonstrates that R-vanzacaftor, but not S -vanzacaftor, is a more efficacious long-term BK potentiator and is expected to meaningfully increase lung function in various airway conditions.

[0016] Also disclosed is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof described herein. Also disclosed are pharmaceutical compositions where the R-vanzacaftor is cleavably linked to a peptide-based nanosponge for enhanced delivery.

[0017] In some aspects, the techniques described herein relate to methods of decreasing mucus concentration and / or mucus viscosity in a patient, including: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

[0018] In some aspects, the techniques described herein relate to methods of increasing mucociliary transport in a patient, including: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

[0019] In some aspects, the techniques described herein relate to methods of treating cystic fibrosis in a patient, including: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.25KU017M-0262724-PCT

[0020] In some aspects, the techniques described herein relate to methods of treating inflammation or assuring the therapy still works in an inflammatory airway environment in a cystic fibrosis, asthma, or COPD patient, including: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

[0021] In some aspects, the techniques described herein relate to methods of treating asthma (including bronchodilation) or COPD (for which no disease modifying therapy exists currently) in a patient, including: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

[0022] In some aspects, the patient has minimal CFTR function. In some aspects, the patient does not have a F508del mutation in CFTR. In some aspects, the patient was previously treated with a CFTR potentiator. In some aspects, R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered as an aerosolized for and / or in a nebulizer, inhaler, or spray through a mucosal membrane (e.g., nose, throat, lungs, mouth, etc.). In some aspects, the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof administered to the patient is from 1 to 20 pM per dose. In some aspects, the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is from 1 to 50 mg / day. In some aspects, the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered for multiple, consecutive days.

[0023] In some aspects, the treatment regimen further comprises administering an LRRC26 potentiator to the patient. In some aspects, the treatment regimen further comprises administering a TMEM16A potentiator to the patient. In some aspects, the treatment regimen further comprises administering nesolicaftor, metformin, or angiotensin blockers to the patient.

[0024] In some aspects, the R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered with a pharmaceutically acceptable delivery vehicle. Preferably, the R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered with a peptide-based nanosponge.

[0025] FIG. 14B shows an exemplary embodiment contemplated for using a novel nanosponge for delivery of the R-vanzacaftor wherein the trimaleimide structure is the core that is attached to a first peptide block of 20 lysine residues and a second peptide block of 10 residues of serine, threonine, aspartic acid / aspartate, or glutamic acid / glutamate (e.g., SEQ ID NO: 1, 2 or 3). Additionally, one of the blocks are capped with a lipid cap (cholesterol), one of the blocks will have a targeting peptide (peptides targeting integrin avP6) and the third position is attached to a signaling sequence that can be either a sequence targeting SIRPa (“don’t eat me” sequence). Depending on the peptides used for the second 10-residue block, R-vanzacaftor25KU017M-0262724-PCTcan be covalently attached to the hydroxyl groups of serine or threonine residues to form a cleavable carbonate bond or covalently attached to the carboxylic acid groups of aspartic acid or glutamic acid groups to form cleavable ester bonds for release of the R-vanzacaftor upon delivery to the targeted site.

[0026] Other objects and features will be in part apparent and in part pointed out hereinafter Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive, as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows an (A) apical loop current based on a model from Cook and Young (FIG. 1A) (Cook, D. I. and Young, J. A. (1989) J. Membr. Biol. 110(2): 139-46; Manzanares, D. et al. (2011) J. Biol. Chem. 286(22): 19830-9); and (B) Cl’ secretion changes with increasing apical K+ conductance. Dashed lines indicate no epithelial sodium channel (ENaC) activity; solid lines indicate with ENaC activity at 35% of the Cl’ conductance. Two different paracellular cation resistances were used in the simulations, 600 Qcm2(black) and 300 Qcm2(red), to show the effect of paracellular conductance on Cl’ secretion.

[0028] FIG. 2 shows replotted and summarized data from table 5 in Burgel et al., (The French Compassionate Program of elexacaftor-tezacaftor-ivacaftor in people with cystic fibrosis with advanced lung disease and no F508del CFTR variant. Eur Respir J. 2023. Epub 20230216) taking only pwCF with two minimal function CFTR variants. (A) 21 pwCF have had on average a small but significant increase in % predicted FEV1. (B) Taking only the 9 pwCF that increased % predicted FEV1, the effect was larger. None had a significant change in sweat chloride. * and **: p<0.05 by t-test.

[0029] FIG. 3 shows BK subunit expressions in large and small airways, created by reanalyzing data from Okuda et al. (Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia. Am J Respir Crit Care Med.2021;203(10): 1275-89.). (A) UMAP projections of major cell types (combed large &small airways). Excluded are fibroblasts, cycling, alveolar, submucosal, and inflammatory cells. Secretory and ciliated subtypes were combined. (B-D) Expression of KCNMA1, LRRC26 and TMEM16A (AN01); additional grey cells were excluded from (A). Expression patterns in (E) large airways and (F) small airways. Patterns: KCNMA1 is found in secretory, ciliated, basal, suprabasal cells and ionocytes. LRRC26 is in secretory, basal, suprabasal, and ciliated cells.25KU017M-0262724-PCTBoth KCNMB2 and KCNMB4 are expressed at low levels. KCNMB4 is not expressed in small airway secretory cells. KCNMB2 is expressed mostly in ciliated cells. AN01 is expressed in secretory cells. Overall, expression patterns indicate that KCNMA1 / LRRC26 BK and TMEM16A are co-expressed in airway secretory cells.

[0030] FIG. 4 show that elexacaftor (E) potentiates BK currents in NHBE and CFBE cells cultured at the ALI, then compared to R-vanzacaftor and S-vanzacaftor (all 5 pM). (A) BK-related AISCquantifications of peak responses in NHBE cell cultures. (B-C) CFTR and BK activity after exposure to elexacaftor, R-vanzacaftor and S-vanzacaftor (all 5 pM) for 24 hours in CFBE homozygous for F508del. (D-E) CFBE cells with minimal function CFTR variants treated with TGF-P I (5 ng / mL) and elexacaftor (5 pM) for 24 hours. (F-J) mRNA expression and BK potentiation. (I- J) Mucociliary transport. n=2-5. Where indicated, one-way ANOVA.

[0031] FIG. 5 shows R-vanzacaftor as a potent BK potentiator with enantiomeric differences. S-vanzacaftor is the most potent acute BK activator with loss of BK potentiation at 24h while modulating F508del CFTR (see FIG. 4, panel B). R-vanzacaftor is a less potent acute BK potentiator but with long lasting BK effects compared to S-vanzacaftor at clinically relevant concentrations. R-vanzacaftor does NOT modulate F508del CFTR (see FIG. 4B). FIG. 5, panels A and C BMI-normal cells (FIG. 5, panels B and D) BMI minimal function CFTR variants cells.

[0032] FIG. 6 shows BK Channel Function. (A) Macroscopic Slol IK at +80 mV in different [Ca2+]i. (B) Normalized GK-V relations in 0 - 100 pM Ca2+from tail currents (C) Log(Po)-V relations with inset single channel currents at -120 mV in 2 pM Ca2+. (D) Horrigan- Aldrich model of BK channel function.

[0033] FIG. 7 shows (A) activators of WT Slol (100 pM phloretin, 10 pM PiMA) shift PO- V to more negative V in 0 Ca. (B-C) V-dependent effects of PiMA and phloretin on Log(Po)- V relations reveal distinct MO As.

[0034] FIG. 8 shows graph plots of the fold-increase in BK channel activity (at -80 mV / 0 Ca) to vehicle control when cells are exposed to different concentrations of S- or R-vanzacaftor. Channels are composed of hSlol or hSlol+LRRC26. Data are from multiple patches (not all measurements are at steady state). Curves are Hill equations with ECso= 2.5 pM and N=l.

[0035] FIG. 9 shows thallium-flux Assays for (A) Slol (F315Y) shows basal activity in DMSO that is potentiated by phloretin and virtually abolished by the inhibitor paxilline. (B) Concentration-response curves for paxilline on WT channels with patch clamp are reproduced by FLIPR. Data for the potent inhibitor IBTX also literature values for WT. Assays ± regulatory subunits reproduce the known subunit- selectivity of (C) Inhibition by IBTX or (D) potentiation25KU017M-0262724-PCTby Lithocholate. (B-9D), mean ± SD (n = 3 wells).

[0036] FIG.10 shows ion channels in ovine airway epithelial cells in vitro are representative of human cells (Id.). (A) BK currents in ovine cells inhibited with 500 nM iberiotoxin. (B) Quantitation of BK inhibition by paxilline (30 pM, high concentration), iberiotoxin (500 nM), and barium (6 mM). n>7 each / >3 sheep. (C) Ovine airway epithelial cells display CFTR activity that can be blocked by CFTRinhl72 and CaCC. (D) Acute treatments of R- and S-vanzacaftor. (E) Chronic treatments of R- and S-vanzacaftor. Distinct letters above bars indicate unique groups, significantly different from each other (p<0.05). Data are mean ± SE.

[0037] FIG. 11 shows TMV assessments (Id.). (A) Inhaled CFTRinhl72 (10 mg) slows TMV and hypertonic saline (HTS) 7% temporarily rescues TMV. All n>3 / >3 sheep. (B) 25 pg TGF- 1 + 10 mg CFTRinhl72 results in sustained slowing of TMV (24 hours). All n=3 / 3 sheep. (C) High dose human neutrophil elastase (HNE; 2380 vs. 1190 mU) slows TMV for 24 hours. All n>3 / >3 sheep. (D) A 3-day challenge with CFTRinhl72 and high HNE leads to prolonged slowing of TMV. Inhaled losartan (50 mg) rescues TMV. n=4 / 4 sheep. (E) TGF- I. n=7 / 3 sheep, and (D) mucus concentrations (% solids) in ovine tracheal secretions. n>21 / 3 sheep. Statistics: (A-D) two-way ANOVA of repeated measures followed by Sidak’s. Baseline TMV was 9.8 ± 0.26 mm / min (n=15 sheep). (C) Stars indicate group assignments of the statistical analysis using Kruskal Wallis followed by Dunn’s (Id.).

[0038] FIG. 12 shows nesolicaftor (NES) improves modulator-corrected F508del-CFTR function in F508del CFBE cells in vitro (Bengtson, C. et al. (2022) Int. J. Mol. Sci. 23(18)). (A) Fully differentiated CFBE homozygous for F508del were treated with elexacaftor (1 pM) / tezacaftor (5 pM) / ivacaftor (1 pM) (ETI) or ETI + NES (10 pM) for 24 hours (all n>6, 3 CF lungs). ISC was measured in Ussing chambers. NES significantly increased CFTR function (B), CaCC (C), expression levels of CFTR (D) and TMEM16A or AN01 mRNA (E). NES reversed reductions in ETI-corrected F508del- CFTR function caused by TGF-pi (5 ng / mL) (F-G). TGF-pi did not significantly impact CaCC (H) but NES increased CaCC. Statistics: * p<0.05, Student’s t-test or ANOVA followed by Holm-Sidak or Friedman after assessing normality with Shapiro-Wilk.

[0039] FIG. 13 shows nesolicaftor rescues TGF-pi-mediated ion channel dysfunction in NHBE cells (still room for BK potentiators). Basolateral exposure of NHBE cells to DMSO, nesolicaftor (NES, 10 pM), TGF-pi (10 ng / ml), or TGF-pi+NES for 24 hours. (A) Representative traces of BK-related Isc. (B) NES improves BK function and TGF-pi -mediated decreases in BK-related Isc. n=14, 8 lungs. (C) Western blot: NES increased LRRC26 expression and rescued TGF-pi -mediated reduction (normalized to P-actin). (D) Summary25KU017M-0262724-PCTdata, n=ll, 9 lungs. (E-F) are NES increased CaCC and ANO1 mRNA trended upwards (preliminary). (G) TGF-pi -mediated decreases in ciliary beat frequency (CBF) are prevented by NES. ACBF between baseline and 24 hours. n=14, 9 lungs. (FIG. 15H) NES reverses TGF-pi-mediated ASL absorption. n=13, 8 lungs. Mean ± SEM. *p <0.05, Friedman test.

[0040] FIG. 14A shows the structure of R-vanzacaftor.

[0041] FIG. 14B shows building blocks of a rationally designed peptide-based nanosponge carrier for delivery of the drug (R-vanzacaftor) comprised of a trimaleimide-based core structure, targeting peptide for integrin avP6 (Jornada, D. H. et al. (2015) Molecules 21(1):42), a “don’t eat me” peptide, cholesterol and covalently linked (esterase-cleavable) to R-vanzacaftor, which should not be taken as limiting of possible nanosponge structures or other possible carriers contemplated herein.

[0042] FIG. 14C shows a mechanism for attachment through a nitrogen.

[0043] FIG. 14D shows a different mechanism for attachment through a different nitrogen in the structure; and as compared to FIG. 14C, it is contemplated that connection to vanzacaftor can proceed by binding to either indicated nitrogen highlighted in FIG. 14C or FIG. 14D.

[0044] FIG. 14E shows a mechanism whereby vanzacaftor can be tethered via a consensus sequence to the K20 blocks (SEQ ID NO: 1, 2, or 3) of rationally designed peptide nanosponges, using the nitrogen highlighted in FIG. 14D.

[0045] FIG. 15 shows in panel A in vivo TriTom imaging of a Rhodamine B labeled PTP-NanoSponge (NS), 24 hours after IP administration shows selective uptake in the area of the pancreatic tumor (white arrows) adjacent to the spleen (yellow arrow). Panel B shows ex vivo IVIS imaging of tissues removed from A, shows the specificity for the pancreatic tumor. Scale based on Radiance (p / sec / cm2 / sr). N=l.

[0046] FIG. 16A shows the H1NMR spectra for R-vanzacaftor.

[0047] FIG. 16B shows the H1NMR spectra for S-vanzacaftor.

[0048] FIG. 16C shows the H1NMR spectra comparisons between the two enantiomers.

[0049] FIG. 16D shows the H1NMR spectra comparisons between the two enantiomers, where the only difference between S- and R-vanzacaftor are the peaks at 2.89 ppm and 2.73 ppm.

[0050] Various objects, aspects, features, and advantages of the disclosure will become more apparent and better understood by referring to the detailed description taken in conjunction with the accompanying drawings, in which like reference characters identify corresponding elements throughout. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements.25KU017M-0262724-PCTDETAILED DESCRIPTION

[0051] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known aspects. Many modifications and other aspects disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain, having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific aspects disclosed and that modifications and other aspects are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0052] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0053] As can be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure.

[0054] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to the arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0055] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any25KU017M-0262724-PCTspecific implementation or combination of implementations of the disclosed methods.

[0056] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0057] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0058] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0059] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another implementation includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another implementation. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0060] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0061] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal implementation. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0062] As used herein, "alkyl" means a straight or branched chain saturated hydrocarbon moi eties such as those containing from 1 to 10 carbon atoms. A “higher alkyl” refers to saturated hydrocarbon having 11 or more carbon atoms. A “C6-C16” refers to an alkyl25KU017M-0262724-PCTcontaining 6 to 16 carbon atoms. Likewise, a “C6-C22” refers to an alkyl containing 6 to 22 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.

[0063] Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or "carbocyclyl" groups. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like.

[0064] “Heterocarbocycles” or “heterocarbocyclyl” groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which can be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quatemized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0065] The term "aryl" refers to aromatic homocyclic (i.e., hydrocarbon) mono-, bi- or tricyclic ring-containing groups preferably having 6 to 12 members such as phenyl, naphthyl and biphenyl. Phenyl is a preferred aryl group. The term "substituted aryl" refers to aryl groups substituted with one or more groups, preferably selected from alkyl, substituted alkyl, alkenyl (optionally substituted), aryl (optionally substituted), heterocyclo (optionally substituted), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkanoyl (optionally substituted), aroyl, (optionally substituted), alkylester (optionally substituted), arylester (optionally substituted), cyano, nitro, amino, substituted amino, amido, lactam, urea, urethane, sulfonyl, and, the like, where optionally one or more pair of substituents together with the atoms to which they are bonded form a 3 to 7 member ring.

[0066] As used herein, “heteroaryl” or “heteroaromatic” refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems can, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl,25KU017M-0262724-PCTisothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl" includes N-alkylated derivatives such as a 1-methylimidazol- 5-yl substituent.

[0067] As used herein, "heterocycle" or "heterocyclyl" refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems can be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like.

[0068] "Alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t-butoxy.

[0069] The terms "halogen" and "halo" refer to fluorine, chlorine, bromine, and iodine.

[0070] The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule can be multiply substituted. In the case of an oxo substituent ("=O"), two hydrogen atoms are replaced.

[0071] The term "optionally substituted," as used herein, means that substitution with an additional group is optional and therefore it is possible for the designated atom to be unsubstituted. Thus, by use of the term “optionally substituted” the disclosure includes examples where the group is substituted and examples where it is not.

[0072] As used herein, “substantially pure” means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), nuclear magnetic resonance (NMR), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), gaschromatography mass spectrometry (GC-MS), and similar, used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Both traditional and modern methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers.

[0073] A “pharmaceutically acceptable” component is one that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and25KU017M-0262724-PCTallergic response) commensurate with a reasonable benefit / risk ratio.

[0074] “Pharmaceutically acceptable salt” refers to a salt that is pharmaceutically acceptable and has the desired pharmacological properties. Such salts include those that may be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium, potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). When two acidic groups are present, a pharmaceutically acceptable salt may be a mono-acid-mono-salt or a di-salt; similarly, where there are more than two acidic groups present, some or all of such groups can be converted into salts.

[0075] “Pharmaceutically acceptable excipient” refers to an excipient that is conventionally useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0076] A “pharmaceutically acceptable carrier” is a carrier, such as a solvent, suspending agent or vehicle, for delivering the disclosed compounds to the patient. The carrier can be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutical carrier. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Many carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oligo / polyethylene glycols in water, oils such as olive oil, soy oil, or injectable organic esters, naturally occurring mono, di-, and polysaccharides, carbohydrates, sugars, proteins, and the like, including any of the following including mixtures thereof: celluloses, derivatives thereof, and microcrystalline forms thereof such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,25KU017M-0262724-PCThydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and the like; other naturally derived polysaccharides such as sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, dextran; mono or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, amylose, and the like; dextrins such as a-, P- or y-cyclodextrin, dimethyl-P-cyclodextrin, dextrin; native starches and their derivatives such as hydroxyethyl or hydroxypropyl starch and carboxymethyl starch; as well as gums such as gum arabic, tragacanth gum and glucomannan. Suitable carriers can further include proteins, such as serum proteins, casein, albumin, and the like.

[0077] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration (topical or injection), optical / ophthalmic administration (topical or injection), intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In some examples, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In some examples, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0078] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for25KU017M-0262724-PCTany particular disease. The desired response to treatment of the disease or condition can also be delaying the onset or even preventing the onset of the disease or condition.

[0079] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration purposes. Consequently, single-dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the disclosure (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons, or virtually any other reasons.

[0080] A response to a therapeutically effective dose of a disclosed compound or composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following the administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied, for example, by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, changing the disclosed compound and / or pharmaceutical composition administered, changing the route of administration, changing the dosage timing, and so on. Dosage can vary and can be administered in one or more doses daily for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0081] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g., human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to a human and constituents thereof.

[0082] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disorder in a subject, particularly a human, and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed25KU017M-0262724-PCTto the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating" can include inhibiting the disease, disorder, or condition or symptoms thereof, e.g., impeding its progress, and relieving the symptoms of disease (but also be disease modifying), disorder, or condition or symptoms thereof, e.g., causing regression of the disease, disorder, and / or condition or symptoms thereof. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0083] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0084] As used herein, the term "diagnosed" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, "diagnosed with cancer" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can treat or prevent cancer. As a further example, "diagnosed with a need for treating or preventing cancer" refers to having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition characterized by cancer or other disease wherein treating or preventing cancer would be beneficial to the subject.

[0085] As used herein, “CFTR” means cystic fibrosis transmembrane conductance regulator.

[0086] As used herein, “pwCF” means people with Cystic Fibrosis.

[0087] As used herein, “mutations” or “variants” can refer to mutations in the CFTR gene or the CFTR protein. A “CFTR gene mutation” refers to a mutation in the CFTR gene, and a “CFTR protein mutation” refers to a mutation in the CFTR protein. A genetic defect or mutation, or a change in the nucleotides in a gene in general results in a mutation in the CFTR protein translated from that gene, or a frame shift(s).

[0088] The term “F508del” refers to a mutant CFTR protein which is lacking the amino acid25KU017M-0262724-PCTphenylalanine at position 508.

[0089] As used herein, “BK channel” refers to large conductance calcium-activated potassium channels.

[0090] The term “TMEM16A” refers to Transmembrane member 16A, interchangeably used with the term “ANO-1” or Anoctamin-1, and is a calcium activated chloride channel.

[0091] As used herein, “MCT” means mucociliary transport.

[0092] As used herein, “chronic” refers to treatments for at least 24h.

[0093] As used herein, “acute” refers to immediate responses to treatment.

[0094] The term “LRRC26” refers to the yl subunit of the BK channel and “KCNMA1” to the gene that encodes the pore forming unit of the BK channel also called Slo-1.

[0095] As used herein, “MO A” means mechanism of action.

[0096] As used herein, the term “modulator” refers to a compound that increases the activity of a biological compound or molecule such as a protein. For example, a CFTR modulator is a compound that increases the activity of CFTR. The increase in activity resulting from a CFTR modulator includes but is not limited to compounds that correct, potentiate, stabilize and / or amplify CFTR.

[0097] As used herein, the term “avP6” refers to a specific integrin, namely avP6.

[0098] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.Methods

[0099] About 35,000 people with Cystic Fibrosis (pwCF) live in the US. Historically, lung disease with respiratory failure caused mortality, driven by mucus hyperconcentration, mucociliary dysfunction, chronic infection, and airway inflammation. CF Transmembrane Conductance Regulator (CFTR) modulator therapy has improved outcomes for eligible pwCF by improving lung function, decreasing sweat chloride, reducing exacerbations, and improving quality of life. However, no similar therapies exist for the -10% of pwCF with two variants not amenable to modulators and the -20% pwCF with poor response to modulators. Finding alternative therapies for those is therefore an important goal for the care of pwCF. Nevertheless, other airway epithelial ion channels could be therapeutically targeted. This includes TMEM16A, for which a potentiator will enter clinical trials soon. Benefits of reducing epithelial sodium channel (ENaC) activity has been recognized from genetic variants. However, clinical trials to block ENaC have failed thus far. Not often considered, large conductance, calcium-activated potassium (BK) channels are responsible for apical loop25KU017M-0262724-PCTcurrents that are critical for apical chloride exit, thereby contributing to airway surface fluid availability with beneficial effects on mucus solids and mucociliary transport (MCT) in CF cells in vitro and in a CF-like sheep model in vivo. It has been shown previously that the clinical CFTR modulator elexacaftor modestly activates BK, as well as we have shown here potentiates BK. More importantly, the novel modulator vanzacaftor potentiates BK effectively, but with differential enantiomeric properties. S-vanzacaftor modulates F508del CFTR (in phase 3 clinical trials) and activates BK but only short-term. Vice versa, R-vanzacaftor does not modulate F508del CFTR but effectively potentiates BK long-term. Thus, the present disclosure shows long-term potentiating BK with R-vanzacaftor or a pharmaceutically acceptable salt thereof improves mucus hydration in pwCF not eligible for approved modulators. Such therapy, however, would also benefit other airway diseases with mucociliary dysfunction, including asthma (where a BK potentiator can also cause bronchodilation) and COPD where significant mucus plugging leads to worsening of the disease for which currently no disease modifying therapy exists.

[0100] Importantly and often overlooked, airway inflammation remains a problem for pwCF and other patients with mucociliary dysfunction. In CF, it has been shown that higher levels of TGF-pi correlate with worse lung function over time. Also previously shown is that higher levels of TGF-pi negatively affect CFTR modulation and BK function, the latter by downregulating expression of LRRC26, the yl subunit required for BK function in non-excitable cells. It is contemplated herein that effective BK potentiation improves mucociliary function in pwCF with minimal function CFTR variants, even in an inflammatory environment. Furthermore, we will avoid severe side effects from BK potentiation outside the lungs using delivery methods that assure airway retention. Finally, it is shown here that nesolicaftor, an mRNA amplifier, rescues BK function and CF secretion in the presence of TGF-pi by increasing LRRC26 and TMEM16A expression, likely via poly(rC)-binding protein 1 (PCBP1). Thus, there is the possibility to overcome inflammation further overcome with nesolicaftor (FIG 1).

[0101] In some embodiments, disclosed herein are pharmaceutical compositions for use as inhalation treatments for pwCF with poor response to modulators. In some embodiments, the pharmaceutical composition includes an R-enantiomer of vanzacaftor, hereafter, R-vanzacaftor. It is shown that the R-enantiomer potentiates BK channels and provide a downstream improvement of mucociliary function. The R-vanzacaftor can be formulated for various routes of delivery and modalities of treatment.

[0102] In some embodiments, a method of treating CF is disclosed. In some embodiments,25KU017M-0262724-PCTthe method comprises administering R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient. In other embodiments, disclosed herein is a method of decreasing mucus concentration and / or mucus viscosity in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient. In other embodiments, disclosed herein is a method of increasing mucociliary transport in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient. In other embodiments, disclosed herein is a method of treating asthma or COPD in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient. In other embodiments, disclosed herein is a method of treating inflammation in a cystic fibrosis patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

[0103] In specific examples, the disclosed methods involve administering substantially pure R-vanzacaftor. That is, the methods, in certain examples, do not involve administering substantial amounts of S-vanzacaftor, e.g., a racemic or scalemic vanzacaftor composition. In other examples, the amount of S-vanzacaftor present in the composition can be less than 10, less than 1, less than 0.5, or even less than 0.05 % based on the total weight of vanzacaftor. The H1NMR spectra and the differences between them for the two enantiomers are shown in FIG. 16A-16D. Preferably, the methods are essentially free of S-vanzacaftor.

[0104] In specific examples, patient can have minimal CFTR function. That is, their CFTR function is reduced as compared to an otherwise health control. In other specific examples, the patient does not have a F508del mutation in CFTR. In still other examples, the patient was previously treated with a CFTR potentiator. That is, patients that have previously undergone treatment for CF by taking CFTR potentiators, especially ones where such treatment was or is no longer effective, are suitable patients for the disclosed methods.

[0105] In specific examples, R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered in a nebulizer, inhaler, or spray. The disclosed compounds can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. When one or more compounds is used in combination with a second therapeutic agent the dose of each compound can be either the same as or differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

[0106] In other examples, R-vanzacaftor can be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The25KU017M-0262724-PCTcompounds can also be administered in their salt derivative forms or crystalline forms.

[0107] R-vanzacaftor can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington’s Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, R-vanzacaftor can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays or aerosols. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99%, and especially, 1 and 15% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0108] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0109] In still other examples, R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered with a nanosponge. In general, these peptide nanosponges are characterized as water-soluble sponge-like supramolecular assemblies or aggregates comprising a plurality of individual peptide-based building blocks (e.g., 1,000-50,000 building blocks) that can125KU017M-0262724-PCTdynamically self-assemble, disassemble, and reassemble into small clusters to facilitate site-selective delivery and uptake of their therapeutic payload into cells. With a suitable nanosponge, R-vanzacaftor is cleavably linked to a peptide nanosponge. For example, the peptide nanosponge can comprise a plurality of peptide-based building blocks. The specific components used in each building block are variable, however, each building block will generally comprise a multi-armed core; a linear peptide sequence having its C-terminal end covalently attached to each arm of the core, directly or via a cysteine residue as a spacer, and its N-terminal end typically capped with a capping moiety (but could be some other kind of terminal moiety, as discussed below). The peptide sequence is a block co-peptide comprising two or more distinct peptide blocks covalently attached to the core via a linking amino acid (C) and spacer residue (G) covalently linking the peptide block at its C-terminus to the core. The peptide blocks can be, for example, a liner peptides of from 2 to 40 lysine residues with optionally 2-40 aspartic acid, arginine, and / or serine residues. The linear peptide sequence is preferably free of any enzymatic consensus sequences and also is preferably not attached to the core via any cleavable consensus sequences. That is, the peptide building block itself is preferably free any cleavable linkages or enzymatic consensus sequences and is therefore resistant to enzymatic degradation. The nanosponge can contain up to 10 wt.% of R-vanzacaftor, e.g., from 0.01, 0.1, 1, 5, or 10 wt.% R-vanzacaftor, with any of the stated values forming the upper or lower endpoint of a range.

[0110] Details of the nanosponges can be found in co-pending PCT / US / 2024 / 049592, filed October 2, 2024, entitled PEPTIDE NANOSPONGES FOR DRUG DELIVERY, published as W02025076082 October 4, 2025, incorporated by reference in its entirety herein.[OHl] In the disclosed methods, the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof can be from 0.01 to 20 pM. In specific examples, R-vanzacaftor or a pharmaceutically acceptable salt thereof can be administered at a concentration of 0.01, 0.1, 1, 5, 10, 15, or 20 pM, where any of the stated values can form an upper or lower endpoint of a range. The method of any of the previous claims, wherein the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is from 1 to 50 mg / day. In specific examples, R-vanzacaftor or a pharmaceutically acceptable salt thereof can be administered at 0.5, 1, 5, 10 15, 20, 25, 30, 35, 40, 45, or 50 mg / day, where any of the stated values can form an upper or lower endpoint of a range.

[0112] In specific examples, the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered for multiple, consecutive days.25KU017M-0262724-PCT

[0113] In specific examples, a LRRC26 rescue medication (e.g., an angiotensin receptor blocker, nesolicaftor, or metformin) and / or a TMEM16A potentiator can also be administered to the patient. In a specific example, nesolicaftor is also administered to the patient.

[0114] Additional advantages of the various embodiments described herein will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present disclosure encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0115] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0116] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the disclosure. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).EXAMPLES

[0117] The following examples are set forth below to illustrate the compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

[0118] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other25KU017M-0262724-PCTreaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described processes. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0119] Scientific rigor. The studies presented herein were performed in oocytes and primary human bronchial epithelial cells from pwCF with minimal function CFTR variants and other primary cells, some transformed to make them semi-immortalized. Given the small number of available donors, female and male differences (sex as a variable) were not assessed. In vitro experiments were confirmed in an expression system and in vivo can be confirmed using a CF-like sheep model, suited for minimal function CFTR variants.

[0120] The following examples address how apical loop currents created by BK (possibly by enhancing chloride secretion of TMEM16A channels) can address mucociliary dysfunction in pwCF with minimal function CFTR variants and patients with other airway diseases including asthma and COPD (in the latter two by interacting with CFTR as well). The explored mechanisms regarding inflammation provide also basis to re-introduce or repurpose variants of clinically approved therapies (e.g., nesolicaftor and angiotensin receptor blockers), with in vitro (oocytes and CFBE cells) and then in vivo studies (CF-like sheep model and sheep models of asthma and COPD). The preclinical in vitro cell culture models have been indispensable for the development of CFTR modulators as they predict clinical outcomes, and we pose they will do the same for BK potentiators. In addition, a time-tested CF-like sheep model is used to confirm findings in a realistic, inflammatory environment for CF, asthma, and COPD.Example 1

[0121] The following example provides mechanisms of differential BK activation or potentiation by S-and R-vanzacaftor and their effects on mucociliary function in primary normal and CF bronchial epithelial cells and in a heterologous expression system (Xenopus oocytes).

[0122] Cellular expression of BK and TMEM16A in airway epithelia. The epithelial / mucus / air interface provides a periciliary layer dominated by membrane-attached mucins offering a polymer brush layer for cilia to beat and an overlying gel-forming mucin layer that moves entrapped particles out of the airways. Inadequate hydration of the gelforming mucus compresses the periciliary layer due to higher osmotic forces from the overlying mucus layer resulting in mucus stasis. An increase in total mucin concentrations in the gelforming layer was previously verified in pwCF, even when structural lung changes or infections were absent in children. Mucus concentrations (% solids: dry to wet weight ratio) can reach more than 8% in CF, invariably resulting in mucus stasis.25KU017M-0262724-PCT

[0123] The recent introduction of single cell RNAseq data from airway epithelia opened a new era of understanding of distinct cell type distributions along the airway axis and identified rare cell types within the epithelium that are critical to its function. These include ionocytes that express CFTR mRNA at the highest level of all airway cell types. Other important ion channels are also expressed in these cells, including KCNMA1 (without LRRC26). Recent evidence indicates that ionocytes mostly absorb chloride and thus water from the airway. Other than in ionocytes, CFTR is mostly expressed in secretory cells together with KCNMA1 / LRRC26 and TMEM16A (see FIG. 3). Secretory cells are present in peripheral airways and their CFTR functionally responds to the nucleotide-purinergic system to secrete chloride. These findings set the stage to explore apical loop currents between functional BK and TMEM16A in secretory cells in the absence of CFTR activity (see FIG. 3).

[0124] It is proposed that vanzacaftor can activate and potentiate BK channels. However, as disclosed herein, vanzacaftor enantiomers have differential effects. BK channels in non-excitable cells require LRRC26 to activate at physiological voltages. In its absence, BK is not conducting appreciable K+but possibly can in the presence of efficacious potentiators. This is consistent with the additionally provided examples — that BK potentiation by elexacaftor in CFBE cells is reduced by TGF-pi, when LRRC26 is downregulated (see Example 2). To assess benefits for pwCF with minimal function CFTR variants, the mechanism of action (MO A) was explored, including potency and efficacy for activating / potentiating BK with auxiliary subunits expressed in secretory airway epithelial cells.

[0125] In one study, in vitro relevance of acute activation and chronic potentiation of BK by S- and R-vanzacaftor in Ussing chambers is shown. Also shown are the possible effects of chronic BK potentiation by S- and R-vanzacaftor on parameters of mucociliary clearance focusing on using primary CF bronchial epithelial (CFBE) cells with minimal function CFTR variants on both alleles and fully differentiated at the air-liquid interface.

[0126] In another study, mechanisms of action of S- and R-vanzacaftor and their dependence on BK channel subunit composition are shown. Additionally, stimulus conditions in heterologous expression systems using patch clamp electrophysiology and high-throughput FLIPR assays, focusing on the pore forming units (Slol encoded by the KCNMA1 gene) and the yl subunit LRRC26.

[0127] In another study, in vivo relevance of acute and chronic BK potentiation by S- and R-vanzacaftor using tracheal mucus velocity (TMV) and mucus concentrations are shown. Additionally shown are inflammatory markers in ovine airway secretions.First Study of Example 1: Determine relevance of acute and chronic BK potentiation by25KU017M-0262724-PCTS- and R-vanzacaftor in CF cells

[0128] Experimental Methods for in vitro Assays with CFBE cells. Air-liquid interface (ALI) cultures from primary airway epithelial cells were used. Cells from a total of three primary pwCF with minimal function CFTR variants as well as semi -immortalized cells with minimal function CFTR variants were used. Additionally, cells from multiple pwCF homozygous or heterozygous for F508del were used to assess overall applicability in CF. Ussing chambers used standard protocols. Ciliary beat frequency (CBF) was measured using SAVA. Airway surface liquid (ASL) volume was estimated by meniscus scanning and ASL height by confocal microscopy. Quantitative PCR (qPCR) and western blot quantification were done as described in the publications. Mucus concentration (% solids) and viscosity were assessed as published. Mucociliary transport (MCT) was measured by tracking fluorescent beads on apical cell surfaces. Cytokine levels were measured from basolateral media using automated ELISA (ELLA, ProteinSimple). RNA immunoprecipitation (RIP) used the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore Sigma). The following potentiators were used: Elexacaftor from Selleck Chemicals (Cat. No. S8851) and its enantiomers from MedChemExpress (HY-111772 / HY-l 11772A). Vanzacaftor enantiomers from MedChemExpress (Cat# HY-145603 / HY-145603A) were used and independently validated by our chemistry core. Supplementally, a Trikafta pill exhibiting yellow color and containing elexacaftor, tezacaftor and ivacaftor was used, confirming the effects of elexacaftor from other vendors to be equal to the Vertex product. The following experiments were conducted at least ten times with cultures from three pwCF with minimal function CFTR variants on both alleles plus the semi-immortalized cells. Cells from at least five pwCF each homozygous or heterozygous for F508del were used. To account for technical repetition with limited donors, random intercept multi-level regression models were used where p<0.05 was the significant criteria.

[0129] Ion channels relevant for mucociliary function in central and peripheral airways (re-analyzed from published data). Ion channel locations in airway epithelia are critical for the design of novel therapies. In a meta-analysis of previous, published data of single cell RNA sequencing from normal airway epithelia, differentiating between central and small airways were reviewed. See FIG. 3, A-F, showing overall expression patterns of ion channels of interest, TMEM16A (or ANO1), KCNMA1 (or Slot, pore-forming subunit of BK), and LRRC26 (the yl subunit of BK critical to its function in non-excitable cells). KCNMB2 and KCNMB4, P-subunits of BK, were detected in whole culture PCR. KCNMB2 was almost absent in large and small airway secretory cells and KCNMB4 was expressed in large airway25KU017M-0262724-PCTsecretory cells. Relevant expressions of KCNMB1 / 3 were not detected. Thus, the following experiments focused on BK channel potentiation composed of Slo 1 / LRRC26 and influence of KCNMB2 / 4 (see FIG. 3).

[0130] Elexacaftor. Elexacaftor is a moderate BK potentiator with no enantiomeric differences regarding BK potentiation. Normal human airway epithelial (NHBE) cells have been exposed basolaterally to 5-10 pM elexacaftor for 24 hours (chronic). In pwCF, these concentrations were reached at steady state (200 mg per day). BK potentiation was measured in Ussing chambers with a basolateral to apical K+gradient: Elexacaftor left-shifted ATP responses, showing BK potentiation. Elexacaftor from different suppliers (Selleckchem and two enantiomers from MedChemExpress) potentiated identically. CF bronchial epithelial (CFBE) cells with minimal function CFTR variants revealed a trend in BK potentiation with elexacaftor. Exposure to 5 ng / mL TGF- pi (over 24 hours) decreased BK currents as expected (likely via LRRC26 reduction) with no elexacaftor rescue (see FIG. 4, panel B). In F508del CFBE cells, elexacaftor neither potentiated BK nor improved mucociliary transport (MCT, FIG. 4, panel E).

[0131] Vanzacaftor. R-vanzacaftor is a superior BK potentiator with enantiomeric differences. As shown in FIG. 4, in NHBE cells, after 24 hours exposure to R-vanzacaftor BK potentiation exhibited a 12-fold increase over the control whereas S-vanzacaftor exhibited no effect. CFBE cells were exposed either acutely by adding vanzacaftor apically in Ussing chambers or for 24 hours (chronic conditions) by basolateral addition of 1-30 pM R- or S-vanzacaftor. Plasma concentrations of close to 3 pM were reached at steady state in pwCF taking 20 mg S-vanzacaftor daily (however, 3 pM is therefore one SD above mean). Chronic S-vanzacaftor modulated CFTR activity in F508del CFBE cells whereas R-vanzacaftor did not exhibit any effect on CFTR (FIG. 4). Vice-versa, chronic R-vanzacaftor potentiated BK whereas S-vanzacaftor did not (FIG. 4). Importantly, only R-vanzacaftor significantly improved mucociliary transport or MCT (FIG. 4). S-vanzacaftor showed some improvement likely due to the modulation of CFTR activity in F508del CFBE. Further analysis in minimal function CFTR variants (BMI-G542X & BMI-W1282X are semi-immortalized CFBE cells) also revealed that acute treatment with S-vanzacaftor potently activated BK (FIG. 5), albeit at a high concentration. However, 24 hours exposure at clinically relevant concentrations significantly diminished S-vanzacaftor’ s BK potentiation (FIG. 5), an effect also seen in BMI-normal HBE cells (FIG. 5). However, acute R-vanzacaftor had a modest activation effect on BK while chronic exposure (24 hours) displayed up to 17-fold increase in BK activity compared to DMSO (FIG. 5).25KU017M-0262724-PCT

[0132] EXPERIMENTAL DESIGN

[0133] Clinical outcomes were predicted by assessing BK potentiation by vanzacaftor enantiomers in cell cultures and exploring their effects on parameters of mucociliary function. This approach is supported by FDA approvals of modulators for CFTR variants solely based on in vitro work.

[0134] A first set of experiments analyzed BK activation (i.e., acute opening of the BK channel) in Ussing chambers with varying concentrations of R- and S-vanzacaftor under acute exposure by adding it into the chamber. Effects of these molecules on apical potassium currents were assessed in fully differentiated normal HBE (NHBE) and CFBE cells (FIG. 5). Assessment followed protocols for other channels. Amiloride was added to inhibit ENaC-mediated contributions to short circuit currents (Isc). Effects of increasing concentrations of vanzacaftor enantiomers on Iscin basolaterally permeabilized cells exposed to a basolateral to apical K+gradient were assessed. Paxilline was added to assure that Iscwas from BK. ECso was calculated, with the expected ECso in the low pM range.

[0135] A second set of experiments analyzed BK potentiation in Ussing chambers using vanzacaftor enantiomers for 24h exposures followed by stimulation with varying ATP concentrations (FIG. 5). Cultures were pretreated with 0, 1, or 5 pM R- and S-vanzacaftor for 24 hours (simulating chronic conditions) and assessed responses to escalating concentrations of ATP.

[0136] A third set of experiments tested requirements of LRRC26 for BK potentiation. The first and second set of experiments were repeated with KCN A1 or LRRC26 knock down (using lentiviral shRNA methods). LRRC26 overexpression was evaluated to determine whether potentiation changed. NHBE cells were used to establish relevant conditions before moving to CFBE cells.

[0137] Additional Results. The current data confirmed that R- and S-vanzacaftor activate BK acutely with an ECso in the pM range. Given the preliminary data with TGF-pi in primary cells (see also below), it was hypothesized that LRRC26 will be required for meaningful BK potentiation (even if potentiation occurs through the a-subunit). Since CFBE cells were limited, primary NHBE cells were also used initially for Ussing chamber experiments to determine optimal conditions before moving to CFBE cells. BK potentiation (24h exposure) was only seen by R-vanzacaftor and translated to increased MCT while S-vanzacaftor had no effect. BK is a Ca2+dependent channel and its regulation might depend on the availability of airwayspecific purinergic stimulation, provided by intermittent shear stress from breathing and ATP release via pannexin. While a decrease in KCNMA1 and LRRC26 reduced the effect of R-25KU017M-0262724-PCTvanzacftor, higher concentrations achieved with nebulization could overcome the KCNMA1 reduction (FIG. 4, panel J).Second Study of Example 1: Determine mechanisms of action of S- and R-vanzacaftor and their dependence on BK channel subunit composition and stimulus conditions in heterologous expression systems

[0138] BK channel Regulation and the Mechanism of Action (MOA) of Potentiators including Vanzacaftor. Macroscopic BK current (IK) can be recorded at different Ca2+concentrations ([Ca2+], FIG. 6, panel A) by patch clamp electrophysiology in the inside-out configuration from Slol channels expressed in Xenopus oocytes. The relationship between channel activity (Po, open probability) and V or [Ca2+] is illustrated by plotting Po-V relations in different [Ca2+], from 0 (with EGTA) to 100 pM (FIG. 6, panel B). Channels are fully activated in 0 [Ca2+], but Ca2+permits activation at physiological V. The MOA of V and [Ca2+] are best understood by using high resolution single channel recordings to extend measurements to Po < 10'2and plotting Log(Po)-V (see FIG. 6). Changes in Log(Po) by Ca2+are V-independent (until Po saturates), indicating that Ca2+and V act independently. BK channel modulation can be described by the Horrigan-Aldrich (HA) model (FIG. 6), used to fit data in FIG. 6. This model accounts for responses to Ca2+and V and is the ‘standard’ for understanding BK channel modulation by small molecule activators, inhibitors, endogenous ligands and auxiliary subunits. In the model, the channel contains three functional domains (gate, V-sensors, and Ca2+-sensors) that interact allosterically and control activity. The closed to open gate transition (C-O) is regulated by V-sensor activation (R-A) and Ca2+-binding (X-X Ca2+), each described by equilibrium constants (K, J, L) and allosteric interactions.

[0139] The HA model predicts that BK modulators perturb either the function of gate, V-sensor or Ca2+-sensor domains, or allosteric coupling. Examples of each MOA are known. To illustrate, the action of two BK activators were compared in 0 Ca2+(FIG. 7). Phloretin and pimaric acid (PiMA) both shift macroscopic Po-V relations to more negative voltages (FIG. 7), but PiMA and phloretin are distinct on Log(Po)-V and Ca2+, indicating they act by distinct MOA (FIG. 7). PiMA shifts the curve with little effect at negative voltages (FIG. 7) whereas phloretin acts primarily at negative voltages, increasing Po ~100-fold (see FIG. 7). Thus, only phloretin alters channel activity at rest and influences the function of non-excitable cells. Consistent with this notion, it was found that 10 pM NS11021, a BK activator that relaxes smooth muscle at rest, produces a 100-fold increase in Po at -80 mV (not shown). Note that while the absolute Po of BK at negative voltages is low (~10‘6to 10'3), this is sufficient to influence the function of many non-excitable cells because BK channels have the largest single25KU017M-0262724-PCTchannel conductance of any K+- channel and can be expressed at high levels, such that their full activation is not required to produce significant K+current or to alter resting membrane potential under conditions where other voltage-dependent channels also have low Po.

[0140] Model analysis predicted effects of potentiators with physiological V and Ca2+, providing mechanistic insights. The HA model fits to data (FIG. 7) indicating that PiMA enhances V-sensor activation ($1) whereas phloretin reduces V-sensor coupling to the gate D), providing clues as to where they bind to the channel. Defining the MOA of BK potentiators here allows for prediction of how S- and R-vanzacaftor act in physiological and pathophysiological conditions. The latter includes effects of auxiliary subunits present in airway epithelia and other tissues because the effects of auxiliary subunits on HA model parameters are known. Heterologous expression of channels in oocytes allows for directly injecting RNA for Slol and different auxiliary subunits.

[0141] Increases in resting BK channel activity (NPo at -80 mV, 0 Ca2+) relative to vehicle control are seen when oocytes are exposed acutely to different concentrations of S- or R-vanzacaftor with channels of hSlol ± LRRC26 (yl; FIG. 8). ECso = 2.5 pM is determined by fitting the data for S-vanzacaftor and hSlol / LRRC26 channels, with the Hill equation (N=l). Other curves fit reasonably well with ECso and N constrained by the same parameters. Importantly, vanzacaftor profoundly enhances resting activity, with a maximal increase > 1000-fold in the case of S-vanzacaftor and hSlol / LRRC26, which is comparable to the effect of saturating Ca2+. Owing to this efficacy, effects of S-vanzacaftor at concentrations as low as 50 nM (9-fold) were detectable. Similarly, R-vanzacaftor produced 2- to 3 -fold increases in the activity of hSlol / LRRC26 at 100 nM, and a 50-fold increase in activity at 5 pM, which is comparable to the effect of NS 11021 and much greater than that of 10 pM elexacaftor (2-3-fold, not shown).

[0142] These preliminary data show that vanzacaftor is a highly effective BK activator, increasing its basal activity. Additional can elucidate that differences exist between S- and R-enantiomers, as described in more detail herein, with R-vanzacaftor being a potentiator, whereas S-vanzacaftor only demonstrates acute BK activation, but not potentiation. The presented results show that vanzacaftor enhances channel activity in non-excitable cells and is more effective in the presence of LRRC26, favoring activity in airway epithelial cells.

[0143] High-throughput assay for BK modulators. To assay BK channel activity in a quantitative high-throughput manner, a fluorescent Tl+-flux assay is used with mammalian cells (U2OS), transiently transfected with BK subunits using BacMam baculovirus. These viruses infect but do not replicate in U2OS, providing -100% transfection efficiency and25KU017M-0262724-PCTexpressing proportionally to virus concentration. To introduce constructs, human Slol and auxiliary subunits (LRRC26, pi, P2, P4) have been subcloned into pFastbac (Invitrogen) vectors, modified with a mammalian CMV promoter, and then recombined into the baculovirus genome using the Bac-to-bac system (Invitrogen) and transfected in SF9 cells for virus production. To facilitate detection of activity at rest, a point mutation (F315 Y) was used in the pore-lining segment of Slol to stabilize the open conformation and increase Po more than 1000-fold, thereby increasing assay sensitivity without activating V- or Ca2+-sensors. It is known that F315Y does not alter modulation of resting activity by the pi subunit, and its activity at -80 mV increases 100-fold by 100 pM phloretin, like the WT (FIG. 7), supporting the that F315Y reproduces the pharmacological response of a resting channel.

[0144] Thallium (Tl+) flux assays. Tl+passes through K+channels. A Tl+-sensitive dye (Thallos, Ion Biosciences) increases fluorescence in the presence of Tl+and is used to detect flux through open K+channels. Initial rate of fluorescence increases by Tl+is proportional to K+conductance. U2OS cells transduced with Slol in 384 well plates were assayed using a FLIPRTETRA kinetic plate reader in the presence of 300 pM ouabain and 2 mM furosemide to inhibit K+transporters. BK channel modulators or vehicle controls were added to wells and incubated for 10 minutes before injecting Tl+solution to measure channel activity. FIG. 9, panel A shows increases in normalized fluorescence produced by 2 mM Tl+in wells treated with 30 pM phloretin, vehicle control (0.3% DMSO), or a 3 pM concentration of paxilline (PAX) sufficient to completely inhibit Slol. Fluorescence was normalized by the basal signal measured prior to Tl+addition and subtracting 1. Tl+-flux was quantified by the initial rate of increase, F’o. Phloretin produced a robust increase in F’o relative to DMSO while PAX reduced it to levels comparable to untransfected controls (not shown). Concentration -response curves for PAX were identical to those for the WT measured by patch clamp electrophysiology: IC50 for IBTX (l.l±0.1 nM) is comparable to literature (1 nM,93 FIG. 9).

[0145] Assays were developed for BK channels with different auxiliary subunits, expressed by mixing virus in a 1 :2 MOI ratio for Slol and auxiliary subunit to assure saturation with the latter (confirmed by patch clamp, not shown). [Tl+] was then adjusted for each variant (0.5 - 4 mM) to yield F’o similar to F315Y alone. Assays for yl (LRRC26) used a 4: 1 ratio to maintain Po«I, such that activators could be detected. The assay accurately reproduced known subunitselectivity of several controls (FIG. 9). Assays were highly reproducible with a coefficient of variation <8%, as reflected by the error bars in FIG. 8; and provided excellent statistics for detection of activators or inhibitors (Z’ = 0.52 to 0.82, SW= 5.3 to 21.8). It has been previously examined if losartan changes association between LRRC26 and KCNMA1 using Tl+-flux.25KU017M-0262724-PCTLosartan (24 hour exposure) does not increase LRRC26 association with Slot.

[0146] EXPERIMENTAL DESIGN

[0147] Potency, efficacy, subunit-selectivity, and MOA of vanzacaftor enantiomers were assessed with FLIPR and patch clamp electrophysiology in oocytes, which was performed as previously described71in the inside-out configuration at room temperature with an Axopatch 200B (Molecular Devices), Patchmaster acquisition software (HEKA), and Igor Pro software (WaveMetrics) for graphing and data analysis.

[0148] A first set of experiments assess potentiation of Slol BK channels ± auxiliary subunits (LRRC26, P2, P4; FIG. 3) under resting cellular conditions using FLIPR Thallium-flux. The FLIPR assay is conducted to measure concentration-response curves for S- and R-vanzacaftor on Slol channels ± auxiliary subunits. Although the therapeutic focus is on Slol / LRRC26 channels (FIG. 3), determining the dependence of potentiator action on auxiliary subunits is directly relevant to understanding how these compounds may act under inflammatory conditions, where channels containing Slol alone may be expressed, or in large airway secretory epithelial cells, where P4 is expressed. Determining the effect on BK channels containing P2 is also relevant; and determining whether potentiators are differentially sensitive to P subunits may provide clues as to sites of action based on homology of these subunits. Concentration-response curves will be acquired from 0.1 - 50 pM. Compounds will be prepared by serial dilution as 3-fold high stock solutions (0.3-150 pM) to be injected by FLIPR into the assay plate to achieve the final concentration, with each concentration tested in replicates of 3-6 wells. Each plate will also include replicate BK channel activators (30 pM phloretin, 30 pM NS1619), inhibitors (3 pM Paxilline, 100 nM IBTX), and vehicle (DMSO) controls, to evaluate reproducibility and assay statistics. Fluorescence is monitored continuously during compound incubation as a counter-screen for cytotoxicity (lysis or altered pH), which produces timedependent changes in fluorescence prior to Tl+injection.

[0149] A second set of experiments will confirm that S - / R-vanzacaftor do not perturb cytoplasmic [Ca2+]i with FLIPR. Compounds will be screened at concentrations used in Thallium-flux for their ability to increase [Ca2+]i in untransfected U2OS cells. A previous report that vanzacaftor activates BK channels in patch clamp experiments implies that they act directly on the channel as most indirect pathways are eliminated by this method.

[0150] A third set of experiments will optimize FLIPR for Slol / LRRC26 / p4 / p2 channels and assess potentiation by S- / R-vanzacaftor. BK p / y subunits bind to different sites in Slol and can co-assemble in the same channel, suggesting that BK channels in large airway secretory cells could contain LRRC26 and P4 / 2, which are expressed with Slol in these cells (FIG. 3, A-25KU017M-0262724-PCTD). To evaluate the effect of BK potentiators on Slol / LRRC26 / p4 channels, the Slol / LRRC26 assay will be modified to include P4 by co-transfecting cells with Slot, LRRC26, and P4 virus in a 1 :2:2 ratio to assure saturation with both auxiliary subunits (to be verified by patch clamp). It is contemplated that optimizing this assay to maintain the same signal amplitude as the Slol / LRRC26 assay should be straight-forward, as it has been found that expression of P-subunits with Slol causes only a modest reduction in signal that can readily and predictably be compensated by increasing [Tl+] in the assay owing to the linear flux-[Tl+] relationship.

[0151] A fourth set of experiments includes patch clamp validation of FLIPR and HA model analysis of S- and R-vanzacaftor action over a wide range of V and Ca2+. FLIPR will be validated and extended with patch clamp recordings in oocytes to define the MOA of potentiators. At a minimum, effects on channels composed of Slol alone and Slol / LRRC26 will be tested, and additional variants with P2 / 4 subunits and LRRC26 will be investigated if FLIPR suggests potentiator action is differentially sensitive to subunits. To define the MOA, the compound effects at a maximally effective (ideally saturating) concentration on the steadystate activation (Po) of BK variants will be determined over a wide range of V and [Ca2+]i. Data will be fit with the HA Model (FIG. 6) to identify changes in parameters associated with functional domains of the channel, as was previously done.78'81Results will provide insight into the biophysical mechanism and potential sites of action of modulators and characterize Ca2+-dependence of their action, which is not assayed by the FLIPR screen.

[0152] Expected results, potential pitfalls, and alternative approaches. Outcomes and alternative approaches have been discussed above. Other p subunits may be important for potential effects on BK channels in other tissues. It is not expected that chronic exposure changes the MOA, but this will be assessing desensitization by analyzing function of BK in oocytes and U2OS cells chronically treated with S- or R-vanzacaftor, including determining changes in HA model parameters compared to acute responses. This will determine if channel function is irreversibly altered (e.g., Slol / LRRC26 interaction is disrupted) or if drug response is altered in its EC50 or MOA. It will also be confirmed that the V-dependence and kinetics don’t change when the ratio of LRRC26 / p4 / p2 to Slol increases.

[0153] Third Study of Example 1: Determine in vivo relevance of acute and chronic BK potentiation by S- / R-vanzacaftor

[0154] Methods using a CF-like sheep model. Adult ewes will be used, and all procedures approved by the Mount Sinai Medical Center IACUC. Sheep are conscious and intubated. Methods for measuring tracheal mucus velocity (TMV) are published. Aerosols are delivered using dosimetry. A respirator delivers aerosols directly during inspiration at 20 breaths / minute25KU017M-0262724-PCTand a tidal volume of 500 mL. CFTRinhl72 is dissolved in DMSO and ethanol. Stock solutions of human neutrophil elastase (HNE; Elastin Product Company) is diluted in PBS (2380 mU of enzyme; Example 2). TGF-P1 (Example 2) and S- / R-vanzacaftor are dissolved in -cyclodextrin in water. Solvents do not influence TMV. A baseline TMV measurement will be obtained (mean has been 9.8 ± 0.26 mm / min in previous experiments) followed by nebulizations of specified agents. Subsequent TMV measures will be obtained as below.

[0155] Suitability of a CF-like sheep model. There is a strong case for the suitability of a non-genetic ovine CF-like model to mimic mucociliary dysfunction due to minimal function CFTR variants. This non-genetic sheep model recapitulates important aspects of CF-related human airway disease: 1) mucociliary dysfunction is caused by rendering CFTR nonfunctional (using CFTRinhl72) as seen with minimal function CFTR variants; 2) CF-related airway inflammation is induced by neutrophil elastase, found abundantly in inflamed CF airways; and 3) muco-ciliary dysfunction is maintained beyond immediate pharmacological challenges. This model lends itself to testing interventions, and measuring multiple outcomes, including clearance, mucus concentration, and airway inflammation at different time points, even over several days. In fact, sheep have often been used for inhalation studies because their lung size and anatomy are similar to humans, because they can be trained for restrained inhalation exposure (while being awake), and because they are suitable for predicting human responses to therapeutics in respiratory diseases. A notable exception of the translation of sheep results to human beings was the failure of ENaC inhibition to improve mucociliary function. The explanation for this failure was that the effective sheep dosing was not used in the human trials, where lower dosing failed (as would have been predicted by the ovine model), and the sheep model used in these studies did not include airway inflammation. Genetic CF animals exist, including the ferret, pig, rabbit, and rat, as well as an intrauterine / newborn sheep model (the lamb does not survive after birth and is therefore not suitable for these studies). Each of these models has contributed to understanding human CF. However, these models are not without challenges due to accessibility, expense, breeding, survivability, and even translatability because of a lack of biological agents specific for the species. Given these caveats, the non-lethal CF-like sheep model proposed here is a reasonable, translatable model with a successful history in drug development.

[0156] We further confirmed that ion channels are similar in ovine and human airway epithelia. Polarized ovine airway epithelial cell cultures reveal functional expression of apical BK, CFTR, and other channels (FIG. 10). Like human cells, S-vanzacaftor activates ovine BK acutely but does not potentiate BK chronically while R-vanzacaftor has little effect acutely but25KU017M-0262724-PCTpotentiates BK ~12-fold chronically (FIG. 10). For assessment of TMV, ewes are challenged with an aerosol of CFTRinhl72. Hypertonic saline improves TMV for 2 hours (FIG. 11). The CFTRinhl72 effect is sustained for 24 hours when high dose human neutrophil elastase (HNE; 2380 vs. 1190 mU), abundantly found in CF airways, is added to the aerosol challenge (FIG. H).

[0157] Improvements in TMV can be measured with inhalation of therapeutics (FIG. 11). Hypertonic saline (HTS; 7%) only temporarily rescues TMV indicating that inhibitors are not washed out with the increased clearance. 2 mg R-vanzacaftor but NOT S-vanzacaftor partially rescues TMV (2 mg total dose in EtOH that itself does not change TMV). This shows that an acute activator of BK has no therapeutic value here, but that a potentiator does. A first set of experiments uses the 3 -day repeat CFTRinhl72+HNE challenge model to test the ability of inhaled S- and R-vanzacaftor to acutely recover induced mucociliary dysfunction on the last day of CFTRinhl72+HNE. Inflammatory markers include TGF-pi and TNF-a, IL-ip, IL-6, IL-8, IL-13, and COX-2 in aspirates from the airways, assessing the effect of vanzacaftor, to confirm that CFTRinhl72+HNE challenge increases TGF-pi concentrations and to see if vanzacaftor reduces these responses.

[0158] A second set of experiments repeated the first set of experiments using chronic vanzacaftor inhalational doses (with each dosing of CFTRinhl72+HNE) and additionally used the same endpoints. Inhaled S-vanzacaftor did not rescue TMV (FIG. 11, panel D). As shown in FIG. 11 and compared with FIG. 8, S-vanzacaftor has an acute activation of the BK Channel, however, this is not sustained. Instead, as shown in FIG. 11 and compared with FIG. 8, R-vanzacaftor, while being a modest BK activator, it demonstrates substantial BK potentiation over time, and therefore has a significant impact on mucociliary function.

[0159] These results will be mirrored by other parameters of mucociliary function such as mucus concentrations. Dosing intervals as well as relevant dosing will be determined to set the stage of explorations into human beings. It is expected that vanzacaftor enantiomers will likely be absorbed, and the results will allow assessment of these levels, which are related to Example 3.

[0160] Previously published experiences with TMEM16A potentiators already predicted translation of in vitro data to this animal model. This is the case here as well (FIGs. 4 and 11) Needs for additional anti-inflammatory medications in addition to vanzacaftor will be explored in Example 2 with nesolicaftor and the full, 3 -day challenge CF-like sheep model.Example 2

[0161] The following example provides mechanisms of nesolicaftor’ s enhancement of BK25KU017M-0262724-PCTpotentiation by R-vanzacaftor.

[0162] Shown in a first study is that nesolicaftor enhances R-vanzacaftor’ s action by rescuing TGF-pi’s detrimental effects via enhanced LRRC26 and TMEM16A expression levels in CFBE cells with minimal function CFTR variants.

[0163] In a second study, the beneficial effects of adding nesolicaftor to R-vanzacaftor in a CF-like sheep model with additional TGF-pi -dominant inflammation is shown.

[0164] CF-associated inflammation by TGF-pi causes BK dysfunction by downregulating LRRC26, thereby reducing mucociliary transport by inhibiting potassium secretion. It has been shown that BK potentiation is reduced in this environment. Therefore, it is important to determine if inflammation that reduces efficacy of BK potentiation can be managed. A relatively new class of modulators, termed amplifiers, increase protein biosynthesis, thereby augmenting other modulators and potentiators and ultimately the function of ion channels. The amplifier nesolicaftor (or PTI-428) increases CFTR mRNA stability and translation (FIG. 12) as well as LRRC26 mRNA (FIG. 13). This is accomplished, at least in part, by the association of nesolicaftor with poly(rC)-binding protein 1 (PCBP1), that binds to consensus sequences in certain mRNAs, thereby promoting mRNA stabilization and protein translation. Nesolicaftor has been investigated in clinical trials along with approved CFTR modulators that do not potentiate BK with reported small clinical benefits. However, clinical trials with effective CFTR modulators have never been conducted. FIG. 12 shows that nesolicaftor can also rescue TGF-pi -mediated inhibition of modulator-corrected CFTR function in primary human CFBE cells, homozygous for F508del. Nesolicaftor reverses TGF-pi -induced reductions in ciliary beating and increases in secreted cytokines indirectly through its effects on apical ion channel function. Thus, amplifiers may be of value to modulators / potentiators that lose their efficacy under inflammatory conditions in the CF airway.First Study of Example 2: Determine if nesolicaftor enhances R-vanzacaftor’s action by rescuing TGF-pi’s detrimental effects via enhanced LRRC26 and TMEM16A expression levels in CFBE cells with minimal function CFTR variants

[0165] Nesolicaftor rescues TGF-pi-mediated ion channel dysfunction (FIG. 13). The mRNA amplifier nesolicaftor (or PTI-428) has been thought to specifically increase CFTR mRNA stability, independent of CFTR genotype. However, shown here that nesolicaftor’ s effects are not restricted to CFTR as it increases mRNA and function of both TMEM16A and BK channels, the latter via LRRC26. This is accomplished by nesolicaftor’ s association with PCBP1 (FIG. 13). The preliminary results show that nesolicaftor normalizes expression of LRRC26 in the presence of TGF-pi but only partially recovering BK function. Thus, additional25KU017M-0262724-PCTBK potentiation will be necessary to realize benefits under inflammatory conditions. The data here is reported from NHBE cells fully differentiated at the air-liquid interface. Nesolicaftor not only enhances CFTR function (FIG. 12) but also leads to a significant increase in BK activity with an increase in LRRC26 mRNA as well as CaCC with an increase in AN01 or TMEM16A mRNA (latter low n), providing evidence that nesolicaftor’ s action on ion channel function is not specific. Further, basolateral TGF-pi (10 ng / ml for 24 hours) causes a significant decrease in BK currents and LLRC26 mRNA (FIG. 13). Nesolicaftor partially rescues TGF- pi -mediated decreases in BK conductance and LRRC26 mRNA. As the modulation of BK activity by nesolicaftor is unreported, we set out to investigate the mechanism by which nesolicaftor enhances BK function. As LRRC26 is critical to maintain BK conductance in airway epithelia we assess nesolicaftor’ s effects on TGF- pi-mediated decreases in LRRC26 expression. In the presence of TGF-pi, nesolicaftor does not change KCNMA1 mRNA but increases LRRC26 protein and restored TGF-pi-mediated reductions in LRRC26. Additionally, it is shown that nesolicaftor rescues parameters of mucociliary function by increasing CBF and ASL volumes (FIG. 13), even in the presence of TGF- pi.

[0166] Mechanistically, nesolicaftor has been shown to increase mRNA stability by binding to PCBP1, which in turn binds to a PCBP1 consensus element within the mRNA. Using RBPmap analysis, it has been identified that PCBP1 binding consensus motifs within the 3 ’UTR of LRRC26 mRNA. Thus, nesolicaftor induces LRRC26 protein and mRNA expression by increased PCBP1 binding at 3 ’UTR. Primers specific for the PCBP1 consensus sequence region within the 3 ’UTR of LRRC26 have been designed to investigate associations between PCBP1 and LRRC26 in RNA immunoprecipitation (RIP) experiments after treating NHBE cells with DMSO (control) or nesolicaftor for 24 hours. Anti-PCBPl precipitates were enriched in LRRC26 mRNA specific to the 3 ’UTR compared to anti-IgG as well as DMSO controls when treated with nesolicaftor. This suggests that PCBP1 directly binds to the consensus motif present in the 3 ’UTR of the LRRC26 mRNA and that this binding is enhanced by nesolicaftor. These data suggest that nesolicaftor augments BK conductance through increased LRRC26 protein expression, an effect mediated by enhanced association of PCBP1 with the consensus motif present on the 3 ’UTR of LRRC26. NES induces expression of PCBP1 immunoprecipitated NHBE RIP lysates. RIP analysis uses PCBP1 antibodies, followed by RT-qPCR detection of LRRC26 expression. LRRC26 mRNA is enriched in NES-treated samples compared to matched IgG controls. n=3, 3 lungs. The data (not shown) was verified with p < 0.05, paired t test.

[0167] EXPERIMENTAL DESIGN25KU017M-0262724-PCT

[0168] A first set of experiments uses R-vanzacaftor at different concentrations, chronically as in Example 1, to determine their continued ability to improve conductance and parameters of mucociliary function with TGF-pi -dominated inflammation (and other cytokines such as IL- pi). All experimental setups and measurements were described above. Basolateral additions of 5 ng / mL TGF-pi or 10 ng / mL IL-pi for 24 hours will be used to induce inflammation as seen in preliminary data as well as our and other publications. Additionally tested will determine whether 1-10 pM nesolicaftor (reachable by inhalation) rescues TGF-pi or IL-pi -induced mucociliary dysfunction in the presence of R-vanzacaftor in CFBE cells with minimal function alleles. Nesolicaftor dosing was derived from clinical pharmacokinetic data, where in steady state, nesolicaftor reaches 3-5 pM in the blood after 50 or 100 mg daily dosing (EC90 = 4.3 pM for CFTR protein folding).

[0169] Second experimental set: We will repeat experiments using our Cloud nebulizer to deposit R-vanzacaftor with nesolicaftor onto the apical surface using the estimated ASL concentration of deposition that worked best basolaterally. We will repeat endpoints above acutely and with long-term exposures (24h). We will use basolateral additions of 5 ng / mL TGF-pi or 10 ng / mL IL-pi for 24h to induce inflammation.

[0170] Third experimental set: We will extend our work on the PCBP1 -binding to LRRC26 and TMEM16 mRNAs using RIP assays under the conditions described above. Expected results, potential pitfalls, and alternative approaches. We expect that nesolicaftor addition will enhance the overall mucociliary function in CFBE cells. It is envisioned that this may provide a novel double combination therapy with “best-in-class” BK potentiator plus nesolicaftor) for pwCF with minimal function mutations. Such combinations could see quick turnarounds into clinical trials. If mucociliary function is not enhanced by nesolicaftor, we will revisit acute exposures of S- and R-vanzacaftor with nesolicaftor.Second Study of Example 2: Adding nesolicaftor to R-vanzacaftor in a CF-like sheep model with TGF-]J-dominant inflammation

[0171] EXPERIMENTAL DESIGN

[0172] Preliminary data have been discussed above and in FIG. 11. To simulate defective CFTR function and neutrophilic inflammation in CF, 1-3-day inhalational challenges with CFTRinhl72 plus human neutrophil elastase (HNE) will be used (as opposed to a simpler model in Example 1). This multi-day inhalation challenges increase airway TGF-pi levels and mucus concentrations (FIG. 11). The sheep will be exposed with nesolicaftor ± R-vanzacaftor chronically (with each dosing of CFTRinhl72+HNE). As before, TMV will be measured, collect secretions for inflammatory markers will be collected, and airways will be brushed25KU017M-0262724-PCTbefore and after challenge for LRRC26 mRNA expression. For all conditions, including controls, 3-5 sheep will used. It is expected that addition of nesolicaftor may further rescue TMV. It is expected that nesolicaftor will not be sufficient alone and that the combination of nesolicaftor + R-vanzacaftor will exhibit superior results as compared to nesolicaftor and R-vanzacaftor alone.Example 3

[0173] The following example shows the effects of chemical modifications to R-vanzacaftor to enhance airway retention.

[0174] Rationally designed peptide nanosponges for advanced drug delivery. The therapeutic target BK is expressed on the apical airway surface. To avoid systemic exposures and possibly side effects from activating BK channels in other parts of the body (e.g. brain), nebulized treatments in vitro and in vivo with modifications of R-vanzacaftor that promote airway retention are explored.

[0175] A peptide nanosponge for advanced drug delivery is developed and comprises a trigonal tris-maleimide linker, a K20D10 (SEQ ID NO:1) or K20S10 (SEQ ID NO:2) peptide building block, and a terminal cholesterol unit responsible for nanosponge formation by means of hydrophobic nano-droplets. These nanosponges deliver drugs to targeted cells. A nanosponge design with an integrin avP6 targeting peptide was chosen and expressed in secretory cells in the airway where BK is expressed (FIG. 3). The targeting sequence is derived from the viral peptide A20FMDV2. Anchoring to the cell surface is followed by micropinocytosis, rapid endosomal escape, and release of the covalently attached drug R-vanzacaftor. Attachment of cholesterol and drugs will be facilitated by esterase-cleavage bonds, which can be rapidly severed by human esterases. To avoid loss by phagocytes, the “Don’t-Eat-Me” peptide sequence (SEQ ID NO:4) kGNYTCEVTELSREGKTVIELKk will be co-attached. This peptide sequence mimics binding of regulatory protein alpha (SIRPa) in phagocytes, which triggers an auto-protective response of CD47. Both peptide sequences bear D-amino acids at both terminal ends to prevent rapid proteolytic degradation. Building principles of the drug delivery nanosponge are shown in FIG. 14A-14E. Rationally designed peptide nanosponges form 30-50 nm supramolecular particles in aqueous solutions.

[0176] An example for pancreatic cancer delivery is shown in FIG. 15. To increase accumulation in pancreatic ductal adenocarcinoma, a nanosponge targeting the cancer surface marker plectin was designed. This nanosponge comprises a trigonal tris-maleimide linker, a S10K20CG (SEQ ID NO:3) peptide building block with cholesterol and the plectin-targeting peptide (SEQ ID NO: 5) KTLLPTP coupled to rhodamine. For in vivo experiments, the25KU017M-0262724-PCTreferenced “Don’t-Eat-Me” sequence was co-attached. Mice with pancreatic ductal adenocarcinoma (21 days of growth) were treated with a plectin-targeting nanosponge. Twelve hours after intraperitoneal inj ection, mice were sacrificed, and various organs and tumor tissues removed and imaged ex vivo to determine localization of the nanosponge. Imaging shows selective uptake of nanosponges in the tumor.First Study of Example 3: Determine effects of modifying R-vanzacaftor by adding airway retention triggers in CFBE cells

[0177] The peptides targeting integrin avP6 and the “Don’t Eat Me” peptide will be synthesized as previously described, and characterized by UPLC-MS. Preparative HPLC will be performed if the purity will not exceed 98%, based on peak integration (optical detection at 1=260 nm).

[0178] In-silico modeling was performed to find peptide sequences that target integrin avP6 using homology models (Uniprot: P18564 ITB6 HUMAN) on the SWISS-MODEL server. The stereochemical quality of the model was ascertained with MolProbity version 4.4. Most favored, additionally allowed, and generously allowed regions were identified using the Ramachandran plot. Protein-peptide docking allowing for peptide flexibility was performed using High Ambiguity Driven protein-protein DOCKing, referred to as HADDOCK (version v2.4-2022.08). The predicted interactions and interface residues for the top-ranking model of each peptide conformer were analyzed on the Proteins, Interfaces, Structures and Assemblies (PDBePISA) server. The resulting binding energy was averaged for four structures contained in the highest ranked cluster obtained from HADDOCK for each of the integrin avP6 binding peptides. A viral peptide ((SEQ ID NO: 6) NAVPNLRGDLQVLAQKVAR) seems optimal, previously identified as suitable for binding to integrin avP6. Based on this information, consecutive point mutations were performed to optimize binding to integrin avP6. A total of 35 peptides were investigated (Table 1), with the most promising peptides, based on free energy rankings, being later assessed in NHBE cells.Table 1.25KU017M-0262724-PCT

[0179] EXPERIMENTAL DESIGN

[0180] For initial experiments, NHBE cells are used and exposed to nebulized R-vanzacaftor nanosponges with the avP6 targeting peptide. BK potentiation is measured chronically (over 24 hours) using de-escalating doses. The transepithelial appearance of vanzacaftor in the basolateral compartment is also determined. These experiments provide a qualitative perspective of which nanosponge will be best for further use. CFBE cells are used to repeat these experiments.

[0181] It is expected that the dosing regimen of R-vanzacaftor coupled to nanosponges will need less drug than the free vanzacaftor dosing as it targets cells of interest specifically and will have less leakage to the basolateral (systemic) compartment. In case there is no BK potentiation, different nanosponges will be subsequently constructed for troubleshooting, including one that has a fluorophore attached to assure appropriate binding to avP6 and internalization.Second Study of Example 3: Determine effects of R-vanzacaftor modifications in a CF-like sheep model

[0182] EXPERIMENTAL DESIGN

[0183] Based on the first study results, the best performing class of nanosponge will be used in sheep and experiments repeated as outlined above. Importantly, it is expected that mucociliary dysfunction will be rescued with these nanosponges with lower dosing requirement in addition to lower systemic exposures of vanzacaftor.

[0184] CONCLUSIONS

[0185] There is a critical knowledge gap in how to address lung disease in pwCF who are not eligible for modulator therapy and other muco-obstructive lung disease such as COPD (and possibly asthma). To close this gap, it is contemplated that potentiating BK specifically with R-vanzacaftor will improve mucus hydration and mucociliary clearance in CF with minimal function CFTR variants (and other muco-obstructive diseases), even in a TGF-P dominated25KU017M-0262724-PCTinflammatory milieu. The foregoing examples and studies included cultures from airway cells of pwCF with minimal function CFTR variants in vitro, oocyte expression systems that dissect contributions of BK subunits to vanzacaftor’ s potentiating ability, and in vivo using a CF-like, non-genetic sheep model that mimics minimal function CFTR variants. Additionally studied was a novel therapy by potentiating BK and a new drug delivery system targeted to airway epithelia. Finally, it is contemplated that the disclosed therapies and drug delivery systems could also be applied to multiple other airway diseases with pathophysiological contributions of abnormal ion channel conductance leading to mucociliary dysfunction, such as asthma and COPD.

Claims

25KU017M-0262724-PCTWHAT IS CLAIMED IS:

1. A method of treating cystic fibrosis in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

2. The method of claim 1, wherein the patient has minimal CFTR function.

3. The method of claim 1 , wherein the patient does not have a F508del mutation in CFTR.

4. The method of claim 1, wherein the patient was previously treated with a CFTR potentiator.

5. The method of any of the previous claims, wherein R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered in a nebulizer, inhaler, or spray.

6. The method of any of the previous claims, wherein R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered with a nanosponge.

7. The method of any of the previous claims, wherein the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is from 1 to 20 pM.

8. The method of any of the previous claims, wherein the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is from 1 to 50 mg / day.

9. The method of any of the previous claims, wherein the therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof is administered for multiple, consecutive days.

10. The method of any of the previous claims, further comprising administering a LRRC26 potentiator to the patient.

11. The method of any of the previous claims, further comprising administering a TMEM16A potentiator to the patient.

12. The method of any of the previous claims, further comprising administering nesolicaftor, metformin, or angiotensin blockers to the patient.

13. A method of decreasing mucus concentration and / or mucus viscosity in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

14. A method of increasing mucociliary transport in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

15. A method of treating asthma or COPD in a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.25KU017M-0262724-PCT16. A method of bronchodilating a patient, comprising: administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

17. A method of treating inflammation in a cystic fibrosis patient, comprising:administering a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof to the patient.

18. A pharmaceutical composition for use in treating a patient according to any of the methods of claims 1-17, the medicament comprising a therapeutically effective amount of R-vanzacaftor or a pharmaceutically acceptable salt thereof.

19. Use of a specific enantiomer, R-vanzacaftor or a pharmaceutically acceptable salt thereof, to treat a patient, such as to increase mucociliary transport, improve bronchodilation, decrease inflammation, or treat cystic fibrosis, asthma, or COPD in a patient in need thereof.

Images