Anion-selective molecular prosthetics for cftr

Abstract

Disclosed are therapeutic methods for treating a disease or condition characterized by decreased expression or reduced function of an ion channel using an amphotericin B derivative, or an amphotericin B derivative and an ion channel modulator. The methods can be used to treat cystic fibrosis.

Description

[0001] UIX-05125

[0002] ANLON-SELECELVE MOLECULAR PROSEffEELCS

[0003] / OR CEER

[0004] RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No.

[0005] 63 / 691,534, filed September 6, 2024.

[0006] GOVERNMENT SUPPORT

[0007] This invention was made with government support under GM118185, and HL152960-02; subaward S02766-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND OF THE INVENTION

[0009] Cystic fibrosis is a well-known autosomal recessive genetic disease that affects multiple organ systems, notably pulmonary and gastrointestinal systems. It occurs in 1 in 3,000 live births and is caused by mutations in the CFTR gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane-expressed chloride channel protein in vertebrates. Cystic fibrosis is typically characterized by chronic and potentially fatal respiratory infections. Many patients benefit from CFTR modulators, but still have a median survival of only 66 years. About 18% of people with CF in the U.S. are unable to benefit from CFTR modulators and currently do not have access to any disease-modifying therapies.

[0010] CFTR is an ABC transporter-class ion channel that conducts chloride ions across epithelial cell membranes. Mutations of the CFTR gene affecting chloride ion channel function lead to dysregulation of epithelial fluid transport in the lung, pancreas and other organs, resulting in cystic fibrosis. Complications include thickened mucus in the lungs with frequent respiratory infections, and pancreatic insufficiency giving rise to malnutrition and diabetes. These conditions lead to chronic disability and reduced life expectancy. In male patients, the progressive obstruction and destruction of the developing vas deferens and epididymis appear to result from abnormal intraluminal secretions, causing congenital absence of the vas deferens and male infertility.

[0011] CFTR functions as a cAMP-activated ATP-gated anion channel, increasing the conductance for certain anions (e.g., CF) to flow down their electrochemical gradient. ATP- - 1 -

[0012] FH13055872.2 UIX-05125 driven conformational changes in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. This functionality is in contrast to other ABC proteins, in which ATP-driven conformational changes fuel uphill substrate transport across cellular membranes. Essentially, CFTR is an ion channel that evolved as a “broken” ABC transporter that leaks when in open conformation.

[0013] The CFTR is found in the epithelial cells of many organs, including the lung, liver, pancreas, digestive tract, reproductive tract, and skin. Normally, the protein moves chloride and thiocyanate ions (with a negative charge) out of an epithelial cell to the covering mucus. Positively charged sodium ions follow passively, increasing the total electrolyte concentration in the mucus, resulting in the movement of water out of the cell by osmosis.

[0014] SUMMARY OF THE INVENTION

[0015] In certain aspects the present disclosure relates to methods of treating or preventing a disease or condition characterized by decreased expression or reduced function of an ion channel, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB-AA:

[0016] (C2'-epiAmB-AA). In further aspects, the present disclosure relates to methods of treating a disease characterized by reduced function or decreased expression of an ion channel, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0017] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0018] - 2 -

[0019] FH13055872.2 UIX-05125

[0020] (C2'-epiAmB-AA); and

[0021] (b) an ion channel modulator.

[0022] In still further aspects, the present disclosure relates to methods of increasing transepithelial current of airway cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0023] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA: (C2'-epiAmB-AA); and

[0024] (b) an ion channel modulator.

[0025] In certain aspects, the present disclosure relates to methods of increasing the pH of airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of: (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0026] - 3 -

[0027] FH13055872.2 UIX-05125

[0028] (C2'-epiAmB-AA); and

[0029] (b) an ion channel modulator.

[0030] In further aspects, the present disclosure relates to methods of increasing bicarbonate secretion into airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0031] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0032] (C2'-epiAmB-AA); and (b) an ion channel modulator.

[0033] In yet further aspects, the present disclosure relates to methods of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0034] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0035] (C2'-epiAmB-AA); and

[0036] (b) an ion channel modulator.

[0037] In still further aspects, the present disclosure relates to methods of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0038] - 4 -

[0039] FH13055872.2 UIX-05125

[0040] (C2'-epiAmB-AA).

[0041] BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A-1H demonstrate that differing ion selectivities for AmB-AA and AmB-

[0042] NAcC channels are associated with different effects in preclinical models for protein channel deficiencies. FIG. 1A depicts the chemical structure of AmB, C3' N-Acetyl ethyl carboxylate (AmB-NAcC) and Cl 6- Aminoethyleneamide AmB (AmB-AA). FIG. IB depicts graphed representative traces of Ussing chamber current clamp ion- selectivity protocol prior to correcting for junction potentials. Step I) Apical media switch to 70 mM NaCl solution for paracellular ion movement determination. Step II) Addition of DMSO bilaterally. FIG. 1C depicts graphed results of Ussing chamber current clamp protocol done with 43 pM addition of AmB resulting in a positive change in voltage (cation selective). FIG. ID depicts graphed results of Ussing chamber current clamp protocol done with 43 pM addition of AmB-NAcC resulting in a positive change in voltage (cation selective). FIG. IE depicts graphed results of Ussing chamber current clamp protocol done with 43 pM addition of AmB-AA resulting in a negative voltage change (anion selective). FIG. IF depicts the estimated relative Pci / PNa for AmB, AmB-NAcC, and AmB-AA across FRT monolayers. FIG. 1G depicts the maximum % growth relative to WT for all three compounds which was determined by comparing the trklAtrk2A and WT yeast growth across a concentration range for each compound. FIG. 1H depicts the ASL pH of CuFi-1 epithelial (dF508 / dF508) treated with 2 pM AmB, AmB-NAcC or AmB-AA. For FIGs. 1F-1G, data are represented as mean ± s.e.m. (f,h), One-Way ANOVA with multiple comparisons. Statistical significance for FIG. 1G was analyzed with an RM One-Way ANOVA with multiple comparisons, ns, not Significant. **** p <0.0001, *** P<0.001 ** P=0.0014, * P=0.01.

[0043] FIGs. 2A-2C demonstrate that AmB-AA outperforms AmB as a Molecular Prosthetic. FIG. 2A depicts a graph obtained in Ussing chamber current clamp protocol with AmB: step I) Apical media switch to 70 mM NaCl solution for paracellular ion movement

[0044] - 5 -

[0045] FH13055872.2 UIX-05125 determination; step II) Switch back to 140 mM NaCl solution; step III) Addition of AmB 43 pM bilateral; step IV) Apical switch to 70 mM + 43 pM AmB. Addition of AmB resulted in a positive voltage change (cation selective). FIG. 2A depicts the ASL pH of CuFi-1 airway epithelial dose response between AmB at 0, 2, 20, 50,100 pM and AmB-AA at 2 pM FIG. 2B depicts the ASL pH of CuFi-1 airway epithelial dose response between AmB-AA at 0, 2, 20, 50, 100 pM and AmB at 2 pM FIG. 2C depicts the results of a conductance study with Fisher rat thyroid (FRT) mounted cells on Ussing chamber using apical 70 pM NaCl (pH=7) modified Ringer’s solution and basolateral 140 mM NaCl (pH=7) modified Ringer's solution treated bilaterally with 2.69, 5.37, 10.75, 21.5, 43, or 100 pM of AmB, AmB-NAcC, and AmB-AA with complete Voltage (mV) and conductance (mS / cm2) traces for 2.6 pM and 100 pM runs. Data are represented as mean ± s.e.m. Statistical significance for FIGs. 2A-2B was analyzed with an unpaired t test. Statistical significant for FIG. 2C was analyzed with a two-sided unpaired Student’s t-test. ns, not Significant. **** P <0.0001, *** P<0.001 ** P=0.0014, * P=0.01. FIGs. 3A-3F demonstrate that AmB-AA outperforms AmB as a Molecular

[0046] Prosthetic. FIG. 3A depicts the change in ASL pH in CuFi-1 airway epithelial dose response between AmB and AmB-AA at 0, 2, 20, 50, and 100 pM. FIG. 3B depicts the intracellular pH in CuFi-1 airway epithelial of AmB versus AmB-AA at 2 pM FIG. 3C depicts ENaC hyperactivity in CuFi-4 airway epithelial for AmB and AmB-AA. FIG. 3D depicts the results of a conductance study with Fisher rat thyroid (FRT) mounted cells on Ussing chamber using apical 70 pM NaCl (pH=7) modified Ringer’s solution and basolateral 140 mM NaCl (pH=7) modified Ringer's solution treated bilaterally with 2.69, 5.37, 10.75, 21.5, 43, or 100 pM of AmB, AmB-NAcC, and AmB-AA. FIG. 3E depicts the ICP-MS of ASL of CuFi-1 treated with AmB-AA at 50 pM with or without Ouabain FIG. 3F depicts Ouabain-sensitive proton flux in primary airway epithelial between AmB and AmB-AA at 2 pM. Data are represented as mean ± s.e.m. ns, not Significant. **** P <0.0001, *** P<0.001 ** P=0.0014, * P=0.01

[0047] FIGs. 4A-4I demonstrate that the toxicity of AmB-AA is mechanistically similar to AmB. FIG. 4A depicts the UV-Vis absorbance of AmB-AA with increasing ratios of cholesterol complexation. FIG. 4B depicts the toxicity of AmB, AmB-AA, C2’epiAmB, and AmB-AA complexed with cholesterol all at 50 pM in CuFi-1 epithelial. FIG. 4C depicts the toxicity of AmB, AmB-AA, C2’epiAmB, and AmB-AA complexed with cholesterol all at 50 pM in CuFi-4 epithelial. FIG. 4D depicts a close-up view of AmB-AA bound to cholesterol. FIG. 4E depicts chloride efflux from POPC liposomes with 10% cholesterol after DMSO,

[0048] - 6 -

[0049] FH13055872.2 UIX-05125

[0050] AmB, C2’epiAmB, and Natamycin addition. FIG. 4F depicts chloride efflux from POPC liposomes with 10% lanosterol after DMSO, AmB, C2’epiAmB, and Natamycin addition. FIG. 4G depicts H14CO3‘ rescue after 1 hour of incubation with ABCI-003 and C2’epiAmB. FIG. 4H depicts the ASL pH rescue of AmB, and C2’epiAmB at 2 pM. FIG. 41 depicts an expanded view of AmB-AA bound to cholesterol. Data are represented as mean ± s.e.m. ns, FIGs. 4A, 4B, 4G, and 4H were analyzed with a One-Way ANOVA with multiple comparisons, ns, not Significant. **** P <0.0001, *** P<0.001 ** P=0.0014, * P=0.01.

[0051] FIGs. 5A-5H demonstrate that Epi-AmB-AA Retains Increased Anion Selectivity and is Less Toxic than AmB. FIG. 5A depicts the synthetic scheme and chemical structure of C2’epiAmB-AA. FIG. 5B depicts the UV-vis absorbance of C2’epiAmB-AA with increasing ratios of cholesterol. FIG. 5C depicts the toxicity of CuFi-1 cells treated with C2’epiAmB-AA, AmB, and AmB-AA at 50 pM. FIG. 5D depicts graphed results of Ussing chamber current clamp ion-selectivity protocol. Step I) Apical media switch to 70 mM NaCl solution for paracellular ion movement determination. Step II) Addition of C2’epiAmB-AA AmB 100 pM bilateral. FIG. 5E depicts the estimated relative permeability of chloride to sodium ions for C2’epiAmB-AA at 200 pM and AmB at 44 pM across FRT monolayers. FIG. 5F depicts H14CO3‘ rescue after 10 minutes of incubation with AmB, AmB-AA, and C2’epiAmB-AA at 2 pM in CuFi-4 airway epithelial. FIG. 5G depicts ASL pH dose response of C2’epiAmB-AA at 2, 20, 50, and 100 pM in CuFi-1 airway epithelial. FIG. 5H depicts ASL pH rescue or primary CF airway epithelial pretreated with 2 and 50 pM of AmB, AmB- AA, and Epi-AA. Data are represented as mean ± s.e.m. FIGs. 5C-5G were analyzed with a One-Way ANOVA with multiple comparisons. FIG. 5H was analyzed with a Two-sided unpaired Student’s t-test. ns, not Significant. **** P <0.0001, *** P<0.001 ** P=0.0014, * P=0.01. FIGs. 6A-6F demonstrate that AmB-NAcC hints at higher cation selectivity than

[0052] AmB in physiological Assays. FIG. 6A depicts a dose response of AmB in yeast rescue assay. FIG. 6B depicts a dose response of AmB-NAcC in yeast rescue assay. FIG. 6C depicts a dose response of AmB-AA in yeast rescue assay. FIG. 6D depicts the Total Cellular K+levels of yeast for AmB, AmB-AA, AmB-NAcC via ICPMS. FIG. 6E depicts the Total Cellular K+levels of trklAtrk2A with AmB-NAcC at varying doses. FIG. 6F depicts the ASL pH dose response for AmB-NAcC.

[0053] FIGs. 7A-7G depict the toxicity profile of AmB -Derivatives in cholesterol containing cell lines. FIG. 7A depicts an RPTEC (primary renal proximal tubule epithelial cells; n=3

[0054] - 7 -

[0055] FH13055872.2 UIX-05125 biological replicates / conc.)) toxicity plot of AmB, AmB-AA, C2’epiAmB, C2’epiAmB-AA, AmB-NAcC. FIG. 7B depicts a HEPG2 (human liver cells; n=3 biological replicates / conc.) toxicity plot of AmB, AmB-AA, C2’epiAmB, C2’epiAmB-AA, and AmB-NAcC. FIG. 7C depicts a Hemolysis Plot of AmB, AmB-AA, C2’epiAmB, and C2’epiAmB-AA. FIG. 7D depicts a CuFi-4 Toxicity of AmB, AmB-AA, C2’epiAmB, C2’epiAmB-AA, and AmB- NAcC, at 2, 50, and lOOpM. FIG. 7E depicts an AmB:Cholesterol UV-Vis binding plot. FIG. 7F depicts a C2’epiAmB:Cholesterol UV-Vis binding plot. FIG. 7G depicts an AmB- NAcGCholesterol UV-Vis binding plot.

[0056] FIGs. 8A-8D demonstrate that POPC Liposomes shows AmB channel formation in non-sterol containing membranes. FIG. 8A depicts the chloride efflux of AmB, C2’epiAmB, Natamycin, AmB-AA, and C2’epiAmB-AA (2 pM) in sterol free liposomes. FIG 8B depicts AmB, C2’epiAmB, Natamycin, and C2’epiAmB-AA (2 pM) in cholesterol containing liposomes (2, 5, 10, and 20%) FIG. 8C depicts the chloride efflux of AmB, C2’epiAmB, Natamycin, and C2’epiAmB-AA (2 pM) in lanosterol containing liposomes (2, 5, 10, and 20%) FIG. 8D depicts the chloride efflux of AmB, C2’epiAmB, and Natamycin (2 pM) in ergosterol containing liposomes (2, 5, 10, and 20%).

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Transmembrane ion channels are essential proteins responsible for the regulation of electrochemical gradients, which are key for healthy biological function. Genetic disorders that disturb their function, categorized as channelopathies, can have severe physiological effects. These effects are increasingly exacerbated and are often lethal in channelopathies caused by loss of function (LOF) mutations, which are often more severe due to limited treatment options. Common treatment options, such as small molecule therapeutics, are rare for LOF mutations, as it is often easier to design function inhibition rather than function rescue. Dravet syndrome (DS), Long QT syndrome (LQS), and Cystic Fibrosis (CF) represent a long list of difficult to treat channelopathies linked to deadly brain, heart, and lung phenotypes. Advancements towards new treatment options for LOF channelopathies, such as gene and modulator therapy, have been made but remain gene-dependent, economically inaccessible, or too early in development. The severity of channelopathies and difficulty in therapeutic development highlights the need for new broad approaches to treating LOF genetic diseases.

[0059] - 8 -

[0060] FH13055872.2 UIX-05125

[0061] Molecular prosthetics (MPs) have been shown to successfully recover function in patients with LOF affected transport proteins. For example, in patients with LOF with respect to: Ferroportin using Hinokitiol as an MP; or Trkl / Trk2 and CFTR using Amphotericin B (AmB) as an MP. In particular, AmB forms a separate, distinct ion channel that recovers ion secretion lost as a result of the defective transport protein. Despite their low metal or ion selectivity, Hinokitiol and AmB were capable of recovering physiology in their respective assays in addition to highlighting their gene-agnostic mechanisms. In such experimental models, it was determined that the level of function required to restore the system out of a disease state is not very high due to the built-in robustness of the model systems. Therefore, it was demonstrated that the first- generation MPs did not need to be a perfect surrogate of the parent protein being replaced to restore functional capacity. Nevertheless, these MPs showed some evidence of being limited by their non-selective nature. A clear example was shown with AmB in its ability to restore growth in trklAtrk2A yeast, which exhibit K+ion channel deficiency and are unable to concentrate potassium or grow in standard media. AmB restored trklAtrk2A yeast growth during a time course as the AmB -treated trklAtrk2A cells took 12 hours longer to reach wild-type growth levels. Additionally, in Airway surface liquid (ASL) pH rescue, AmB was discovered to have a narrow therapeutic window and to not reach wildtype levels of pH. It was rationalized that the non-selective nature of the ion channels formed by AmB could be limiting their physiological recovery. As is often found in biological ion channels, mutations affecting ion selectivity can lead to a disease state. In aldosterone- producing adrenal adenomas (APAs) two somatic mutations near the selectivity filter of KCNJ5, a potassium selective ion channel, have been identified to cause loss of K+- selectivity. This loss of K+-selectivity correlated with an increase in Na+- selectivity which was attributed to cellular depolarization and hypertension in patients. The limitations of current MPs and the necessity of finely tuned ion channels cannot be overlooked. Inspiration from biological complexity can be learned from and translated to the field of Molecular prosthetics (MPs).

[0062] It was hypothesized that improved recovery of physiology can be achieved by increasing protein likeness of AmB compared to its biological counterpart, specifically with respect to ion selectivity and conductance. In order to test this hypothesis electrostatic interactions were selected to guide the design model of new protein-like AmB derivatives, as they are key in biological ion channel selectivity. With this design principle in mind, two charged derivatives of AmB were synthesized and studied, C3' N-Acetyl ethyl carboxylate

[0063] - 9 -

[0064] FH13055872.2 UIX-05125

[0065] (AmB-NAcC, having two carboxylates)) and C16-Aminoethyleneamide AmB (AmB-AA, having two amines).

[0066] The mechanism of ion channel self-assembling process of AmB is not settled, though certain aspects of the mechanism are accepted. AmB can penetrate the lipid bilayer creating pores through its dimerization with other AmB monomers. Additionally, it is accepted that the bilayer is penetrated with the C35-OH group, leaving the C16-carboxylate and C3'-amine present in the channel opening or head space. In some embodiments, this disclosure demonstrates that modification of AmB at C16 and C3’ positions can be used to modify the channel's conductance and ion selectivity without killing its channel-making properties. Through optimization of previously reported synthetic pathways, two key derivatives,

[0067] AmB-NAcC and AmB-AA were prepared. The newly introduced charged species contained either two negatively charged carboxylates, for AmB-NAcC, or two positive charged amines for AmB-AA. The ability of AmB-NAcC and AmB-AA to form cation and anion selective channels was explored. The impact of these charges on channel self-assembly, or bioavailability was also explored.

[0068] The C16-carboxylic acid and C3’ -amine in the headspace were leveraged to introduce a non-zero charge to the self-assembled ion channel. By acylating and blocking the positive C3’ -amine, while also introducing another carboxylic acid to the base structure, a net negative AmB-derivative was prepared. Similarly, by amidation of the carboxylic acid, with an amine nucleophile that could introduce another amine species, ethylenediamine, a net positive AmB-derivative was prepared. Given their net charges, AmB-NAcC could be more cation selective and AmB-AA more anion selective than AmB. The charges on the molecules could also reduce channel self-assembly, or rather bioavailability, resulting in a reduction of conductance, an effect previously seen with cholesterol pre-complexed AmB. In some embodiments, the present disclosure relates to modification of the ion selectivity and conductance of AmB to more closely resemble the replaced proteins studied in previous models. In some embodiments, the present disclosure relates to two low conductive and ion selective AmB derivatives, cation-selective AmB-NAcC and anionselective derivative AmB-AA. AmB-NAcC demonstrated an increased rescue of physiology compared to AmB in in a potassium-dependent yeast growth rescue assay. In contrast, compared to AmB, AmB-AA was a poor MP surrogate in a potassium-dependent yeast growth rescue assay. However, in a bicarbonate dependent airway surface liquid (ASL) pH rescue assay, the opposite effects were reported with AmB-NAcC retaining the low rescue

[0069] - 10 -

[0070] FH13055872.2 UIX-05125 that AmB has demonstrated in the past and AmB-AA showing an improved physiological rescue compared to AmB. Additionally, the increased protein-likeness of AmB-AA demonstrated to negate negative effects previously seen in AmB, such as ATP12A overexpression, which limited AmB’s therapeutic window. These improved effects of AmB- AA further translated to primary airway culture epithelia. Key models to test the restoration of host defenses such as ASL viscosity, height, and pH all showed AmB-AA with a WT-level of recovered physiology. In a stressed system, mimicked by testing the restored ASL antibacterial properties against .S'. Aureus AmB-AA demonstrated a 75% improvement.

[0071] The therapeutic approach of leveraging small molecules with protein-like function, molecular prosthetic (MPs), to treat genetic diseases is a growing field with significant potential. Amphotericin B (AmB), a non-anion-selective channel forming small molecule, has demonstrated the potential of this approach by effectively replacing missing protein function and restoring physiology in two distinct channelopathy models. Ion channels formed by AmB, which conduct both monovalent cation and anions with a preference for the former, was shown to rescue K+-dependent yeast growth in a Trkl / Trk2 deficient yeast model. Additionally, despite their lack of anion selectivity AmB-based channels were found to restore HCO f secretion, airway surface liquid (ASL) pH, ASL hydration, and antibacterial properties in cultured cystic fibrosis (CF) airway epithelial. These promising results paved the way for a dry powder formulation of AmB with cholesterol, CM001, currently undergoing clinical trials for the treatment of CF.

[0072] The versatility demonstrated by AmB proved that an MP could effectively restore physiology despite lacking perfect protein-like function, such as ion selectivity. However, in the absence of cholesterol, the therapeutic range of AmB is narrowed. At high concentrations of AmB the ASL acidifies. This was recently shown to be caused by an increase in K+efflux through the non-anion-selective AmB-based channels leading to an increase H+secretion by ATP12a, a non-gastric H+ / K+-ATPase that secretes protons into the ASL. It was thus hypothesized that a CFTR-like, anion selective, AmB derivative could have improved functional recovery and extended the therapeutic range in CF models bypassing the limitations of the first- generation MP, AmB. The ion selectivity of biological ion channels is often guided by a complex variation and combination of factors such as amino acid sequences, tertiary structures, and pore size to name a few. Among these interactions, electrostatic interactions are often highlighted as primary contributors to ion selectivity. As is the case with CFTR, charged residues such as

[0073] - 11 -

[0074] FH13055872.2 UIX-05125 lysine and arginine populate the pore lining of the anion selective ion channel resulting in a PNa / Pci of 0.03. Inspired by nature’s designed principles, it was hypothesized that the ion selectivity of AmB channels would shift towards being anion- selective through the addition of positively charged residues in or near the pore of the small molecule ion channels. As no single accepted model of the AmB ion channel exists, the design principles to make an anion selective derivative was guided by the previous results with synthetic derivatives of AmB. In the past, derivatives that replaced the C3-OH for a proton were synthesized, and studies suggested that the polyol region of AmB was within the lining of the self-assembled pore. These models also positioned the charged carboxylate and mycosamine ring towards the channel heads. Most recently, modifications aimed towards these two groups, such as the amidation of the C41-carboxilate, provided derivatives with retained channel activity in ergosterol containing organisms. These derivatives provided a foundation for a rough model that could be used alongside the nature inspired designed principles to design an anion- selective derivative. Based on the electrostatic designed principle guiding anion- selectivity, it was deduced that a modification of the C41-carboxilate could introduce another positively charged amine to the structure of AmB. Additionally, this modification was predicted to retain ion channel activity based on our experience with similar derivatives.

[0075] The non-anion selective molecular prosthetic (MP) amphotericin B (AmB) is demonstrating promising results and a good safety profile in undergoing clinical trials in people with cystic fibrosis (CF). A superior next generation MP was speculated to be possible as CFTR, the missing protein responsible for CF, is known to be anion- selective. Guided by this protein-likeness design principle and the synthetic understanding of the AmB ion channels, electrophysiological studies and channelopathy models were used to confirm this speculation. The electrostatic design principles alongside the quantitative electrophysiological studies led to the development of AmB-AA, an anion-selective MP.

[0076] The amidation of the C41 position of AmB with ethylene yielded the next generation MP, AmB-AA. Remarkably, the anion selectivity, Pci / PNa, calculated for AmB-AA was found to be more than 50 times higher than that of AmB when experimentally tested in Ussing chamber assays on FRT monolayers. This change in permeability to anions such as chloride resulted in a channel selectivity that is much more similar to CFTR, which has an estimated Pci / PNa around 10-20. In contrast, when a bis-negatively charged derivative was made, AmB- NAcC, the ion selectivity was not changed significantly which is thought to be due to the already high cation selectivity of AmB. However, AmB-NAcC was more effective at

[0077] - 12 -

[0078] FH13055872.2 UIX-05125 restoring trklAtrk2A yeast growth which is dependent on K+movement with trklAtrk2A yeast cultures incubated with AmB-NAcC having a higher [K+] intracellular. This strategy of incorporating electrostatic charges to the AmB channel derivative is versatile and has an effect on the recovery in both cation- and anion-deficient model systems. This property of anion selectivity for AmB-AA channels allows them to permeabilize the apical membrane of CF airway epithelia to HCO f more efficiently with minimal cation flux disrupting the ion homeostasis. The therapeutic window of AmB was previously found to be narrow because a high [AmB] increases H+secretion by ATP 12a. thereby negating the alkalization produced by AmB. AmB-AA did not cause ATP 12a overactivity, so there was no additional acidification which led to the increased ASL pH recovery up to lOOpM. This basified apical surface liquid seen when AmB-AA is pretreated on cultured CF airway epithelia corresponds to a more acidic intracellular pH showing that there is a measurable effect as HCCF' is secreted from the cell. The improved ASL pH for AmB-AA was also tested in primary cultured airway epithelia from people with CF, a more clinically relevant cell line. The safety profile of AmB-AA, although similar to that of AmB in various cholesterol- containing cell lines, was significantly worse in cultured CF airway epithelia. With the intent to design a clinically translatable CF therapeutic, this toxicity mechanism was investigated to provide insight on developing a safer anion- selective MP. In previous studies, the toxicity of AmB was found to be a direct result of cholesterol extraction which prompted the prediction of a similar explanation for AmB-AA. Similarly, XPLOR-NIH simulated annealing and molecular dynamics showed various similarities between AmB and AmB-AA aggregates, which held void volumes allowing for the housing of sterols. Additionally, AmB- AA showed to have the characteristic shift in UV-Vis spectra in the presence of cholesterol, previously seen by AmB-cholesterol complexes. The complexation of AmB-AA with cholesterol showed to mitigate the toxicity of the MP in cultured CF airway epithelia, effectively confirmed its toxicity mechanism. Leveraging these findings alongside synthetic design principles known to reduce cholesterol binding, the safer anion- selective MP, C2’- epiAmB-AA was effectively prepared.

[0079] In summary, C2’epiAmB-AA represents the first of a next-generation of anion- selective MPs rationally designed to better address the loss of CFTR function. Given the infancy of this therapeutic approach, our findings may also provide evidence that similarities to the parent protein matter when designing or selecting other MPs towards addressing other genetic diseases.

[0080] - 13 -

[0081] FH13055872.2 UIX-05125

[0082] Cystic fibrosis (CF) is a progressive disease caused by mutations in the CFTR anion channel. The ion channel-forming small molecule natural product, AmB, can serve as a molecular prosthetic for CFTR to partially restore HCO f and Cl" secretion, airway surface liquid (ASL) pH, ASL hydration, and antibacterial properties in cultured CF airway epithelia, but only at low concentrations. Unlike CFTR, AmB -based ion channels favor cation permeability. It was hypothesized that this feature may cause AmB to lose its ability to increase ASL pH at high concentrations in cultured CF airway epithelia. Here it is shown that derivatives of AmB can be designed to have an improved relative permeability to anions, and that this can lead to more robust improvements in ASL pH in cultured CF airway epithelia which are maintained at both low and high concentrations.

[0083] The ion selectivity of biological protein channels is determined by factors such as pore diameter and electrostatic interactions, the latter often highlighted as a primary contributor. In the case of CFTR, there are two key aspects that influence the anion selectivity. The cytosolic entry through the CFTR pore is lined with many positively charged lysine and arginine residues such as R128, K190, R303, K370, K1041, and R1048. This creates an anion conducting pathway that stabilizes anions through electrostatic interactions. In addition, there is a narrow selectivity filter at the extracellular ends of transmembrane helices 1, 6, and 8 that contains residues G103, R334, F337, T338, and Y914 with the R334 residue being positively charged. These two features collectively influence the anion selectivity of this protein channel [permeability of chloride relative to sodium (Pci / PNa) ~10- 30],

[0084] Computational modeling predicts the Cl -Cl 3 polyol regions of multiple AmB molecules line an ion conducting pore. These models also position the negatively charged C41 carboxylate near the channel entrance with the positively charged C3’ amine somewhat further away (FIG. 1A). A previous series of studies based on functional group deletions and / or other modifications of AmB provide support for this general channel architecture. It was also found that many AmB derivatives can still form channels. It was thus reasoned that converting the negatively charged C41 -carboxylate group into a positively charged variant would yield a derivatives that improves relative permeability to anions. Specifically, a positively charged ethylene diamine appendage was added to the C41 position of AmB (FIG. 1A), thereby mimicking the positive charges found lining anion channels.

[0085] CFTR obtains its anion selectivity through positively charged amino acid residues that line the ion conduction pathways. A positively charged ethylene diamine moiety was

[0086] - 14 -

[0087] FH13055872.2 UIX-05125 added near the putative small molecule channel entrance. This resulted in AmB-AA and C2’epiAmB-AA forming small molecule ion channels with increased relative permeability to anions.

[0088] There are three potential advantages for these new derivatives over AmB as molecular prosthetics to treat CF. First, AmB-AA increases ASL pH more effectively than AmB in cultured CF airway epithelia. Second, AmB-AA increases ASL pH even at high concentrations where AmB fails. Third, C2’epiAmB-AA should not extract native cholesterol from membranes and thus it should avoid potential disruptions in the activities of many transmembrane protein ion channels that depend on cholesterol for their functions. C2’epiAmB-AA represents a next-generation molecular prosthetic for CFTR with improved relative permeability to anions compared to AmB. Therefore, AmB-AA may provide greater benefit to people with CF including those that cannot benefit from CFTR modulators. More broadly, these findings may have implications for designing optimized molecular prosthetics for other types of genetic diseases. Accordingly, in certain aspects, the present disclosure relates to methods of treating or preventing a disease or condition characterized by decreased expression or reduced function of an ion channel, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB-AA:

[0089] (C2'-epiAmB-AA).

[0090] In certain embodiments, the ion channel is an anion channel. In further embodiments, the ion channel is a chloride channel. In yet further embodiments, the ion channel is a CFTR channel. In still further embodiments, the ion channel is an HCO f channel. In certain embodiments, the disease or condition is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease (COPD), non-cystic fibrosis bronchiectasis (NCFB), hyperkalemic periodic paralysis, paramyotonia congenita, potassium aggravated myotonia, generalized epilepsy with febrile seizures plus (GEFS+), episodic

[0091] - 15 -

[0092] FH13055872.2 UIX-05125 ataxia, familial hemiplegic migraine, spinocerebellar ataxia type 13, long QT syndrome, Brugada syndrome, mucolipidosis type IV, and Dravet syndrome. In further embodiments, the disease or condition is selected from the group consisting of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis. In yet further embodiments, the disease or condition is cystic fibrosis.

[0093] In still further embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered systemically. In certain embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject. In further embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol. In yet further embodiments, the subject is a human. In still further embodiments, the human is less than 12 years old. In certain embodiments, the human is at least 12 years old or at least 6 years old.

[0094] In further aspects, the present disclosure relates to methods of increasing the pH of airway surface liquid, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB-AA:

[0095] (C2'-epiAmB-AA), thereby increasing the pH of airway surface liquid in the subject; wherein the administration is pulmonary.

[0096] In yet further aspects, the present disclosure relates to methods of increasing bicarbonate secretion into airway surface liquid, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB-AA:

[0097] - 16 -

[0098] FH13055872.2 UIX-05125

[0099] (C2'-epiAmB-AA), thereby increasing bicarbonate secretion into airway surface liquid in the subject; wherein the administration is pulmonary. In still further aspects, the present disclosure relates to methods of increasing expiratory volume in one second (FEV1), comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB-AA: (C2'-epiAmB-AA), thereby increasing the subject’s FEV1; wherein the administration is pulmonary.

[0100] In certain embodiments, the subject’s FEV1 is increased by about 3% to about 20%. In further embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject. In yet further embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol. In still further embodiments, the subject is a human less than 12 years old. In certain embodiments, the subject is a human at least 12 years old or at least 6 years old.

[0101] In certain aspects, the present disclosure relates to methods of treating a disease characterized by reduced function or decreased expression of an ion channel, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0102] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0103] - 17 -

[0104] FH13055872.2 UIX-05125

[0105] (C2'-epiAmB-AA); and

[0106] (b) an ion channel modulator.

[0107] In certain embodiments, the disease is cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease. In further embodiments, the disease is cystic fibrosis.

[0108] In further aspects, the present disclosure relates to methods of increasing transepithelial current of airway cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of: (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0109] (C2'-epiAmB-AA); and

[0110] (b) an ion channel modulator. In yet further aspects, the present disclosure relates to methods of increasing the pH of airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0111] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0112] - 18 -

[0113] FH13055872.2 UIX-05125

[0114] (C2'-epiAmB-AA); and

[0115] (b) an ion channel modulator.

[0116] In still further aspects, the present disclosure relates to methods of increasing bicarbonate secretion into airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0117] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0118] (C2'-epiAmB-AA); and

[0119] (b) an ion channel modulator.

[0120] In certain embodiments, the subject has cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease. In further embodiments, the subject has cystic fibrosis.

[0121] In certain aspects, the present disclosure relates to methods of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0122] (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0123] - 19 -

[0124] FH13055872.2 UIX-05125

[0125] (C2'-epiAmB-AA); and

[0126] (b) an ion channel modulator.

[0127] In certain embodiments, the ion channel modulator is a CFTR modulator. In further embodiments, the ion channel modulator is selected from the group consisting of elexacaftor, tezacaftor, ivacaftor, lumacaftor, and forskolin, and any combination thereof. In yet further embodiments, the subject has two mutations in the CF transmembrane conductance regulator (CFTR) anion channel, each copy of the CFTR gene contains one of the two mutations, and wherein the two mutations are each independently selected from the group consisting of:

[0128] - 20 -

[0129] FH13055872.2 UIX-05125

[0130] -21 -

[0131] FH13055872.2 UIX-05125

[0132] In still further embodiments, the cystic fibrosis is refractory to treatment with ivacaftor. In certain embodiments, the cystic fibrosis is refractory to treatment with the combination of elexacaftor, tezacaftor, and ivacaftor.

[0133] - 22 -

[0134] FH13055872.2 UIX-05125

[0135] In yet further embodiments, cholesterol is not co-administered with the C2'-epiAmB- AA.

[0136] In still further aspects, the present disclosure relates to methods of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

[0137] (C2'-epiAmB-AA).

[0138] In certain embodiments, the cystic fibrosis is refractory to treatment with ivacaftor. In further embodiments, the cystic fibrosis is refractory to treatment with the combination of elexacaftor, tezacaftor, and ivacaftor. In yet further embodiments, the ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject. In still further embodiments, the ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol. In certain embodiments, the subject is a human less than 12 years old. In further embodiments, the subject is a human at least 12 years old or at least 6 years old. In yet further embodiments, cholesterol is not coadministered with the ion channel prosthetic. In still further embodiments, the subject has two mutations in the CF transmembrane conductance regulator (CFTR) anion channel, each copy of the CFTR gene contains one of the two mutations, and wherein the two mutations are each independently selected from the group consisting of:

[0139] - 23 -

[0140] FH13055872.2 UIX-05125

[0141] -24-

[0142] FH13055872.2 UIX-05125

[0143] -25 -

[0144] FH13055872.2 UIX-05125

[0145] Active pharmaceutical ingredients (APIs) in accordance with the invention can be combined with other therapeutic agents. The API and other therapeutic agent or agents may be co-administered simultaneously or sequentially. When the other therapeutic agent or agents are administered simultaneously, they can be administered in the same or separate formulations, but they are administered substantially at the same time as the API. The other therapeutic agent or agents are administered sequentially with one another and with the API when the administration of the other therapeutic agent or agents is temporally separated from the administration of the API. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.

[0146] Examples of other therapeutic agents include other antifungal agents, including AmB, as well as other antibiotics, anti-viral agents, anti-inflammatory agents, immunosuppressive agents, and anti-cancer agents.

[0147] As stated above, an “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular API being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular API and / or other therapeutic agent or agents without necessitating undue experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to some medical judgment.

[0148] Multiple doses per day may be contemplated to achieve appropriate systemic levels of

[0149] - 26 -

[0150] FH13055872.2 UIX-05125 compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and “dosage” are used interchangeably herein.

[0151] Generally, daily intravenous doses of API, e.g., the compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, will be, for human subjects, similar to or less than usual daily intravenous doses of AmB. Similarly, daily other parenteral doses of API, e.g., the compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, will be, for human subjects, similar to or less than usual daily other parenteral doses of AmB. In one embodiment, intravenous administration of an API may typically be from 0.1 mg / kg / day to 20 mg / kg / day. In one embodiment, intravenous administration of API may typically be from 0.2 mg / kg / day to 10 mg / kg / day. In one embodiment, intravenous administration of an API may typically be from 0.5 mg / kg / day to 5 mg / kg / day. In one embodiment, intravenous administration of an API may typically be from 1 mg / kg / day to 10 mg / kg / day. Intravenous dosing thus may be similar to, or advantageously, may be less than maximal tolerated doses of AmB.

[0152] Generally, daily oral doses of API will be, for human subjects, about 0.01 milligrams / kg per day to 1000 milligrams / kg per day. It is expected that oral doses in the range of 0.5 to 50 milligrams / kg, in one or more administrations per day, will yield therapeutic results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from one order to several orders of magnitude lower dose per day. In the event the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

[0153] For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents (e.g., AmB). Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy

[0154] - 27 -

[0155] FH13055872.2 UIX-05125 based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

[0156] Formulations of the API are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0157] For use in therapy, an effective amount of the API can be administered to a subject by any mode that delivers the API to the desired location or surface. Administering the pharmaceutical composition of the API may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), pulmonary (e.g., instillation or inhalation), and topical.

[0158] For intravenous and other parenteral routes of administration, the API, e.g., the compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, generally may be formulated similarly to AmB. For example, the compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof can be formulated as a lyophilized preparation with desoxycholic acid, as a lyophilized preparation of liposome- intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a cholesteryl sulfate complex. Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.

[0159] For oral administration, the compounds (i.e., API and other therapeutic agent or agents) can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or poly vinylpyrrolidone (PVP). If desired, disintegrating agents

[0160] - 28 -

[0161] FH13055872.2 UIX-05125 may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers. Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts”, In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al., J Appl Biochem 4:185-9 (1982). Other polymers that could be used are poly-l,3-dioxolane and poly-1, 3,6- tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

[0162] For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the API or by release of the biologically active material beyond the stomach environment, such as in the intestine.

[0163] To ensure full gastric resistance a coating impermeable to at least pH 5.0 is desirable. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

[0164] A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make

[0165] - 29 -

[0166] FH13055872.2 UIX-05125 the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used. The therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

[0167] Colorants and flavoring agents may all be included. For example, the therapeutic (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

[0168] One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, oc-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast- Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0169] Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0170] Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

[0171] An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between

[0172] - 30 -

[0173] FH13055872.2 UIX-05125 the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

[0174] Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

[0175] To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfo succinate and dioctyl sodium sulfonate. Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride. Potential non-ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation or derivative either alone or as a mixture in different ratios.

[0176] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such micro spheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0177] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0178] For administration by inhalation, the API may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.

[0179] - 31 -

[0180] FH13055872.2 UIX-05125

[0181] Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the API and a suitable powder base such as lactose or starch.

[0182] Also contemplated herein is pulmonary delivery of the API. The API is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining, e.g., to the blood stream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565- 569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5): 143- 146 (1989) (endothelin-1); Hubbard et al., Armal Int Med. 3:206-212 (1989) (ex 1 -antitrypsin); Smith et al., 1989, J Clin Invest 84:1145- 1146 (a- 1 -proteinase); Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 (incorporated by reference), issued Sep. 19, 1995 to Wong et al.

[0183] Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. In some embodiments, a dry powder inhaler (DPI) is used for the pulmonary delivery of the compound represented by structural formula (I) or (II).

[0184] Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

[0185] All such devices require the use of formulations suitable for the dispensing of API. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and / or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified API may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

[0186] - 32 -

[0187] FH13055872.2 UIX-05125

[0188] Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise API dissolved in water at a concentration of about 0.1 to 25 mg of biologically active API per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for API stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the API caused by atomization of the solution in forming the aerosol.

[0189] Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the API suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including 1, 1,1,2- tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

[0190] Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing API and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 99% by weight of the formulation. The API should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (pm), most preferably 0.5 to 5 pm, for most effective delivery to the deep lung.

[0191] Nasal delivery of API is also contemplated. Nasal delivery allows the passage of an API to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextrin.

[0192] For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the API in solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the API. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

[0193] Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal

[0194] - 33 -

[0195] FH13055872.2 UIX-05125 inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

[0196] The APIs, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The APIs may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0197] Pharmaceutical formulations for parenteral administration include aqueous solutions of the API in water-soluble form. Additionally, suspensions of the API may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0198] Alternatively, the API may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0199] The API may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0200] In addition to the formulations described above, the API may also be formulated as a depot preparation. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0201] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical

[0202] - 34 -

[0203] FH13055872.2 UIX-05125 compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and / or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249:1527-33 (1990).

[0204] The API and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0205] Suitable buffering agents include: acetic acid and a salt (1-2% w / v); citric acid and a salt (1-3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

[0206] Pharmaceutical compositions of the invention contain an effective amount of an API and optionally another therapeutic agent or agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

[0207] The therapeutic agent(s), including specifically but not limited to the API, may be provided in particles. Particles as used herein means nanoparticles or microparticles (or in

[0208] - 35 -

[0209] FH13055872.2 UIX-05125 some instances larger particles) which can consist in whole or in part of the API or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the API in a solution or in a semi-solid state. The particles may be of virtually any shape.

[0210] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly (octadecyl acrylate).

[0211] The therapeutic agent(s) may be contained in controlled release systems. The term “controlled release” is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term “sustained release” (also referred to as “extended release”) is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term “delayed release” is used in its conventional sense to refer to a drug formulation in which there is a

[0212] - 36 -

[0213] FH13055872.2 UIX-05125 time delay between administration of the formulation and the release of the drug there from. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”

[0214] Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. “Long-term” release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above. Definitions

[0215] The terms “treat” and “treating” as used herein refer to performing an intervention that results in (a) preventing a condition or disease from occurring in a subject that may be at risk of developing or predisposed to having the condition or disease but has not yet been diagnosed as having it; (b) inhibiting a condition or disease, e.g., slowing or arresting its development; or (c) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease. In one embodiment the terms “treating” and “treat” refer to performing an intervention that results in (a) inhibiting a condition or disease, e.g., slowing or arresting its development; or (b) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease. A “subject” or “patient” as used herein refers to a living mammal. In various embodiments, a patient is a non-human mammal, including, without limitation, a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, cat, dog, pig, horse, cow, or non-human primate. In certain embodiments, a patient is a human.

[0216] “Effective amount” as used herein refers to any amount that is sufficient to achieve a desired biological effect.

[0217] “Therapeutically effective amount” as used herein refers to any amount that is sufficient to achieve a desired therapeutic effect, e.g., treating CF.

[0218] A “yeast or fungal infection” as used herein refers to an infection with a yeast or fungus as defined herein. As used herein, “administering” has its usual meaning and encompasses administering by any suitable route of administration, including, without limitation, intravenous, intramuscular, intraperitoneal, subcutaneous, direct injection (for example, into a tumor), mucosal, pulmonary (e.g., by inhalation or instillation (lavage)), oral, and topical.

[0219] - 37 -

[0220] FH13055872.2 UIX-05125

[0221] In one embodiment, the administration is systemically administering.

[0222] In one embodiment, the administration is locally administering.

[0223] As used herein, the phrase “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. A “therapeutically effective amount” is an amount that is sufficient to achieve a desired therapeutic effect, e.g., to treat a disease or condition characterized by decreased expression or reduced function of an ion channel.

[0224] "Active ingredient", "therapeutically active ingredient", "active agent", "drug" or "drug substance" as used herein means the active ingredient of a pharmaceutical, also known as an active pharmaceutical ingredient (API). "Amorphous" as used herein refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically, such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid-like properties occurs at a “glass transition”, typically defined as a second-order phase transition.

[0225] "Crystalline" as used herein refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterized by a phase change, typically a first-order phase transition ("melting point"). In the context of the present disclosure, a crystalline active ingredient means an active ingredient with crystallinity of greater than 75%. In certain embodiments, the crystallinity is suitably greater than 90%. In other embodiments, the crystallinity is greater than 95%. In other embodiments, the crystallinity is less than 10%, or less than 5%. "Drug Loading" as used herein refers to the percentage of active ingredient(s) on a mass basis in the total mass of the composition.

[0226] "Mass median diameter" or "MMD" or “X50” as used herein means the median diameter of a plurality of particles, typically in a polydisperse particle population, i.e., consisting of a range of particle sizes. The X50 values as reported herein are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany), unless the context indicates otherwise.

[0227] "Tapped densities" or ptaPPed as used herein were measured in a fashion similar to Method I, as described in USP <616> Bulk Density and Tapped Density of Powders. Tapped

[0228] - 38 -

[0229] FH13055872.2 UIX-05125 densities represent a closer approximation to particle density than poured bulk densities, with measured values that are approximately 20% less than the actual particle density.

[0230] "Mass median aerodynamic diameter" or "MMAD" as used herein refers to the median aerodynamic size of a plurality of particles, typically in a polydisperse population. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity, generally in air, as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle composition in terms of its settling behavior. The aerodynamic particle size distributions (APSD) and MMAD are determined herein by cascade impaction, using a NEXT GENERATION IMPACTOR™ (Copley Scientific). In general, if the particles are aerodynamically too large, fewer particles will reach specific regions of the lungs. If the particles are too small, a larger percentage of the particles may be exhaled. In contrast, darepresents the aerodynamic diameter of a single particle.

[0231] "Nominal Dose" or "ND" as used herein refers to the mass of drug loaded into a receptacle (e.g., capsule or blister) in a non-reservoir based dry powder inhaler. ND is also sometimes referred to as the metered dose.

[0232] "Emitted Dose" or "ED" as used herein refers to an indication of the delivery of dry powder from an inhaler device after an actuation or dispersion event from a powder unit. ED is defined as the ratio of the dose delivered by an inhaler device to the nominal or metered dose. The ED is an experimentally determined parameter and may be determined using an in vitro device set-up which mimics patient dosing. ED is also sometimes referred to as the delivered dose (DD).

[0233] "Fine particle fraction” (FPF) as used herein, refers to the percentage of active ingredient in the emitted dose with an aerodynamic size less than 5 pm. The aerodynamic particle size distributions (APSD) is determined herein by cascade impaction, using a Next Generation Impactor™.

[0234] "Solids Content" as used herein refers to the concentration of active ingredient(s) and excipients dissolved or dispersed in the liquid solution or dispersion to be spray-dried.

[0235] As used herein, an “airway of a subject” refers to any or all of the following pulmonary structures: trachea, bronchi, and bronchioles. The term “about” refers to variations in numerical values typically encountered by one of skill in the art of respirable compositions, including variations of plus or minus 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a numerical value described herein.

[0236] - 39 -

[0237] FH13055872.2 UIX-05125

[0238] Throughout this specification and in the claims that follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", should be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Unless otherwise stated, or clear from the context, numerical ranges include both the endpoints and any value between.

[0239] EXAMPLES

[0240] Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

[0241] General Materials and. Methods

[0242] Materials: Commercially available materials were purchased from Sigma-Aldrich Co., GoldBio, Fisher Scientific, and Enamine and used without further purifications unless noted otherwise. Triethylamine was freshly distilled under nitrogen from CaH2. Water was double distilled or obtained from a Millipore MilliQ water purification system.

[0243] Reactions: All manipulations of Amphotericin B were carried out under low light and stored under an anaerobic atmosphere at -20 °C. All reactions were monitored by RP- HPLC using an Agilent 1260 Infinity II series HPLC with 6530 LC / Q-TOF system equipped with an Ecplise XDB-C18 5 micron 4.6 x 150 mm column with UV detection at 406 nm.

[0244] Cell lines and Growth conditions: CuFi-1 and FRT cells (Welsh Laboratory, University of Iowa) were grown from cyostock on Cell culture Treated 75cm2flasks (Thermo Scientific BioLite). The flasks for CuFi-1 cells were previously coated with 3 mL 50 pg / mL human placental collagen type IV (Sigma- Aldrich) for a minimum of 18 hours at room temperatures, then rinsed twice with PBS, and dried prior to seeding. Cells were cultured with 12 mL of Bronchial Epithelial Cell growth Medium (BEGM) with BulletKit (Lonza CC- 3170) including eight SingleQuots of supplements (bovine pituitary extract (BPE), 2 mL; hydrocortisone, 0.5 mL; hEGF, 0.5 mL; adrenaline, 0.5 mL; transferrin, 0.5 mL; insulin, 0.5 mL; retinoic acid, 0.5 mL; triiodothyronine, 0.5 mL). The gentamycin-AmB supplement was discarded and was instead supplemented with 50 pg / mL penicillin-streptomycin (Corning Cellgro), 50 pg / mL gentamycin (Sigma-Aldrich G1397), and 2 pg / mL fluconazole (Sigma- Aldrich). Flasks for FRT cells were used without collagen pre-treatment. Cells were cultured

[0245] - 40 -

[0246] FH13055872.2 UIX-05125 with 12 mL of Ham’s F-12, IX (Modified) with L-glutamine media (Corning 10-080-CV) supplemented with 50 pg / mL penicillin-streptomycin (Corning Cellgro), 50 pg / mL gentamycin (Sigma- Aldrich G1397), and 2 pg / mL fluconazole (Sigma- Aldrich). Both cell lines were grown to 90% confluence at 37 °C in 5% CO2, changing the medium every two days, and then trypsinized with 4 mL 0.25% trypsin containing 1 mM EDTA (Gibco 25200- 056). Trypsin was inactivated with 10 mL HEPES -buffered saline solution (Lonza CC-5024) with 1% bovine calf serum. Cells were spun down in an Eppendorf Centrifuge 5430R at 1,500 r.p.m. for 5 mins and resuspended in BEGM medium for passaging or Ultroser G medium for seeding. Seeding was done through resuspension of cells in 1:1 DMEM:Ham’s F-12 supplemented with 2% v / v Ultroser G (Crescent Chemical). CuFi-1 cell lines were seeded at 1.5 x 104cell per cm2on 24 well Falcon plates with six companion membranes 0.4-pm transparent PET membranes (Coming 3470). Membranes were coated with collagen similar to the flask protocol above. The cells were matured at an air-liquid interface by aspirating the apical surface and continuing media change for 14 days to reach full differentiation. Upon maturation, the cells were never used beyond 1.5 months. FRT cell lines were seeded at 1.1 x 105cell per cm2in non-collagen treated membranes on 24 well Falcon plates with six companion membranes 0.4-pm Polycarbonate membranes (Coming 3413). The cells were matured for 7 days while maintaining 150 pL of Ultroserg G medium on the apical membrane fresh. Upon maturation, the cells were never used beyond 4 days.

[0247] Yeast Growth Conditions. Isogenic Saccharomyces cerevisiae BY4741(WT) and BYT12(trklAtrk2A) were maintained on 15mM KC1 YPAD agar plates at 4°C. Yeast were streaked 2-4 weeks from glycerol stocks. Overnight media was 100 mM KC1 Yeast Nitrogen Base (YNB) pH 5.8 prepared from 4 g of (NH4 SO4, 1.63 g of translucent K+ free YNB (ForMedium, CYN7505), 1.285 g of BSM (ForMedium, DBSM225), and 7.45g KC1 L'1. Media pH was adjusted to 5.8 with citric acid and ammonium hydroxide before autoclaving. Subsequently, 50mL of sterile 40% w / v dextrose was added per liter (dextrose solutions were filter- sterilized using a 0.22pm filter). The same formulation was followed for K+-limiting media (15mM KC1 final), with 1.11g of KC1 added per liter. Small-Molecule Rescue Assay of Potassium-Transporter-Deficient Yeast. All

[0248] Experiments were done under sterile conditions. Overnight cultures of three to five cultures were grown to saturation over 12-24h, 200XRPM, 30 °C in 100 mM KC1 YNB pH 5.8 media. Afterwards, 50mL of cell suspension was pelleted at lOOOx g, 23°C for 5 min. Supernatant

[0249] - 41 -

[0250] FH13055872.2 UIX-05125 was removed, and cells were resuspended in 50mL of sterile Milli-Q H2O. Cells were centrifuged and washed 2 more times. After removing the supernatant, cells were resuspended in 15 mM KC1 YNB, pH 5.8 and diluted with 15 mM KC1 YNB media to OD6OO=0.090-0.110. Fresh stock solutions of Amphotericin B and Amphotericin B derivatives were prepared in DMSO and concentrations were determined by UV-vis. UV- vis specifications in methanol AmB compounds include Z,max=406nm, a = I 64.000M'1cm'1. A 40-fold concentrated small molecule solution was prepared in DMSO for each treatment dose, and the small-molecule solution was delivered as a 1:40 v / v ratio for small molecule to cell suspension. Per tested concentration, lOOmL of cell suspension was treated with either DMSO control or small molecule in sterilized 250mL flasks. Cells were grown protected from light for 12h at 200XRPM, 30°C.

[0251] Quantification of Total Cellular K+Content via Inductively Coupled Plasma- Mass Spectroscopy. After 24 h, 1 mL aliquots of each sample were utilized for ODeoo values by UV-vis. Subsequently, samples were vacuum filtered through 0.8 pm membrane filters (EMD Millipore) and washed with 25 mL of ice-cold 20 mM MgCh (used ultrapure, molecular grade biology water). Yeast samples were resuspended in 5 mL of 20 mM MgCh and transferred into metal-free conical tubes, flash frozen, and lyophilized (>72 h). Next, under argon, dried samples were weighed and sealed in metal-free centrifuge tubes before inductively-coupled mass spectroscopy. Ussing Chamber Dose-Response and Ion Selectivity Studies - Procedure A: Two

[0252] NaCl solutions were prepared, one with a high [NaCl], and one with a low [NaCl]. The high [NaCl] solution contained 140mM of NaCl, 5 mM HEPES, 1.2 mM calcium gluconate, 1.1 mM magnesium gluconate, and 5 mM glucose. The low [NaCl] Ringer’s solution contained was the same except for having a 70mM NaCl concentration. Solutions were titrated to 7.40 at 37°C with NMDG and osmolarity was adjusted to 305-310 mOsm with mannitol using an Advanced Instruments Model 3250 Osmometer.

[0253] Mature FRT cells, prepared as mentioned above, were mounted in a Ussing chamber (Warner U2500) with 3M KC1 agar bridges connected to amplifiers recording open-circuit trans epithelial voltage (Vt) with intermittent current injections of ±0.5 nA. Membranes were mounted on the Ussing Chamber using the culture cup insert for Transwell adaptor, 6.5 mm (Warner U9924T-06). After completing dilution potential experiments on FRT monolayers, the monolayer was lysed with water and junction potentials induced by ion dilutions were assessed then subtracted from obtained dilution potentials.

[0254] - 42 -

[0255] FH13055872.2 UIX-05125

[0256] For dose-response conductance studies (FIG. 2D): The cells were treated to 5 mL Apical low [NaCl] solution and 5 mL Basolateral High [NaCl] prior to starting conductance recording and allowed to equilibrate prior to the addition of DMSO, AmB, AmB-NAcC, or AmB-AA at selected concentrations of 2.69, 5.37, 10.75, 21.5, 43, or 100 pM. Conductance readings for each compound and concentration were summarized in FIG. 2D by determining the difference in conductance post and prior compound addition.

[0257] For ion selectivity studies (FIG. IB- IF and FIG. 5D): The cells were treated to 5 mL bilateral High [NaCl] buffer. After equilibration, the apical solution was aspirated and replaced with 5 mL low [NaCl] buffer (FIG. 1B-1F and FIG. 5D step I). After equilibration of FRT paracellular ion flow, the cells were treated with either DMSO, AmB, AmB-NAcC, or AmB-AA at 43 pM and allowed to equilibrate (FIG. 1B-1F and FIG. 5D step II).

[0258] Ussing Chamber Dose-Response and Ion Selectivity Studies - Procedure B:

[0259] Matured FRT cells, prepared as mentioned above, then mounted in a dual-channel Ussing chamber (Warner U2500) using the culture cup insert for Transwell adaptor, 6.5 mm (Warner U9924T-06). The membraned was bilaterally washed with a high 5 mL of NaCl- modified Ringer’s solution (containing 140 mM NaCl, 5 mM HEPES, 1.2 mM Calcium Gluconate, 1.1 mM Magnesium Gluconate, and 5 mM glucose pH 7.4 and OSM ~ 310) and voltage zeroed in preparation for a current clamp experiment. The apical solution was aspirated and 5 mL of a low NaCl modified Ringer’s solution (similar to high NaCl solution only with reduced NaCl, to 70 mM) was flushed through. Conductance was tracked and baseline for 5- 10 minutes prior to 50 uL bilateral addition of stock solutions (1000X) of AmB, AmB-AA, or AmB-NAcC in DMSO.

[0260] A similar setup as described above was done and used for Ion selectivity studies with additional recordings to determine membrane potential (mV) effects based on compounds used. Bilateral 140 mM NaCl Ringer’s solution was used as a baseline prior to (I) fast apical solution aspiration and replacement with 70 mM NaCl solution to determine the paracellular ion movement background. (II) Baseline was restored with fast apical solution aspiration and replacement back to 140 mM NaCl Ringer’s solution. (Ill) Bilateral Addition of AmB, AmB- AA, or AmB-NAcC was done, and the baseline was allowed to zero before continuing. (IV) Another fast apical solution aspiration and replacement with 70 mM NaCl (with AmB, AmB-

[0261] AA, or AmB-NAcC) was performed to determine the ion-dependent flow. Sodium and Chloride Activity Coefficients: Determining the activity coefficients (aion) for sodium and

[0262] - 43 -

[0263] FH13055872.2 UIX-05125 chloride were calculated using the ionic strength of each solution and the extended Debye- Hiickel equation, which is applicable for physiological concentrations: Equation 1

[0264] Where y is the activity coefficient of the ion, z is the ionic charge, g is the ionic strength of the solution, and a is the effective diameter of the hydrated ion. The constants -0.522 and 3.31 were used because experiments were performed at 37°C. The ion activity was then calculated by the equation:

[0265] ^ion = 7 * [c]

[0266] Equation 2 Where a is the ion activity, y is the activity coefficient (a.Na =0.735872606 and aci =0.71537313), and [c] is the ion concentration (140 mM).

[0267] Permeability calculations: The FRT monolayer was modeled as an electrical circuit so that:

[0268] Equation 3

[0269] Where AVt is the total measured dilution potential in the presence of small molecule, GERT is the trans epithelial conductance of the untreated FRT monolayer, GFRT + GAHIB is the trans epithelial conductance of the FRT monolayer treated with an AmB-based small molecule, VFRT is the measured dilution potential of the FRT monolayer prior to applying the AmB channel, GAHIB is obtained from subtracting the untreated monolayer Gt(GFRT) from the AmB- treated monolayer (GFRT + GAmB). and VAmB is the calculated the contribution of the small ion AmB pathway to Vt. VAmB then can be used to calculate the relative permeability (Pci / PNa) of the AmB small molecule using the Goldman-Hodgkin-Katz equation63,64:

[0270] - 44 -

[0271] FH13055872.2 UIX-05125

[0272] Equation 4

[0273] Approximating the constants at 37°C simplifies to:

[0274] Equation 5

[0275] Where F is Faraday’s constant, R is the gas constant , T is temperature, A VAmB is the dilution potential for the contribution of the small ion AmB pathway to Vt, and Pcf / Na+ is the relative permeability Cl’ to Na+.

[0276] Data Analysis: Electrophysiological data was analyzed using a custom graphical user interface coded in MATLAB version R2018b (Mathworks). Equations were solved in MATLAB version R2018b (Mathworks) using custom code.

[0277] Airway Surface pH Studies: Determination of ASL pH rescue was performed as described in Muraglia, K. A. et al., Nature 2019, 567 (7748), 405-408.

[0278] Measurement of ASL pH in cell line and. primary cultures of airway epithelia

[0279] Small-diameter NuLi, CuFi and primary cultured epithelia were used for this experiment (0.33 cm2). The ratiometric pH indicator SNARF-conjugated dextran (Molecular Probes) was used to measure ASL pH. SNARF powder was suspended via sonication in perfluorocarbon- 72 (Sigma-Aldrich FC-72) and distributed onto the apical surface. ASL pH was measured 2 h later. SNARF was excited at 488 nm and emission was recorded at 580 nm and 640 nm using a Zeiss LSM 800 microscope at x40 water immersion for cell line cultures and a Zeiss LSM 510 microscope for primary cultures. To generate a standard curve for pH determination, SNARF was dissolved in colorless pH standards and fluorescence ratios were converted to pH.

[0280] In all experiments, ASL pH of compound-treated epithelia was measured and compared to the results from vehicle-treated epithelia.

[0281] For apical administration, cultured airway epithelia were incubated for 48 h at 37 °C before measurement of ASL pH.

[0282] - 45 -

[0283] FH13055872.2 UIX-05125

[0284] CuFi-l / CuFi-4 alamarBlue Toxicity Studies: All experiments were run less than two months after seeding. Fresh USG medium was added to the basolateral side before experimentation. To cultured CuFi-1 or CuFi-4 epithelial 2.5 pL of a solution of desired compound in DPBS (400 pM stock; for a final concentration of 50 pM) was added to the apical membrane. The cells were then incubated for 48 h at 37 °C in a 5% CO2 atmosphere. After the end of the treatment period 2.5 pL of alamarBlue (ThermoFisher) was added to the apical membrane. Cells treated with 20 pL of Triton X were used as a control for 100% cytotoxicity. Using a plate reader, the emission at 585 nm was tracked using an excitation wavelength of 555 nm. The percent viability was quantified by using the difference between compound viability, 100% viability (blank control), and 0% viability (Triton-X 100 control).

[0285] Liposomes preparation: A 7 mL vial was charged with 640 pL of POPC solution (25mg / mL of POPC in CHCh) (For sterol free liposomes). For 10% cholesterol loaded liposomes a 230 pL solution of cholesterol (4mg / mL of cholesterol in CHCh) was added. For 10% lanosterol loaded liposomes a 230 pL solution of cholesterol was added instead. NOTE: This ratio was modified accordingly for the different percentages of sterol used for the dose response. To have enough liposomes to run two replicates 3, 7 mL, vials were prepared. The solvent was removed with gentle stream of nitrogen. The films were stored under high vacuum for 8+ hours prior to use. The films were then resuspended in 1 mL of a 250 mM NaCl, 40 mM HEPES buffer (pH 7.5) solution and vortexed for 3 minutes to get multilamellar vesicles (MLVs). The suspension was pulled into a Hamilton (Reno, NV) 1 mL gastight syringe. This syringe was put in a mini-extruder where the lipid solution was passed through a 0.20 pm Millipore (Billeric, MA) polycarbonate filter 21 times to isolate LUVs. These steps were repeated with every prepared vial. Each vial corresponding to one type of liposome were combined after extraction. The combined liposome solutions, sterol-free, cholesterol-loaded, or lanosterol-loaded, were then purified via dialysis using 3,500 dialysis cassettes. The solutions were dialyzed three times against 600 mL of a 62.5 mM MgSCL, 40 mM HEPES buffer solution (pH 7.3). The first two dialysis were 2 hours long meanwhile the last one was performed overnight.

[0286] Liposomes quantification: 10 pL of the individual LUVs were added to 7 mL vials (in triplicate). 450 uL of H2SO4 (8.9 M) was added to each vial, including to 3 additional 7 mL vials with no sample (blank control). The vials were then added to a 225 °C heated aluminum block for 25 minutes (WITHOUT CAPS) and then moved to room temperature and allowed to cool for 5 minutes. After cooling 150 uL of 30% w / v aqueous hydrogen

[0287] - 46 -

[0288] FH13055872.2 UIX-05125 peroxide was added to every vial and returned to the 225 °C heated aluminum block for 30 minutes (WITHOUT CAPS). Samples were again moved to room temperature and allowed to cool for 5 minutes. Once cooled 3.9 mL of MilliQ water and 500 uL of 2.5% w / v ammonium molybdate solution was added along with 500 uL 10% w / v ascorbic acid. The vials were sealed with a PTFE-lined cap and vortex vigorously for 5 minutes before moving to a 100 °C heated aluminum block for 7 minutes. The vials were then moved to room temperature and allowed to cool for 15 minutes before 200 uL were taken, added into a 96- well plate and analysis by UV / Vis spectroscopy (820 nm absorbance) was done using a plate reader. The concentration of liposomes were quantified by generating a standard curve by adding 0 (blank), 20, 40, 60, 80, 100, 120, 140 uL of a 0.65 mM phosphorus standard solution (Sigma) to 7 mL vials (in triplicate) and repeated all previous steps. Using this standard curve the concentration of phosphorous was quantified for all liposomes, for 3 vials per liposomes batch the concentration was around 13-17 mM.

[0289] Chloride Efflux experiments: Desired liposomes were diluted to 1 mM using a 62.5 mM MgSO4, 40 mM HEPES buffer solution (pH 7.3). 4 mL of this solution was added to a 20 mL vial (with 40 uL of ISA) and stirred. A chloride ion selective electrode (Fischer) was calibrated using standard Fischer protocols and then added into the stirring mixture. Using the StarCom software, the baseline chloride ppm were recorded over a period of 2 minutes, followed by the addition of 40 uL of either DMSO of desired compound (0.2 mM compound stock solution in DMSO for a final concentration of 2 uM) and recording the ppm change for a period of 10 minutes. To finish the readings, 40 uL of 30% v / v Triton X-100 was added to the stirring mixture and the data was collected for an additional 3 minutes before stopping (total run time of 15 minutes). %Chloride efflux was quantified by baselining the data using the first 2 minutes of the run and quantifying the change in ppm using the post Triton X-100 readings as 100% chloride efflux.

[0290] System Preparation and Molecular Dynamics Simulations: All molecular simulations were performed using NAMD3 and subsequent visualization done with VMD. The system was prepared using the lowest-energy AmB-Erg lattice (FIG. 1A) from previously reported data. The structure for the original AmB-Erg latticed was used as a template for modeling the AmB-AA + Sterol lattices. To maintain the structure of the experimentally resolved sponge, the macrolide region of all AmB molecules were constrained using the fixedAtoms flag in NAMD3 and all sterol molecules left unconstrained. AmB parameters were obtained from CHARMM General Force Field and sterols parameters

[0291] - 47 -

[0292] FH13055872.2 UIX-05125 were obtained from CHARMM36 forcefield. After constructing each AmB-AA system, the sponge was solvated using the TIP3P water model and ionized at 150mM NaCl. An initial energy minimization was done for 10000 steps and a Ins NPT equilibration run allowing water molecules to relax and system size to converge. For equilibration and production runs, the Nose-Hoover constant pressure method was used to maintain constant pressure while temperature was maintained through langevin dynamics with a damping coefficient of 0.5 ps-1. The simulation was carried out using an isothermal-isobaric ensemble at 310 K with a pressure of 1.0 atm. A 12- A cut-off was used for nonbonded interactions with a smoothing function implemented farther than 10 A. The integration time step of 2 fs was used, and the bond distances of the hydrogen atoms were constrained using the SHAKE algorithm. For long-range electrostatic calculations, the particle mesh Ewald method was used, with a grid density of less than 1 A-3. Production runs were 500ns long with 3 replicates each.

[0293] Characterization Methods

[0294] Cholesterol Content. Cholesterol content was determined by RP-HPLC with detection at 210 nm. Samples were analyzed with an Agilent 1260 Infinity II HPLC system (Wilmington, DE, USA). Separation was achieved with Haisil Clipeus™ C18 column (5 pm) using an isocratic method (aceto nitrile / isopropanol, 1.0 v / v). Cholesterol was quantified using a single-point calibration using Cholesterol HP, Ph. Eur / USP-NF raw material (Carbogen Amcis, Beuvry-la-Foret, France). Primary Particle Size Distributions. Primary particle size distributions were determined via laser diffraction (Sympatec GmbH, Clausthal-Zellerfeld, Germany). The Sympatec H3296 unit was equipped with an R2 lens, an ASPIROS micro dosing unit, and a RODOS / M dry powder-dispersing unit. Approximately 2 mg to 5 mg powder was filled into an ASPIROS tube and fed at 5 mm / s into a RODOS operated with 4 bar dispersion pressure and 65 mbar vacuum. Powders were introduced at an optical concentration of approximately 1% to 5% and data were collected over a measurement duration up to 15 seconds. Particle size distributions were calculated by the instrument software using the Fraunhofer model. Reported values represent the mean of three independent measurements for each collector.

[0295] Tapped Density. Tapped densities (ptapped were determined using a cylindrical cavity of known volume (0.593 cm3). Powder was filled into this sample holder using a microspatula. The sample cell was then gently tapped on a countertop. As the sample volume decreased, more powder was added to the cell. The tapping and addition of powder steps were repeated until the cavity was filled, and the powder bed no longer consolidated with

[0296] - 48 -

[0297] FH13055872.2 UIX-05125 further tapping. The tapped density is defined as the mass of this tapped bed of powder divided by the volume of the cavity.

[0298] Example 1: Ion selectivity and conductance Ussing Chamber studies

[0299] Electrophysiological studies of AmB versus AmB-NAcC and AmB-AA using Fischer rat Thyroid (FRT) cells were conducted with bilateral addition of the tested compounds. Fischer rat thyroid (FRT) cells have been used to study CFTR and drug candidates via the Ussing chamber for decades. These cells form tightly packed, high polarize, and high transepithelial resistance monolayers that have minimal background noise which make them good electrophysiological models. Using this FRT model, electrophysiological studies of AmB, AmB-NAcC, and AmB-AA were performed to identify any change in conductance based on the modifications. In this assay, AmB demonstrated a dose response in conductance, sharply increasing about 2.5 mS / cm2at 2.6 pM to 11 mS / cm2at 10.8 pM and then retain that conductance up to 100 pM. Unlike AmB, AmB-NAcC showed a mild dose response with no notable signs of conductance at 2.6 pM that slowly increases to 4.6 mS / cm2at 100 pM . In a similar manner, the effect of AmB-AA was dose dependent with its conductance increasing about 0.3 mS / cm2at 2.6 pM up to 10.28 mS / cm2at 43 pM and retaining a similar conductance up to 100 pM. The difference in conductance between AmB-NAcC and AmB-AA despite their relatively similar structures was attributed to their sterol binding ability. AmB toxicity is a direct result of sterol binding which is key for channel formation. AmB-NAcC was less toxic, and AmB-AA was more toxic compared to AmB as later shown in yeast models.

[0300] Within a similar system used to test the compounds’ conductance, a new protocol to detect their effect on the membrane potential and discern quantitatively their chloride over sodium selectivity (Pci / PNa) was employed. Surprisingly, it was found that AmB and AmB- NAcC did not differ significantly in selectivity with average Pci / PNa values of -0.1178 and 0.4102, respectively. Both remained highly cation selective despite AmB-NAcC also having very low conductivity. In contrast, AmB-AA was found to have a significant change in selectivity compared to AmB, being very anion-selective, with an average Pci / PNa of 1.5.

[0301] In the past, trklAtrk2A yeast rescue assays have been essential at identifying and studying the properties of AmB as a MP. In the plasma membrane of yeast, Trkl and Trk2 potassium transporters are responsible for moving K+from a low concentration outside the cell to a much greater concentration inside the cell. The genetic deletion of these channels results in a lack of cell growth and decreased intracellular concentration of K+(abbreviated

[0302] - 49 -

[0303] FH13055872.2 UIX-05125

[0304] [K+]i) under standard media conditions. With the now determined high cation selectivity, AmB channels have been shown to restore the cell growth and partially recover [K+]i.

[0305] Despite AmB-NAcC demonstrating the same cation selectivity as AmB in Us sing chamber studies, it performed significantly better in its ability to rescue trklAtrk2A yeast growth. In particular, while comparing the max rescue of all three compounds, AmB-NAcC showed a higher significant change compared to AmB. Despite showing similar cation selectivity in Ussing Chamber studies, AmB-NAcC showed better performance in this phenotypic assay. Due to this assay’s potassium dependence, AmB-AA, which was determined to be anion selective, was worse at rescuing yeast compared to AmB. These differences in yeast growth rescue between AmB, AmB-NAcC, and AmB-AA were confirmed by measuring the [K+]i of each compound via ICP-MS. For AmB-NAcC, the concentration of 0.25 pM showed to have the highest yeast growth rescue that was comparable to wild type levels. However, the [K+]i values showed that concentrations of 0.10 pM and 0.25 pM transported the same amount of K+inside the cell. The sensitivity of this assay prevented high concentrations, at or above 0.10 pM, to be tested for AmB and AmB- AA since toxicity would result in large deviations in the results. At a lower concentration of 0.05 pM, AmB-AA demonstrated no significant rescue of yeast growth or [K+]i levels compared to AmB. This proves that AmB-AA struggles to restore trklAtrk2A yeast growth because its anion selectivity prevents significant K+flow needed for the yeast to grow. Example 2: AmB-AA increased ASL pH rescue in airway epithelium

[0306] Cystic fibrosis (CF) is a fatal genetic disorder stemming from over 2000 unique mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) anion selective channel. These mutations lead to the loss of bicarbonate secretion resulting in loss of host airway epithelial defenses, such as ASL pH. Partial recovery of bicarbonate secretion with AmB has been demonstrated to restore ASL pH to a functional capacity, serving as a standard model to test the effectiveness of MPs. Additionally, at high concentrations, AmB has shown to negate ASL pH rescue by increasing the expression of a biomarker for ion homeostasis, ATP12A. Tracking both ASL pH and ATP12A expression with the proposed derivatives demonstrated the importance of designing a MP for CF with increased anion selectivity. Unlike in the potassium-dependent assays, AmB-AA was more efficient over the cation- selective AmB and AmB-NAcC in anion-dependent assays. ASL-pH rescue is an assay used for demonstrating the effectiveness and limitations of AmB as a MP and therefore is useful for comparing the new derivatives. At concentrations lower than 25 pM AmB was

[0307] - 50 -

[0308] FH13055872.2 UIX-05125 capable of rescuing ASL pH to a significant level. AmB-NAcC also did rescue ASL pH, but unlike AmB it only did so at concentrations above 50 pM and as effectively as AmB. Additionally, its extended therapeutic window of efficacy was attributed to its lower conductance. Similar to how cholesterol complexation extended AmB’s dose-response through the reduction in conductance as an effect of bioavailability, lower conductance of AmB-NAcC expanded the concentration range of its efficacy. In contrast, AmB-AA addition resulted in a higher ASL pH rescue compared to AmB at concentrations of 2 uM to 25 uM likely due to the higher anion selectivity and much lower conductance of AmB-AA at this range. Despite its similar conductance to AmB at concentrations above 43 pM, the therapeutic range of AmB-AA was extended beyond AmB’ s 25 pM limit and it did not show any signs of dropping off even at concentrations as high as 100 pM. AmB-AA is likely capable of having a high conductance while preventing a loss in ASL pH rescue due to its high anion selectivity, or rather lower potassium selectivity.

[0309] As a direct read out of the ASL pH rescue, H14CO3‘ was used to quantify the levels of bicarbonate effluxed by both derivatives in human airway epithelial. AmB-NAcC did not increase the levels of bicarbonate any higher than AmB at 50 pM, but surprisingly they were found to be the same as the vehicle. Higher cation selectivity than AmB, or rather worse anion selectivity than AmB, could be driving this result but as previously seen with lower concentration of all three compounds (2 pM), conductance can impact efflux. Given that AmB-AA has a similar conductance as AmB at 50 pM, the rate of efflux was comparable, and AmB-AA demonstrated an increased level of bicarbonate secretion compared to AmB.

[0310] The increased proton secretion and therefore negated ASL pH basification seen with high concentrations AmB addition was reproduced and shown to be caused by the overexpression of the apical channel ATP12A, a proton-potassium antiporter. Linked to excessive efflux of potassium by AmB at high concentrations, this effect has been demonstrated to be muted when AmB is complexed with cholesterol. The result of such complexation reduced the conductance of AmB, preventing the fast potassium efflux required for ATP12A overexpression to occur. It is proposed that that in a similar manner AmB-AA can prevent a loss in ASL pH rescue due to its high anion selectivity, or rather increased CFTR-like lower potassium selectivity. Using qPCR sequencing of the airway epithelial used in the ASL pH assay, it was demonstrated that, unlike with AmB, no significant increase of ATP12A expression was seen by AmB-AA when incubated at 2 pM or 50 pM.

[0311] - 51 -

[0312] FH13055872.2 UIX-05125

[0313] Example 3. AmB-AA increases primary human airway epithelial defenses

[0314] AmB, AmB-NAcC, and AmB-AA were additionally tested in their ability to restore key epithelial defenses in primary human airway epithelial. A low concentration of 2 pM and high concentration of 50 pM were used for all compounds as representations of the compound’s therapeutic window. As seen in cultured epithelial, AmB-NAcC did not restore a significant level of ASL pH either at 2 pM compared to AmB but did show a change at 50 pM unlike AmB. This improvement at higher concentrations by AmB-NAcC was attributed to its low conductance at high concentrations. Similar to the cultured epithelial model, AmB- AA restored ASL pH by about 0.4 at both 2 pM and 50 pM. In cultured CF epithelial, ASL viscosity is notability increased. The viscosity has been previously shown to be reduced back to WT-levels by AmB. Unlike AmB, AmB-NAcC did not significantly decrease ASL viscosity at 2 pM or 50 pM. As expected, AmB-AA did reduce ASL viscosity back to WT-levels similar to AmB. Similarly, ASL height is affected in CF epithelial but restored to wild type by AmB. This effect is not reproduced by AmB- NAcC but is demonstrated by AmB-AA at 2 pM and 50 pM.

[0315] Reduced mucus clearance, ASL height, and antibacterial properties which lead to infections have all been linked to ASL acidification. Commonly, .S'. Aureus infections become problematic in CF patients since their acid secretion continuously combats any resistance by the cell or therapeutic option. The antibacterial properties restored by AmB were reproduced here, showing up to 50% restoration of antibacterial properties. Given its higher cation selectivity, AmB-NAcC showed a reduced response at both 2 uM and 50uM. All tested compounds showed no antibacterial properties against .S'. Aureus alone.

[0316] Example 4: Designing and Testing Ion Selectivity of AmB -Derivatives

[0317] The zwitterionic nature of AmB was synthetically modified by coupling the C41- COOH with ethylene diamine. This reaction served to block the negative charge of AmB and introduce another positive charge to the AmB scaffold, yielding the bis-positively charged derivative, AmB-AA. Similarly, the bis-negatively charged derivative, AmB-NAcC, was prepared by acylating the C3’ -amine using succinic anhydride to be used as a control. Given their net charges, AmB-NAcC was expected to be more cation selective and AmB-AA more anion selective than AmB.

[0318] Fischer rat thyroid (FRT) cells have been used to study CFTR and drug candidates via the Us sing chamber for decades. These cells form tightly packed, highly polarized, and high transepithelial resistance monolayers that have minimal background noise which make

[0319] - 52 -

[0320] FH13055872.2 UIX-05125 them good electrophysiological models. Using this FRT model, electrophysiological studies of AmB, AmB-NAcC, and AmB-AA were performed to detect the effect of the compound in membrane potential (online Methods) and calculate their permeability of sodium over chloride ions (Pci / PNa) and chloride over sodium ions (PNa / Pci). AmB was found to be highly cation- selective, with an average PNa / Pci of 39.8144. Similarly, AmB-NAcC, designed to also be cation selective demonstrated a PNa / Pci of 31.7027. Given the high cation selectivity of AmB, we believe this assay was not sensitive enough to discern between two highly cationselective molecules such as AmB and AmB-NAcC. In contrast, AmB-AA successfully inverted the ion-selective seen by AmB, generating anion- selective ion channels, as seen by the average Pci / PNa of 3.0024).

[0321] Example 5: The Effect of Ion Selectivity on Rescuing Physiology in Channelopathy Models

[0322] Having confirmed AmB-AA is anion- selective and AmB-NAcC retains AmB-like properties despite the synthetic modifications, their effects were tested on the previously established physiological models. Using yeast with the genetic deletion of Trkl and Trk2 K+transporters the effectiveness of AmB in a K+-dependent model was reproduced in a modified trklAtrk2A yeast growth rescue assay. AmB-NAcC demonstrated a similar ability to rescue yeast growth compared to AmB. As expected, the anion- selective AmB-AA MP showed to be substantially worse at rescuing trklAtrk2A yeast growth compared to AmB and AmB- NAcC. These results also tracked with a decreased capacity for the anion selective channels formed by AmB-AA to increase intracellular K+ of yeast cells. Similarly, using cultured cystic fibrosis (CF) airway epithelial homogeneous for the AF508 mutation (CuFi-1) the effectiveness of AmB to rescue ASL pH at low concentrations (2 pM) was reproduced. Unexpectedly, AmB-NAcC lost the ability to rescue ASL pH at 2 p M and at higher concentrations. Remarkably, AmB-AA resulted in a higher ASL pH rescue compared to AmB demonstrating additional evidence towards protein-likeness resulting in improved functional recovery.

[0323] Example 6: Efficacy of AmB-AA in Cystic Fibrosis Models

[0324] Encouraged by the increased efficacy of AmB-AA over AmB in the ASL pH study, the extent of functional recovery provided by the increased anion selectivity was next studied.

[0325] An ASL pH dose response of both compounds demonstrated that AmB-AA retained its high ASL pH recovery up to 100 pM, while, as previously reported, AmB did not rescue ASL pH

[0326] - 53 -

[0327] FH13055872.2 UIX-05125 at or above 50 |aM. TO further support and understand the improved basification of the ASL, CuFi-1 epithelial were treated with BCECF fluorescent dye which allowed for the quantification of the intracellular pH (pHint). As expected, CF epithelial treated with 2 pM AmB also showed a decrease in pHint, correlating with the increase of ASL pH. Additionally, CF epithelial pretreated with AmB-AA (2 p M) showed a larger reduction of pHint compared to AmB. These complementary improved results were attributed to the improved efflux of HCO f and Cl’ out of the cell provided by anion selective AmB-AA.

[0328] Missing or altered CFTR activity has been linked to various altered homeostatic mechanisms as a result of loss of anion secretion. Among them, ENaC hyperactivity has been shown to be a result of reduced extracellular anions. Therefore, by using an Ussing chamber to track ENaC dependent short circuit current (pA / cm2) changes as a response to amiloride inhibition, the effectiveness of these MPs to replace CFTR was studied. Mounting non-CF epithelial (NuLi) into the Ussing chamber resulted in a change of 5 pA / cm2, a rather small change compared to mounted CF-epithelial heterogeneous for the AF508 and G551D mutations (CuFi-4), which showed a change of 14 pA / cm2. Pretreatment of CuFi-4 cells with AmB, at 2 and 50 pM, showed no rescue of ion homeostasis, with non- significant changes of 13 and 15 pA / cm2respectively. The lack of change was attributed to the poor anion selectivity of AmB, demonstrating to be a poor replacement for CFTR as the extracellular anion counterbalance required for reduced ENaC activity was not reached by AmB alone. In contrast, CuFi-4 cells pretreated with AmB-AA, at 2 and 50 pM, showed to restore ion homeostasis back to NuLi levels, 5 and 7 pA / cm2respectively, providing further evidence of AmB-AA being a more CFTR-like MP.

[0329] The aforementioned ATP12a overexpression is another example of altered homeostatic mechanisms. However, this is one caused by high concentrations of AmB leading to the excess efflux of K+and therefore the reduced therapeutic range of ASL pH rescue previously reported and reproduced here. Cholesterol complexation prevented this limitation by reducing the conductance of AmB at high concentrations allowing for acceptable levels of K+efflux into the ASL which did not promote ATP12a overactivity. As AmB-AA did not show such limitation, it was first investigated if the improved effect was a result of reduced ion conductance. Using the FRT cells, the conductance of AmB and AmB- AA were compared up to 100 pM, although AmB-AA did have reduced conductance at concentrations below 50 pM, at or above 50 pM the conductance matched that of AmB. This conductance dose response study demonstrated that extended window of ASL pH rescue

[0330] - 54 -

[0331] FH13055872.2 UIX-05125 provided by AmB-AA was not caused by a similar effect as AmB-cholesterol complexation. Given this result, increased anion-selectivity seemed the most likely reason for this effect. This was confirmed by quantifying the concentration of K+via IPC-MS in the ASL of CuFi- 1 cell pre-treated with AmB-AA, which unlike previously shown with AmB, did not show increased levels of K+. Similarly, in non-CF primary human airway epithelial pretreated with AmB and AmB-AA at 50 pM, the levels of ouabain-sensitive ATP12a mediated H+flux was shown to be significantly lower for AmB-AA compared to AmB. CF primary human airway epithelial were then obtained from two donors representing mutations of AF508 / AF508. The cells were pretreated with AmB or AmB-AA at 2pM and 50 p M. RT-qPCR analysis of these cell showed no increased ATP12a expression for any concentration of AmB-AA unlike AmB which increased at 50 pM. These studies collectively showed that the increased anion selective AmB-AA results in a more effective MP compared to AmB for the treatment of CF.

[0332] Example 7: Toxicity of AmB-AA follows the mechanism shown by AmB

[0333] Despite AmB-AA outperforming AmB in CF models and a similar safety profile in various cholesterol containing cells, toxicity studies in cultured CuFi-1 and CuFi-4 airway epithelial pretreated with AmB and AmB-AA 48 hours in advance demonstrated increased toxicity of AmB-AA compared to AmB at 50 pM. As a dry powder formulation, AmB, has shown a promising safety profile in ongoing clinical trials in healthy and CF individuals, hence it was endeavored to synthetically reduce the toxicity of AmB-AA to that of or below AmB. Previously, the toxicity of AmB has been demonstrated to be linked to its ability to bind and extract cholesterol forming a sponge aggregate, therefore it was first aimed to confirm that this was also true for AmB-AA. Treating CuFi-1 and CuFi-4 airway epithelial with the AmB derivative, C2’epi-AmB, indicated that the cells were sensitive to cholesterol extraction, as C2’epiAmB showed no significant toxicity compared to AmB or AmB-AA. To further support the similarities in toxicity mechanism between AmB and AmB-AA, AmB- AA was complexed with cholesterol (AmB-AA:Chol), which has previously been shown to mitigate the toxicity of AmB. The pre-complexed AmB-AA demonstrated no toxicity compared to its non-complex counterpart in culture airway epithelial. The successful AmB- AA:Chol complexation was confirmed by UV-Vis spectroscopy which further supported the binding of cholesterol to AmB-AA. XPLOR-NIH simulated annealing and molecular dynamics yielded a high-resolution structure of AmB-AA which shows to retain the large void volumes that are present in AmB aggregates that are similar in size to sterols. These computational models of AmB-AA further supported this cholesterol dependent toxicity.

[0334] - 55 -

[0335] FH13055872.2 UIX-05125

[0336] Given these results, it was predicted that the toxicity of AmB-AA could be mitigated by reducing cholesterol binding through the synthetic epimerization of the C2’-0H. Despite showing no cholesterol binding C2’epiAmB has been shown to successfully form ion channels in the presence of ergosterol, but it is not known if channel formation in cholesterol containing cells is possible. Attempts to detect ion channel formation in assays which were limited to short incubation periods or requiring high conductive ion channels, such as the FRT conductance, FRT ion selective assay, or radioactive bicarbonate assay failed to show any evidence of Epi-AmB channel formation. However, providing a long incubation period such as the 48 hours required by the H14CO3‘ and ASL pH rescue assay demonstrated that Epi-AmB was able to successfully rescue ASL pH. It was deduced that C2’epiAmB must be able to form ion channels. As there is no consensus in the literature if AmB requires cholesterol binding to form ion channels, the possibility that C2’epiAmB may be forming ion channels without requiring cholesterol binding cannot be dismissed. However, the possibility that C2’epiAmB has some residual UV-Vis undetectable cholesterol binding allowing for slow channel formation over a period of 48 hours also cannot be dismissed.

[0337] Example 8: Epi- AmB-AA is a safer MP and as effective as AmB-AA

[0338] Amidation of the C2’epiAmB with ethylenediamine provided the bis-positively charged, C2’-0H epimerized MP, C2’epiAmB-AA. As expected, C2’epiAmB-AA showed no detectable cholesterol binding via UV-Vis studies which resulted in lower toxicity in cholesterol containing cells and most importantly in cultured airway epithelia compared to AmB-AA and AmB. Unlike Epi at high enough concentrations, such as 200 pM, C2’epiAmB-AA demonstrated quantifiable conductivity allowing for ion-selectivity to be tested and showing a Pci / PNa of 1.12 indicating to be more anion selective than AmB. This detectable conductance in a similar manner translated to showing C2’epiAmB-AA capable of moving bicarbonate in CuFi-4 epithelial via the H14CO3‘ assay at 50 pM. To conclusively determine if C2’epiAmB-AA retained the efficacy of AmB-AA, CuFi-1 cells were pretreated with 2, 20, 50 and 100 pM C2’epiAmB-AA and compared to AmB-AA, in a control dose of 50 pM. The ASL pH dose response with C2’epiAmB-AA showed that this new safer MP retained similar efficacy and extended therapeutic concentrations previously shown by AmB- AA. Remarkably, using the previously obtained CF primary human airway epithelial and pretreating them with 2 and 50pM AmB, AmB-AA, and C2’epiAmB-AA showed a significantly improved ASL pH of C2’epiAmB-AA compared to that of both AmB and AmB- AA.

[0339] - 56 -

[0340] FH13055872.2 UIX-05125

[0341] Example 9: AmB derivatives that favor anion flux

[0342] Towards the goal of identifying a molecular prosthetic that improves the relative permeability to anions, AmB was amidated with ethylenediamine to convert the negatively charged carboxylate at C41 into a positively charged amino ethyl appendage, yielding the bis-positively charged derivative, AmB-AA (FIG. 1A). As a control, a bis-negatively charged counterpart was also synthesized by acylating the C3’ amine with succinic anhydride yielding AmB-NAcC (FIG. 1A)

[0343] The relative permeability to Cl’ and Na+(Pci / PNa) for ion channels formed by AmB was estimated, and these two synthetic derivatives by measuring dilution potentials (FIGs. 1B-1F). Specifically, a transepithelial conductance (Gt) value was first obtained for an untreated Fischer rat thyroid (FRT) monolayer. Next, the apical [NaCl] was reduced to obtain a dilution potential for the monolayer. Then, the ion channel-forming small molecule was added to both sides of the monolayer to form conduits for ion flow and repeated the experimental protocol to obtain Gtand a dilution potential for monolayer treated with AmB derivative. With the Gtvalues and dilution potential values from this series of experiments, the Gtcontribution by the small molecule was calculated and the electrical relationship described by Equation 3 was used to calculate the dilution potential produced by the small molecule. Finally, Pci / PNa was calculated for each small molecule-based ion channel by substituting the small molecule dilution potentials and known [NaCl] into the Goldman- Hodgkin-Katz equation (Equation 4). With this approach, it was estimated that AmB-based ion channels have a Pci / PN of 0.06 ± 0.06 (FIGs. 1C, IF). The negative control, AmB-NAcC, showed a similar estimated Pci / PNa of 0.07 ± 0.06 (FIGs. ID, IF). In contrast, the bis- positively charged derivative AmB-AA yielded a small molecule channel with a Pci / PNa of 3.0 + 1.6 (FIGs. IE, IF). Example 10: Impacts of ion selectivity in channelopathy models

[0344] Having estimated that channels formed by AmB and AmB-NAcC favor cation permeability and those formed by AmB-AA have improved relative permeability to anions, their effects were tested in models of cation- and anion-channel deficiencies. In trklAtrk2A yeast that lack K+transporting Trkl and Trk2 proteins. AmB showed a maximum of 51.9% ± 4.85% growth relative to wild type (FIG. 1G). The AmB-NAcC ion channels were even more effective in restoring the maximum growth of trklAtrk2A yeast to 68.4% + 6.03% of WT (FIG. 1G and FIG. 6). In contrast, AmB-AA, which forms channels that have increased relative permeability to anions, was much less effective at recovering the growth of this cation

[0345] - 57 -

[0346] FH13055872.2 UIX-05125 channel-deficient yeast strain (maximum 35.2% ± 2.48% growth relative to WT) (FIG. 1G). AmB-AA also demonstrated a decreased capacity to increase intracellular K+in trklAtrk2A yeast (FIG. 6).

[0347] In cultured CF airway epithelial homozygous for the AF508 CFTR mutation (CuFi- 1), AmB increased ASL pH at low concentrations (2 pM) (FIG. 1H). The bis-anionic derivative AmB-NAcC failed to increase ASL pH in this assay (FIG. 1H and FIG. 6). However, the bis-cationic derivative, AmB-AA, caused a more substantial increase in ASL pH relative to AmB (FIG. 1H).

[0348] While AmB increases ASL pH in cultured CuFi- 1 epithelia at low concentrations, this activity is lost at concentrations at or above 20 pM (FIG. 2A). It was hypothesized that AmB may lose its capacity to increase ASL pH at high concentrations due to its preference for cation permeability. In contrast, the anion- selective channel forming variant AmB-AA retained its capacity to increase ASL pH across the full range of tested concentrations (up to 100 pM) (FIG. 2B). It was noted that the net conductance for AmB- and AmB-AA-based channels at high concentrations is similar (FIG. 2C). These results collectively support the conclusion that AmB-AA-based channels, likely by increasing relative permeability to anions, outperform AmB-based channels as molecular prosthetics for CFTR in cultured CF airway epithelia.

[0349] Example 11: Rationally Minimizing Toxicity An orally inhaled dry powder formulation containing AmB and cholesterol, ABCL

[0350] 003, was well-tolerated in clinical trials in healthy volunteers, and in people with CF on and off modulators. These results support the use of AmB as a meaningful benchmark for minimum targeted tolerability in cultured airway epithelia. Although AmB-AA shows similar tolerability to AmB in a variety of mammalian cells (FIG. 7), toxicity studies in cultured CuFi- 1 and CuFi-4 airway epithelial treated with AmB and AmB-AA demonstrated increased toxicity for the latter (FIGs. 4A, 4B, and 7D). It was thus sought to mitigate this increased toxicity while keeping the anion selectivity of AmB-AA.

[0351] The toxicity of AmB to human cells is linked to its ability to form an extramembraneous sterol sponge that binds and extracts cholesterol from mammalian lipid bilayers. It was first aimed to determine whether this sterol sponge mechanism was also primarily responsible for the toxicity of AmB-AA. Formation of an AmB-AA:Chol complex was confirmed by UV-Vis spectroscopy (FIG. 4A). Treating CuFi-1 and CuFi-4 airway epithelial with AmB-AA pre-complexed with cholesterol (AmB-AA:Chol) eliminated the

[0352] - 58 -

[0353] FH13055872.2 UIX-05125 toxicity to cultured airway epithelia (FIG. 4B and 4C). As expected, C2’epiAmB, a derivative known to not bind or extract cholesterol (FIG. 7F), also caused no toxicity in cultured airway epithelia cell lines and showed reduced toxicity in other mammalian cell lines compared to AmB (FIGs. 4A, 4B, and 7A-7D). Building on an extensive series of recently reported biophysical and structural studies with AmB, XPLOR-NIH simulated annealing and molecular dynamics yielded a high- resolution structure of the AmB-AA sponge which showed the presence of a large void volume capable of accommodating the presence of a C16-ethylamine chain (FIG. 5D). These computational models further supported the conclusion that AmB-AA similarly kills mammalian cells via a sterol sponge-mediated cholesterol binding-dependent mechanism.

[0354] Guided by our prior studies that epimerizing the C2’-0H of AmB mitigated the cholesterol binding and reduced the toxicity of AmB, it was tested whether the toxicity of AmB-AA could be reduced by adding the same modification at C2’. Notably, while C2’epiAmB has been shown to form ion channels in the presence of ergosterol, it was not clear if C2’epiAmB or its derivatives that likewise do not detectably bind cholesterol could form ion channels in cholesterol containing cells.

[0355] Ussing chamber studies with FRT-derived monolayers failed to produce a Gt response with C2’epiAmB. However, in 10% cholesterol containing liposomes, 2 pM C2’epiAmB effluxed chloride as evidenced by an ion- selective electrode (FIG. 4E) although at a much lower level than AmB. Natamycin (NAT), a non-channel forming polyene macrolide natural product, showed no evidence of chloride efflux in this assay. This suggested that C2’epiAmB was forming ion channels in cholesterol-containing liposomes despite having no detectable binding to cholesterol. This was next tested in sterol-free liposomes and it was found that both AmB and C2’epiAmB retained ion channel activity. The level of channel activity caused by AmB was significantly reduced relative to that seen in 10% cholesterol-containing liposomes. However, C2’epiAmB retained similar activity in the presence or absence of cholesterol (FIG. 8). By repeating these efflux studies in 2, 5, 10, and 20% cholesterol-loaded liposomes, a clear cholesterol-dependent increase in permeabilizing activity for AmB was shown while C2’epiAmB was more or less unaffected by changes in membrane cholesterol content (FIG. 8). These results suggested some capacity for such polyene macrolides to form ion channels in the absence of cholesterol binding.

[0356] It was recognized, however, that sterol free liposomes can have different biophysical properties than their cholesterol-containing counterparts. Thus, the same experiment was

[0357] - 59 -

[0358] FH13055872.2 UIX-05125 performed with 10% lanosterol containing liposomes which have been shown previously to have similar biophysical properties to ergosterol and cholesterol-containing liposomes. Lanosterol does not detectably bind to AmB or C2’epiAmB, yet both of these polyene macrolides caused similar efflux as seen for each of them in sterol free liposomes (FIG. 4F and FIG. 8). These results collectively support the conclusion that sterol binding is not required for ion channel formation by AmB and C2’ epi AmB.

[0359] CuFi-4 airway epithelia were next treated with ABCI-003 and C2’epiAmB at 2 pM and quantified secretion of H14CO3‘ (FIG. 4G). This confirmed that C2’epiAmB can transport H14CO3‘ to the ASL. We also treated cultured CuFi-1 airway epithelia with 2 pM C2’epiAmB and AmB. C2’epiAmB successfully restored ASL pH to similar levels to that of AmB (FIG. 4H). These studies show that C2’epiAmB, which cannot detectably bind cholesterol, is able to form ion channels in cultured CF airway epithelia.

[0360] Example 12: C2’epiAmB-AA is a well-tolerated, more anion permeable MP for CFTR

[0361] The aforementioned studies suggested that epimerizing the C2’ position of AmB- AA might mitigate its toxicity while preserving its increased relative permeability to anions. Accordingly, amidation of the C2’epiAmB with ethylenediamine provided the bis-positively charged hybrid, C2’epiAmB-AA (FIG. 5A). As predicted, C2’epiAmB-AA showed no detectable cholesterol binding via UV-Vis spectroscopy (FIG. 5B) and, compared to AmB- AA and AmB, reduced toxicity in cholesterol containing cells (FIG. 7) including cultured CF airway epithelia (FIG. 5C and FIG. 7). Unlike C2’epiAmB, C2’epiAmB-AA increased Gtin Ussing chamber studies. Dilution potential measurements in FRT monolayers revealed a higher Pci / PNa for C2’epiAmB-AA (1.12 ± 0.16) compared to that of AmB (0.16 ± 0.04). C2’epiAmB-AA increased bicarbonate secretion across CuFi-4 epithelia (FIG. 5F) resulting in an increase in ASL pH across all tested concentrations (FIG. 5G). Finally, primary airway epithelia cultured from people with CF were treated with 2 or 50 pM AmB, AmB-AA, or C2’epiAmB-AA. C2’epiAmB-AA caused a substantial increase in ASL pH (FIG. 5H).

[0362] INCORPORATION BY REFERENCE

[0363] All patents and published patent applications mentioned in the description above are incorporated by reference herein in their entirety.

[0364] - 60 -

[0365] FH13055872.2 UIX-05125

[0366] EQUIVALENTS

[0367] Having described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

[0368] - 61 -

[0369] FH13055872.2

Claims

UIX-05125CLAIMSWe claim:

1. A method of treating or preventing a disease or condition characterized by decreased expression or reduced function of an ion channel, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB- AA:(C2'-epiAmB-AA).

2. The method of claim 1, wherein the ion channel is an anion channel.

3. The method of claim 1 or 2, wherein the ion channel is a chloride channel.

4. The method of any one of claims 1-3, wherein the ion channel is a CFTR channel.

5. The method of any one of claims 1-2, wherein the ion channel is an HCO f channel.

6. The method of any one of claims 1-5, wherein the disease or condition is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease (COPD), non-cystic fibrosis bronchiectasis (NCFB), hyperkalemic periodic paralysis, paramyotonia congenita, potassium aggravated myotonia, generalized epilepsy with febrile seizures plus (GEFS+), episodic ataxia, familial hemiplegic migraine, spinocerebellar ataxia type 13, long QT syndrome, Brugada syndrome, mucolipidosis type IV, and Dravet syndrome.FH13055872.2UIX-051257. The method of claim 6, wherein the disease or condition is selected from the group consisting of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis.

8. The method of claim 7, wherein the disease or condition is cystic fibrosis.

9. The method of any one of claims 1-8, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered systemically.

10. The method of any one of claims 1-8, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject.

11. The method of claim 10, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol.

12. The method of any one of claims 1-11, wherein the subject is a human.

13. The method of claim 12, wherein the human is less than 12 years old.

14. The method of claim 12, wherein the human is at least 12 years old or at least 6 years old.

15. A method of increasing the pH of airway surface liquid, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'-epiAmB- AA:(C2'-epiAmB-AA),- 63 -FH13055872.2UIX-05125 thereby increasing the pH of airway surface liquid in the subject; wherein the administration is pulmonary.

16. A method of increasing bicarbonate secretion into airway surface liquid, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'- epiAmB-AA:(C2'-epiAmB-AA), thereby increasing bicarbonate secretion into airway surface liquid in the subject; wherein the administration is pulmonary.

17. A method of increasing expiratory volume in one second (FEV1), comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is C2'- epiAmB-AA:(C2'-epiAmB-AA), thereby increasing the subject’s FEV1; wherein the administration is pulmonary.

18. The method of claim 17, wherein the subject’s FEV1 is increased by about 3% to about 20%.- 64 -FH13055872.2UIX-0512519. The method of any one of claims 15-18, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject.

20. The method of claim 19, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol.

21. The method of any one of claims 15-20, wherein the subject is a human less than 12 years old.

22. The method of any one of claims 15-20, wherein the subject is a human at least 12 years old or at least 6 years old.

23. A method of treating a disease characterized by reduced function or decreased expression of an ion channel, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:(C2'-epiAmB-AA); and (b) an ion channel modulator.

24. The method of claim 23, wherein the disease is cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease.

25. The method of claim 24, wherein the disease is cystic fibrosis.- 65 -FH13055872.2UIX-0512526. A method of increasing transepithelial current of airway cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:(C2'-epiAmB-AA); and (b) an ion channel modulator.

27. A method of increasing the pH of airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:(C2'-epiAmB-AA); and (b) an ion channel modulator.

28. A method of increasing bicarbonate secretion into airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of: (a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:- 66 -FH13055872.2UIX-05125(C2'-epiAmB-AA); and (b) an ion channel modulator.

29. The method of any one of claims 26-28, wherein the subject has cystic fibrosis, non- cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease.

30. The method of claim 29, wherein the subject has cystic fibrosis.

31. A method of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:(C2'-epiAmB-AA); and (b) an ion channel modulator.

32. The method of any one of claims 23-31, wherein the ion channel modulator is a CFTR modulator.- 67 -FH13055872.2UIX-0512533. The method of any one of claims 23-32, wherein the ion channel modulator is selected from the group consisting of elexacaftor, tezacaftor, ivacaftor, lumacaftor, and forskolin, and any combination thereof.

34. The method of any one of claims 25, 30, or 31, wherein the subject has two mutations in the CF transmembrane conductance regulator (CFTR) anion channel, each copy of the CFTR gene contains one of the two mutations, and wherein the two mutations are each independently selected from the group consisting of:- 68 -FH13055872.2UIX-05125-69-FH13055872.2UIX-0512535. The method of claim 34, wherein the cystic fibrosis is refractory to treatment with ivacaftor.

36. The method of claim 34 or 35, wherein the cystic fibrosis is refractory to treatment with the combination of elexacaftor, tezacaftor, and ivacaftor.

37. The method of any one of claims 1-36, wherein cholesterol is not co-administered with the C2'-epiAmB-AA.- 70 -FH13055872.2UIX-0512538. A method of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of an ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof, wherein the ion channel prosthetic is C2'-epiAmB-AA:

39. The method of claim 38, wherein the cystic fibrosis is refractory to treatment with ivacaftor.

40. The method of claim 38 or 39, wherein the cystic fibrosis is refractory to treatment with the combination of elexacaftor, tezacaftor, and ivacaftor.

41. The method of any one of claims 38-40, wherein the ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject.

42. The method of claim 41, wherein the ion channel prosthetic or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol.

43. The method of any one of claims 38-42, wherein the subject is a human less than 12 years old.

44. The method of any one of claims 38-42, wherein the subject is a human at least 12 years old or at least 6 years old.

45. The method of any one of claims 38-44, wherein cholesterol is not co-administered with the ion channel prosthetic.- 71 -FH13055872.2UIX-0512546. The method of claim 38, wherein the subject has two mutations in the CF transmembrane conductance regulator (CFTR) anion channel, each copy of the CFTR gene contains one of the two mutations, and wherein the two mutations are each independently selected from the group consisting of:- 72 -FH13055872.2UIX-05125-73-FH13055872.2UIX-05125-74-FH13055872.2

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