Restoring physiology with small molecule mimics of missing proteins

Abstract

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

Description

[0001] RESTORING PHYSIOLOGY WITH SMALL MOLECULE MIMICS OF MISSING PROTEINS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 527,684, filed July 19, 2023; and U.S. Provisional Application No. 63 / 539,430, filed September 20, 2023.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under R35GM118185 and P01 HL152960-02; Subaward: S02766-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] 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. Typically characterized by chronic and potentially fatal respiratory infections, cystic fibrosis currently has only symptomatic treatments, and patients treated with CFTR modulators have a median survival of only 66 years.

[0008] 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.

[0009] CFTR functions as a cAMP-activated ATP -gated anion channel, increasing the conductance for certain anions (e.g., CL) to flow down their electrochemical gradient. ATP- 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.

[0010] 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.

[0011] SUMMARY OF THE INVENTION

[0012] In certain embodiments the present disclosure relates to a method of treating 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 thereof, wherein the compound is represented by structural formula I or structural formula IE

[0013] In certain embodiments, the present disclosure relates to a pharmaceutical composition, comprising:

[0014] (i) a compound represented by structural formula I or structural formula IE

[0015] or a pharmaceutically acceptable salt or hydrate thereof;

[0016] (ii) calcium chloride (CaCh);

[0017] (iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and, optionally,

[0018] (iv) cholesterol (Choi).

[0019] In some embodiments, the present disclosure relates to 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 the pharmaceutical composition according to any of the embodiments disclosed herein.

[0020] In some embodiments, the present disclosure relates to a method of treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, wherein the administration is pulmonary.

[0021] In some embodiments, the present disclosure relates to a method of increasing the pH of airway surface liquid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, thereby increasing the pH of airway surface liquid in the subject; wherein the administration is pulmonary. In some embodiments, the present disclosure relates to a method of increasing bicarbonate secretion into airway surface liquid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, thereby increasing bicarbonate secretion into airway surface liquid in the subject; wherein the administration is pulmonary.

[0022] In some embodiments, the present disclosure relates to a method of increasing expiratory volume in one second (FEV1), comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, thereby increasing the subject’s FEV1; wherein the administration is pulmonary.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A depicts a schematic demostrating the robustness hypothesis diagram with first generation molecular prosthetic (MP) resulting in partial recovery of functional capacity and increased protein-like MP resulting in increased recovery of physiology.

[0025] Figure IB depicts a schematic demonstrating simplified amphotericin B (AmB), AmB-NAcC (a derivative of AmB comrising two carboxyl groups), and AmB-AA (a derivative of AmB comprising two amino groups) ion channel models guiding the design in their charged modifications with predicted ion selectivity.

[0026] Figure 2A depicts chemical structures of amphotericin B (AmB), the archetypical ion channel-forming small molecule, C3' N- Acetyl ethyl carboxylate (AmB-NAcC) and Cl 6- Aminoethyleneamide AmB (AmB-AA).

[0027] Figure 2B depicts a plot showing conductance of AmB, AmB-AA, and AmB-NAcC in Fischer rat thyroid (FRT) cells Ussing chamber assay performed with Apical 70 uM NaCl (pH=7) modified Ringer’s solution and basolateral 140 mM NaCl (pH=7) modified Ringer's solution treated bilaterally with AmB, AmB-AA, and AmB-NAcC.

[0028] Figure 2C 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 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). Figure 2D depicts a graph obtained in Ussing chamber current clamp protocol done with 43 pM addition of AmB-AA as described for Figure 2C resulting in a negative voltage change (anion selective).

[0029] Figure 2E depicts a graph obtained in Ussing chamber current clamp protocol done with 43 pM addition of AmB-NAcC as described for Figure 2C resulting in a positive change in voltage (cation selective).

[0030] Figure 2F depicts a plot demonstrating calculated Pci / PNa of AmB and AmB-AA based on graphed Ussing chamber voltage change normalized to the data’s paracellular movement (Step I).

[0031] Figure 2G depicts a plot demonstrating calculated Pci / PNa of AmB and AmB-NAcC based on graphed Ussing chamber voltage change normalized to the data’s paracellular movement (Step I).

[0032] Figure 3A depicts a plot demonstrating concentration-dependence of AmB-mediated rescue of cell growth observed upon addition of AmB to trkl Atrk2A yeast cells.

[0033] Figure 3B depicts a plot demonstrating concentration-dependence of AmB-AA- mediated rescue of cell growth observed upon addition of AmB-AA to trkl Atrk2A yeast cells.

[0034] Figure 3C depicts a plot demonstrating concentration-dependence of AmB-NAcC- mediated rescue of cell growth observed upon addition of AmB-NAcC to trklAtrk2A yeast cells.

[0035] Figure 3D is a graph depicting comparison of trklAtrk2A yeast cell growth restoration for AmB, AmB-AA, and AmB-NAcC, which was determined by comparing the maximum rescue concentration for each compound AmB at 0.063 pM, AmB-AA at 0.125 pM, and AmB-NAcC at 0.25 pM. **** P <0.0001, *** P0.001, ** P=0.0014.

[0036] Figure 3E is a graph depicting comparison of trklAtrk2A yeast cell growth restoration for AmB, AmB-AA, and AmB-NAcC at various concentrations. **** P <0.0001, *** P0.001, ** P=0.0014.

[0037] Figure 3F is a graph depicting total cellular K+, in wild type S. cerevisiae, untreated trklAtrk2A cell, AmB-treated trklAtrk2A cells, AmB-AA-treated trklAtrk2A cells, and AmB-NAcC-treated trklAtrk2A cells at various concentrations. **** P <0.0001, *** P0.001, ** P=0.0014.

[0038] Figure 4 is a plot depicting concentration-dependent change in ASL pH for AmB, AmB-AA, and AmB-NAcC. ***** P <0.0001, *** P0.001, ** P=0.0014. Figure 5A is a graph depicting OD600 rescue ratio for AmB-NAcC at 0.00 pM, 0.10 pM, and 0.25 pM by ICP-MS. **** P<0.0001, *** P<0.001, ** P<0.01.

[0039] Figure 5B is a graph depicting total cellular K+for AmB-NAcC at 0.00 pM, 0.10 pM, and 0.25 pM as determined by ICP-MS. *** P<0.001, ** P<0.01, * P<0.05.

[0040] Figure 6 displays a phase diagram of fully hydrated mixtures of dipalmitoylphosphatidylcholine and cholesterol.

[0041] Figure 7 depicts a simplified AmB ion channel model guiding the design of electrostatic modifications to shift its ion selectivity.

[0042] Figure 8A 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 AmB 43 pM bilateral.

[0043] Figure 8B 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).

[0044] Figure 8C 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).

[0045] Figure 8D depicts the ASL pH of CuFi-1 epithelial (dF508 / dF508) treated with 2 pM AmB, AmB-NAcC or AmB-AA. Data are represented as mean ± s.e.m. ns, not Significant. **** p <0.0001, *** P0.001 ** P=0.0014, * P=0.01

[0046] Figure 9A 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. Data are represented as mean ± s.e.m. ns, not Significant. **** P <0.0001, *** P0.001 ** P=0.0014, * P=0.01

[0047] Figure 9B depicts the intracellular pH in CuFi-1 airway epithelial of AmB versus AmB-AA at 2 pM. Data are represented as mean ± s.e.m. ns, not Significant. **** P <0.0001, *** P0.001 ** P=0.0014, * P=0.01

[0048] Figure 9C depicts ENaC hyperactivity in CuFi-4 airway epithelial for AmB and AmB-AA. Data are represented as mean ± s.e.m. ns, not Significant. **** P <0.0001, *** P0.001 ** P=0.0014, * P=0.01

[0049] Figure 9D depicts 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. Data are represented as mean ± s.e.m. ns, not Significant. **** P <0.0001, *** P<0.001 ** P=0.0014, * P=0.01 Figure 9E depicts ICP-MS results of ASL of CuFi-1 treated with AmB-AA at 50 pM with or without Ouabain. Data are represented as mean ± s.e.m. ns, not Significant. **** P <0.0001, *** P0.001 ** P=0.0014, * P=0.01

[0050] Figure 9F 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, *** P0.001 ** P=0.0014, * P=0.01

[0051] Figure 10A depicts the toxicity of AmB, AmB-AA, C2'-epiAmB, and AmB-AA complexed with cholesterol all at 50 pM in CuFi-1 epithelial cells.

[0052] Figure 10B depicts the toxicity of AmB, AmB-AA, C2'-epiAmB, and AmB-AA complexed with cholesterol all at 50 pM in CuFi-4 epithelial cells.

[0053] Figure 10C depicts the UV-Vis absorbance of AmB-AA with increasing ratios of cholesterol complexation.

[0054] Figure 10D depicts a model of AmB-AA bound to cholesterol.

[0055] Figure 10E depicts ASL pH rescue of AmB, AmB-AA, and C2'-epiAmB 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.

[0056] Figure HA depicts graphical representations of various yeast phenotypes and their reaction to treatment with AmB and AmB-AA.

[0057] Figure 11B depicts the change in ASL pH in cells treated with increasing concentrations of AmB-NAcC.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] 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.

[0060] 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 (Figure 1 A). 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 wild-type 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).

[0061] 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 (AmB-NAcC, having two carboxylates)) and Cl 6- Aminoethyleneamide AmB (AmB-AA, having two amines) (Figure 2A).

[0062] 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.

[0063] Through optimization of previously reported synthetic pathways, two key derivatives, AmB-NAcC and AmB-AA were prepared (Figure 2A). 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 (Figure IB). The impact of these charges on channel self-assembly, or bioavailability was also explored.

[0064] 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.

[0065] 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 (Figures IB and 2A). In some embodiments, the present disclosure relates to two low conductive and ion selective AmB derivatives, cation-selective AmB-NAcC and anion-selective derivative AmB-AA (Figures 2A-2E). 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 that AmB has demonstrated in the past and AmB-AA showing an improved physiological rescue compared to AmB. Additionally, the increased proteinlikeness 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.

[0066] 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-ani on-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 HCCL" 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.

[0067] The versatility demonstrated by AmB proved that an MP could effectively restore physiology despite lacking perfect protein-like function, such as ion selectivity (Figure 1A). 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 ATP 12a, 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 (Figure 1A). 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 lysine and arginine populate the pore lining of the anion selective ion channel resulting in a P\a / Pci of 0.03 (Figure 7). 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 (Figure 7).

[0068] 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 C35-OH or 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 (Figure 7). 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 ani on-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 (Figure 7).

[0069] 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 ani on- 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 along side the quantitative electrophysiological studies led to the development of AmB-AA, an anion-selective MP.

[0070] The amidation of the C41 position of AmB with ethylene yielded the next generation MP, AmB-AA. Remarkably, the anion selectivity, Pci / P\a, 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 restoring trklAtrk2A yeast growth which is dependent on K+movement with trklAtrk2A yeast cultures incubated with AmB-NAcC having a higher [K+]intraceiiuiar. 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.

[0071] This property of anion selectivity for AmB-AA channels allows them to permeabilize the apical membrane of CF airway epithelia to HCCK 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 ATP12a. 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 HCCK 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.

[0072] 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. Collectively, these results demonstrate that designed protein-likeness can lead to increased recovery of physiology. Although trkltrk2-like AmB-NAcC did not demonstrate conclusively increase in cation selectivity compared to AmB, its reduced conductance, another key property of biological channels, could be leveraged in designing a MP for treating a cation- selective channelopathy. The CFTR-like molecular prosthetic, AmB-AA, could present a future drug candidate or initial model for future SAR studies to improve CF treatment.

[0073] Accordingly, in some embodiments, the present disclosure relates to a method of treating 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 thereof, wherein the compound is represented by structural formula I or structural formula II:

[0074] In some embodiments, the compound is the compound represented by structural formula I or a pharmaceutically acceptable salt thereof.

[0075] In some embodiments, the ion channel with decreased expression or reduced function is an anion channel. For example, in certain embodiments, the ion channel is a chloride channel. Alternatively, the ion channel is a bicarbonate (HCO3 ) channel. In some embodiments, the ion channel is the CFTR channel. In some embodiments, the compound is the compound represented by structural formula I or a pharmaceutically acceptable salt thereof; and the ion channel is an anion channel. For example, in certain embodiments, the ion channel is a chloride channel. Alternatively, the ion channel is an HCCh' channel. In some embodiments, the ion channel is the CFTR channel.

[0076] In some embodiments, the compound is the compound represented by structural formula II or a pharmaceutically acceptable salt thereof.

[0077] In some embodiments, the ion channel with decreased expression or reduced function is a cation channel. For example, in certain embodiments, the ion channel is a K+channel.

[0078] In some embodiments, the compound is the compound represented by structural formula II or a pharmaceutically acceptable salt thereof; and the ion channel is a cation channel. For example, in certain embodiments, the ion channel is a K+channel.

[0079] In some 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 ataxia, familial hemiplegic migraine, spinocerebellar ataxia type 13, long QT syndrome, Brugada syndrome, mucolipidosis type IV, and Dravet syndrome. For example, in certain embodiments, the disease or condition is selected from the group consisting of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis. For example, in certain embodiments, the disease or condition is cystic fibrosis. In some embodiments, the disease or condition is cystic fibrosis; and the compound is represented by structural formula I.

[0080] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered systemically.

[0081] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered to an airway of the subject. For example, in certain embodiments, the compound or a pharmaceutically acceptable salt thereof is administered as an aerosol to an airway of the subject, such as the bronchial airway of the subject.

[0082] In some embodiments, the subject is a human.

[0083] In some embodiments, the human is an adult. In some embodiments, the human is a juvenile. In some embodiments, the human is less than 12 years old. Alternatively, the human is at least 12 years old. For example, in some embodiments the human is at least 6 years old. In certain embodiments, the present disclosure relates to a pharmaceutical composition, comprising:

[0084] (i) a compound represented by structural formula I or structural formula II: or a pharmaceutically acceptable salt or hydrate thereof;

[0085] (ii) calcium chloride (CaCh);

[0086] (iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and, optionally,

[0087] (iv) cholesterol (Choi).

[0088] In certain embodiments, the pharmaceutical composition comprises about 0.5% to about 25% w / w of a compound represented by structural formula I or structural formula II. In certain embodiments, the pharmaceutical composition comprises about 1% to about 22% w / w of a compound represented by structural formula I or structural formula II. For example, in certain embodiments, the pharmaceutical composition comprises about 0.5% to about 25%, about 1% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 12% to about 20%, about 14% to about 20%, about 16% to about 20%, about 18% to about 20%, about 1% to about 15%, about 3% to about 15%, about 5% to about 15%, about 7% to about 15%, about 9% to about 15%, about 11% to about 15%, about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 1% to about 8%, about 2% to about 8%, about 3% to about 8%, about 4% to about 8%, about 5% to about 8%, or about 6% to about 8% w / w of a compound represented by structural formula I or structural formula II. In certain embodiments, the pharmaceutical composition comprises about 2% to about 16% w / w a compound represented by structural formula I or structural formula II. In certain embodiments, the pharmaceutical composition comprises about 20% w / w, about 19% w / w, about 18% w / w, about 17% w / w, about 16% w / w, about 15% w / w, about 14% w / w, about 13% w / w, about 12% w / w, about 11% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w of a compound represented by structural formula I or structural formula II. In certain embodiments, the pharmaceutical composition comprises about 14% w / w a compound represented by structural formula I or structural formula II. In certain embodiments, the pharmaceutical composition comprises about 5% w / w of a compound represented by structural formula I or structural formula II.

[0089] In certain embodiments, the pharmaceutical composition comprises about 0.1% to about 8% w / w Choi. In certain embodiments, the pharmaceutical composition comprises about 0.5% to about 3% w / w Choi. In certain embodiments, the pharmaceutical composition comprises about 0.3% to about 6% w / w Choi. For example, in certain embodiments, the pharmaceutical composition comprises about 0.1% to about 8%, about 0.2% to about 8%, about 0.3% to about 8%, about 0.4% to about 8%, about 0.5% to about 8%, about 0.6% to about 8%, about 0.7% to about 8%, about 0.8% to about 8%, about 0.9% to about 8%, about 1% to about 8%, about 2% to about 8%, about 3% to about 8%, about 4% to about 8%, about 5% to about 8%, about 6% to about 8%, about 0.1% to about 6%, about 0.2% to about 6%, about 0.3% to about 6%, about 0.4% to about 6%, about 0.5% to about 6%, about 0.6% to about 6%, about 0.7% to about 6%, about 0.8% to about 6%, about 0.9% to about 6%, about 1% to about 6%, about 2% to about 6%, about 3% to about 6%, about 4% to about 6%, about 0.1% to about 3%, about 0.2% to about 3%, about 0.3% to about 3%, about 0.4% to about 3%, about 0.5% to about 0.6%, about 0.7% to about 3%, about 0.8% to about 3%, about 0.9% to about 3%, about 1% to about 3%, about 0.2% to about 4%, about 0.6% to about 4%, about 0.8% to about 4%, or about 1% to about 4% w / w of Choi. In certain embodiments, the pharmaceutical composition comprises about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, about 1% w / w, about 0.9% w / w, about 0.8% w / w, about 0.7% w / w, about 0.6% w / w, about 0.5% w / w, about 0.4% w / w, about 0.3% w / w, about 0.2% w / w, or about 0.1% w / w of Choi.

[0090] In certain embodiments, the pharmaceutical composition comprises about 1% to about 10% w / w CaCh. In certain embodiments, the pharmaceutical composition comprises about 4% to about 7% w / w CaCh. For example, in certain embodiments, the pharmaceutical composition comprises about 0.5% to about 15%, about 1% to about 15%, about 2% to about 15%, about 3% to about 15%, about 4% to about 15%, about 5% to about 15%, about 6% to about 15%, about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, about 11% to about 15%, about 12% to about 15%, about 13% to about 15%, about 0.5% to about 12%, about 1% to about 12%, about 2% to about 12%, about 4% to about 12%, about 6% to about 12%, about 8% to about 12%, about 10% to about 12%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10 %, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 0.1% to about 8%, about 0.2% to about 8%, about 0.3% to about 8%, about 0.4% to about 8%, about 0.5% to about 8%, about 0.7% to about 8%, about 0.8% to about 8%, about 0.9% to about 8%, about 1% to about 8%, about 2% to about 8%, about 3% to about 8%, about 4% to about 8%, about 5% to about 8%, about 6% to about 8%, about 0.2% to about 6%, about 0.4% to about 6%, about 0.6% to about 6%, about 0.8% to about 6%, about 1% to about 6%, about 2% to about 6%, about 3% to about 6%, about 4% to about 6%, or about 5% to about 6% w / w of CaCh. In certain embodiments, the pharmaceutical composition comprises about 12% w / w, about 11% w / w, 10% w / w, about 9% w / w, 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, about 1% w / w, about 0.9% w / w, about 0.8% w / w, about 0.7% w / w, about 0.6% w / w, about 0.5% w / w, about 0.4% w / w, about 0.3% w / w, about 0.2% w / w, or about 0.1% w / w of CaCh.

[0091] In certain embodiments, the pharmaceutical composition comprises about 60% to about 95% w / w phospholipids. In certain embodiments, the pharmaceutical composition comprises about 70% to about 90% w / w phospholipids. In certain embodiments, the pharmaceutical composition comprises about 75% to about 90% w / w phospholipids. For example, in certain embodiments, the phospholipids are present in the pharmaceutical composition in an amount of about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, or about 70% to about 75% w / w / . In certain embodiments, the phospholipids are present in the pharmaceutical composition in an amount of about 65% w / w, about 70% w / w, 75% w / w, about 80% w / w, 85% w / w, about 90% w / w, or about 95% w / w.

[0092] In certain embodiments, the weight ratio of Choi to phospholipids is about 0.001 : 1 to about 0.1:1. In certain embodiments, the weight ratio of Choi to phospholipids is about 0.005:1 to about 0.05:1. For example, in certain embodiments Choi and the phospholipids are present in a weight ratio of about 0.05:1. In some embodiments, Choi and the phospholipids are present in a weight ratio of about 0.001 : 1 to about 0.01 : 1, about 0.005: 1 to about 0.01 : 1, about0.01:l to about 0.05:1, aboutO.005:1 to about 0.05:1, or about 0.001:1 to about 0.005:1. In some embodiments, Choi and the phospholipids are present in a weight ratio of about 0.001:1, about 0.005:1, about 0.01:1, 0.05:1, or about 0.1:1.

[0093] In certain embodiments, HSPC and DSPG are present in a weight ratio of about 2:1 to about 19: 1. In certain embodiments, HSPC and DSPG are present in a weight ratio of about 7:1 to about 12:1. In certain embodiments, HSPC and DSPG are present in a weight ratio of about 1 : 1 to about 3 : 1. In some embodiments, HSPC and DSPG are present in a weight ratio of about 2.25:1. In some embodiments, HSPC and DSPG are present in a weight ratio of about 9:1. In some embodiments, HSPC and DSPG are present in a weight ratio of about 0.1:1 to about 20:1, about 0.2:1 to about 20:1, about 0.5:1 to about 20:1, about 0.7:1 to about 20:1, about 1:1 to about 20:1, about 2:1 to about 20:1, about 3:1 to about 20:1, about 4:1 to about 20:1, about 5:1 to about 20:1, about 6:1 to about 20:1, about 7:1 to about 20:1, about 8:1 to about 20:1, about 9:1 to about 20:1, about 10:1 to about 20:1, about 12:1 to about 20:1, about 14:1 to about 20:1, about 16:1 to about 20:1, about 0.1:1 to about 16:1, about 0.5:1 to about 16:1, about 1:1 to about 16:1, about 2:1 to about 16:1, about 4:1 to about 16:1, about 6:1 to about 16:1, about 8:1 to about 16:1, about 10:1 to about 16:1, about 12:1 to about 16:1, about 0.5:1 to about 12:1, about 1:1 to about 12:1, about 3:1 to about 12:1, about 5:1 to about 12:1, about 7:1 to about 12:1, about 9:1 to about 12:1, about 0.5:1 to about 9:1, about 1:1 to about 9:1, about 3:1 to about 9:1, about 5:1 to about 9:1, about 7:1 to about 9:1, about 0.5:1 toabout7:l, about 1:1 toabout7:l, about3:l toabout7:l, about5:l toabout7:l, about0.5:l to about 5:1, about 1 : 1 to about 5:1, about 3 : 1 to about 5:1, about 0.5 : 1 to about 3 : 1 , or about 1:1 to about 3:1. In some embodiments, HSPC and DSPG are present in a weight ratio of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1 to about 20:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, or about 20:1,

[0094] In certain embodiments, the molar ratio of Choi to the compound represented by structural formula I or structural formula II is about 0.05:1 to about 1.2:1. In certain embodiments, the molar ratio of Choi to the compound represented by structural formula I or structural formula II is about 0.4:1 to about 1.2:1. In certain embodiments, the molar ratio of Choi to the compound represented by structural formula I or structural formula II is about 0.05:1 to about 0.4:1. In certain embodiments, the molar ratio of Choi to the compound represented by structural formula I or structural formula II is about 0.4:1. In certain embodiments, the molar ratio of Choi to the compound represented by structural formula I or structural formula II is about 0.01:1 to about 1.2:1, about 0.02:1 to about 1.2:1, about 0.03:1 to about 1.2:1, about 0.05:1 to about 1.2:1, about 0.07:1 to about 1.2:1, about 0.09:1 to about 1.2:1, 0.1:1 to about 1.2:1, about 0.2:1 to about 1.2:1, about 0.3:1 to about 1.2:1, about 0.5:1 to about 1.2:1, about 0.7:1 to about 1.2:1, about 0.9:1 to about 1.2:1, about 1:1 to about 1.2:1, about 1.1:1 to about 1.2:1, about 0.01:1 to about 1:1, about 0.02: 1 to about 1:1, about 0.03 : 1 to about 1:1, about 0.05:1 to about 1:1, about 0.07:1 to about 1:1, about 0.09:1 to about 1:1, about 0.1:1 to about 1:1, about 0.2:1 to about 1:1, about 0.3:1 to about 1:1, about 0.5:1 to about 1:1, about 0.7:1 to about 1:1, about 0.9:1 to about 1:1, about 0.01:1 to about 0.8:1, about 0.02:1 to about 0.8:1, about 0.03:1 to about 0.8:1, about 0.05:1 to about 0.8:1, about 0.07:1 to about 0.8:1, about 0.1:1 to about 0.8:1, about 0.2:1 to about 0.8:1, about 0.3:1 to about 0.8:1, about 0.5:1 to about 0.8:1, about 0.7:1 to about 0.8:1, about 0.01:1 to about 0.6:1, about 0.02:1 to about 0.6:1, about 0.03:1 to about 0.6:1, about 0.05:1 to about 0.6:1, about 0.07:1 to about 0.6:1, about 0.08:1 to about 0.6:1, about 0.09:1 to about 0.6:1, about 0.1:1 to about 0.6:1, about 0.2:1 to about 0.6:1, about 0.3:1 to about 0.6:1, about 0.5:1 to about 0.6:1, about 0.01:1 to about 0.5:1, about 0.02:1 to about 0.5:1, about 0.03:1 to about 0.5:1, about 0.05:1 to about 0.5:1, about 0.07:1 to about 0.5:1, about 0.08:1 to about 0.5:1, about 0.09:1 to about 0.5:1, about 0.1:1 to about 0.5:1, about 0.2:1 to about 0.5:1, about 0.3:1 to about 0.5:1, about 0.1:1 to about 0.4:1, about 0.2:1 to about 0.4:1, about 0.3:1 to about 0.4:1, about 0.1:1 to about 0.3:1, about 0.2:1 to about 0.3:1, or about 0.2:1 to about 0.3:1. In certain embodiments, the molar ratio of Choi to the compound represented by structural formula I or structural formula II is about 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, or 1.2:1.

[0095] In certain embodiments, the molar ratio of phospholipids to CaCh is about 4:1 to about 2:1. In certain embodiments, the molar ratio of phospholipids to CaCh is about 2:1. In certain embodiments, the molar ratio of phospholipids to CaCh is about 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, or 1:2.

[0096] In certain embodiments, the compound represented by structural formula I or structural formula II has a crystallinity greater than about 75%. In certain embodiments, the compound represented by structural formula I or structural formula II has a crystallinity greater than about 85%. In certain embodiments, the compound represented by structural formula I or structural formula II has a crystallinity greater than about 95%. In certain embodiments, the compound represented by structural formula I or structural formula II has a crystallinity of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[0097] In some embodiments, the pharmaceutical composition comprises Choi. In some embodiments, the pharmaceutical composition does not comprise Choi.

[0098] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0099] (i) about 14.0% w / w of the compound represented by structural formula I or structural formula II;

[0100] (ii) about 2.3% w / w Choi;

[0101] (iii-a) about 70.3% w / wHSPC;

[0102] (iii-b) about 7.8% w / wDSPG; and

[0103] (iv) about 5.52% w / w CaCh.

[0104] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0105] (i) about 14.0% w / w of the compound represented by structural formula I or structural formula II; (ii) about 6.81% w / w Choi;

[0106] (iii-a) about 51.2% w / wHSPC;

[0107] (iii-b) about 22.8% w / DSPG; and

[0108] (iv) about 5.2% w / w CaCh.

[0109] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0110] (i) about 3.4% w / w of the compound represented by structural formula I or structural formula II;

[0111] (ii) about 0.57% w / w Choi;

[0112] (iii-a) about 80.72% w / wHSPC;

[0113] (iii-b) about 8.97% w / w DSPG; and

[0114] (iv) about 6.34% w / w calcium chloride (CaCh).

[0115] In certain embodiments, the pharmaceutical composition comprises:

[0116] (i) Choi and the compound represented by structural formula I or structural formula

[0117] II in a molar ratio about 0.2 to about 1.0;

[0118] (ii) Choi and the phospholipids in a weight ratio of less than about 0.05;

[0119] (iii) HSPC and DSPG in a weight ratio of about 2.0 to about 10; and

[0120] (iv) the phospholipids and CaCh in a molar ratio of about 2: 1.

[0121] In certain embodiments, the pharmaceutical composition comprises:

[0122] (i) Choi and the compound represented by structural formula I or structural formula

[0123] II in a molar ratio 0.4;

[0124] (ii) Choi and the phospholipids in a weight ratio of less than about 0.05;

[0125] (iii) HSPC and DSPG in a weight ratio of about 9:1; and

[0126] (iv) the phospholipids and CaCh in a molar ratio of about 2: 1.

[0127] In certain embodiments, the compound is represented by structural formula I.

[0128] In certain embodiments, the compound is represented by structural formula II.

[0129] In certain embodiments, the compound represented by structural formula I or structural formula II and Choi are not complexed; and the compound represented by structural formula I or structural formula II is not encapsulated in liposomes.

[0130] In certain embodiments, the compound represented by structural formula I or structural formula II is coated with a porous shell of phospholipids and Choi.

[0131] In certain embodiments, the pharmaceutical composition is formulated as a dry powder. In certain embodiments, the tapped density of the powder particles is about 0.03 to about 0.4 g / mL.

[0132] In certain embodiments, the tapped density of the powder particles is about 0.06 to about 0.2 g / mL.

[0133] In certain embodiments, the Carr’s index of the powder particles is about 20 to about 32.

[0134] In certain embodiments, the main transition temperature (Tm) of the shell is at least 70 °C, at least 80 °C, or at least 90 °C. In certain embodiments, the main transition temperature (Tm) of the shell is about 50 °C to about 100 °C, about 70 °C to about 100 °C, about 80 °C to about 100 °C, or about 85 °C to about 95 °C.

[0135] In certain embodiments, the water content of the powder is about 1.5 to about 6% w / w.

[0136] In certain embodiments, the composition is formulated for pulmonary administration or airway administration.

[0137] In certain embodiments, the composition is formulated for aerosol administration.

[0138] In certain embodiments, the composition is formulated for aerosol administration as a dry powder.

[0139] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell of phospholipids (PL) and Choi, wherein the crystallinity of the compound represented by structural formula I or structural formula II is at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0140] In certain embodiments, the mass median diameter, Xso, of the powder particles is about 0.5 pm to about 5.0 pm, about 1.0 pm to about 4.0 pm, or about 1.5 pm to about 3.5 pm.

[0141] In certain embodiments, the powder particles have an X90 of about 1.5 pm to about 10.0 pm, such as about 2.0 pm to about 8.0 pm.

[0142] In certain embodiments, the mass median aerodynamic diameter (MMAD) of the powder particles is about 1.5 pm to about 4.0 pm.

[0143] In certain embodiments, the mass median aerodynamic diameter (MMAD) of the powder particles is about 2.0 pm to about 3.5 pm.

[0144] In certain embodiments, a carrier-free dry powder composition of the compound represented by structural formula I or structural formula II, Choi, and PL is provided, wherein the compound represented by structural formula I or structural formula II is not encapsulated in the lipids, and the compound represented by structural formula I or structural formula II and lipids are phase separated in their own domains.

[0145] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell of PL and Choi, wherein the Chol / compound represented by structural formula I or structural formula II ratio is about 0.05 to about 1.2 mol / mol, such as about 0.2 to about 0.6 mol / mol.

[0146] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell of PL and Choi, wherein the Chol / PL is less than 0.10 w / w, or less than 0.05 w / w.

[0147] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell comprising PL and Choi. In some embodiments, the PL comprises hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine

[0148] (DPPC), distearoylphosphatidylglycerol (DSPG), or a combination thereof.

[0149] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell of PL and Choi, wherein the PL comprises a mixture of (1) HSPC or DSPC, and (2) DSPG in a w / w ratio of about 2.3 w / w (i.e., 7 / 3 w / w) and about 19.0 w / w (i.e., 95 / 5 w / w), such as about 8 w / w to about 18 w / w.

[0150] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the PL / Ca ratio is about 2.0 mol / mol to about 4.0 mol / mol, about 2.0 mol / mol to about 3.0 mol / mol, or about 2.0 mol / mol. The PL / Ca ratio should not decrease below about 2.0 mol / mol.

[0151] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the compound represented by structural formula I or structural formula II drug particles coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the percentage of the compound represented by structural formula I or structural formula II in the composition is less than 60% w / w, such as less than 30% or less than 20% w / w. In some embodiments, the drug loading is about 0.5% to about 25% w / w, and the nominal Chol / compound represented by structural formula I or structural formula II ratio is about 0.05 to about 1.2 mol / mol.

[0152] In certain embodiments, a dry powder composition of engineered particles is provided that includes spray-dried core-shell particles of fine crystalline particles of the compound represented by structural formula I or structural formula II (about 14.0% w / w) coated with a porous shell of PL and Choi, wherein the Choi / compound represented by structural formula I or structural formula II ratio is about 0.4 mol / mol, the Chol / PL ratio is about 0.03 w / w, the PL / Ca ratio is about 2 mol / mol, and the ratio of PC / PG in the PL is about 9.0 w / w.

[0153] In certain embodiments, a dry powder composition of engineered particles is provided that includes spray-dried core-shell particles of fine crystalline particles of the compound represented by structural formula I or structural formula II (about 3.4% w / w) coated with a porous shell of PL and Choi, where the Chol / compound represented by structural formula I or structural formula II ratio is about 0.4 mol / mol, the Chol / PL ratio is about 0.006 w / w, the PL / Ca ratio is about 2 mol / mol, and the ratio of PC / PG in the PL is about 9.0 w / w.

[0154] In certain embodiments, a dry powder composition of engineered particles is provided that includes spray-dried core-shell particles of fine crystalline particles of the compound represented by structural formula I or structural formula II (14.0% w / w) coated with a porous shell of PL and Choi, where the Chol / compound represented by structural formula I or structural formula II ratio is about 0.4 to about 1.2 mol / mol, the Chol / PL ratio is less than 0.05 w / w, the PL / Ca ratio is about 2 mol / mol, and the ratio of PC / PG in the PL is between about 2.3 and about 9.0 w / w.

[0155] In certain embodiments, the maximum Chol / compound represented by structural formula I or structural formula II ratio is about 1.2 mol / mol, but this high ratio may be acceptable only for lower drug loadings (e.g., no more than 10.0% w / w) where the lipids are maintained in a highly ordered so phase. Decreases in the Chol / compound represented by structural formula I or structural formula II to 0.4 mol / mol may allow higher drug loadings (e.g., no more than 22% w / w) within the so phase.

[0156] In certain embodiments, the compositions described herein include an HSPC / DSPG ratio of about 2.3 to about 9.0 w / w, and a PL / Ca2+ratio of about 2.0 mol / mol.

[0157] In certain embodiments, selection of the compositions described herein is driven by maintenance of the lipids in a single phase (i.e., the gel phase (so) with a Tmthat is more than 50°C above an accelerated storage temperature of 40°C). In some embodiments, selection of the compositions described herein is driven by maximal increases in ASL pH that are maintained across a wide range of concentrations of the compound represented by structural formula I or structural formula II. In certain embodiments, selection of the compositions described herein is driven by decreased hygroscopicity relative to compositions with HSPC / DSPG < 9.0. In some embodiments, selection of the compositions described herein is driven by increased manufacturing yield. In certain embodiments, selection of the compositions described herein is driven by improved powder flowability. In some embodiments, selection of the compositions described herein is driven by improved aerosol performance relative to powders containing the lo phase.

[0158] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the nominal dose is about 0.01 mg to about 50 mg, about 0.1 mg to about 10.0 mg, about 0.5 mg, about 1.0 mg, about 2.0 mg, about 4.0 mg, or about 6.0 mg.

[0159] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the mass median diameter (Xso) of the particles is about 1.0 to about 5.0 pm, such as about 1.5 to about 4.0 pm.

[0160] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the X90 of the particles is about 3 pm to about 10 pm, such as about 3.5 pm to about 7 pm.

[0161] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the tapped density of the particles is about 0.03 to about 0.40 g / mL, such as about 0.06 to about 0.20 g / mL.

[0162] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the water content in the powder is about 1.0% to about 10.0%, preferably about 2.0% to about 5.0%.

[0163] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the mass median aerodynamic diameter (MMAD) is about 1.0 pm to about 6.0 pm, such as about 2.0 pm to about 4.0 pm, when administered from a portable dry powder inhaler.

[0164] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of the drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the fine particle fraction less than 5 pm expressed as a percentage of the nominal dose is at least 30% w / w, at least 50%, or at least 60% w / w, when administered with a portable dry powder inhaler.

[0165] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the powder is produced by spray drying a liquid feedstock comprising fine crystals of the compound represented by structural formula I or structural formula II suspended in an oil-in-water emulsion stabilized by a monolayer of the mixture of lipids described herein.

[0166] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the lipids have a main transition temperature (Tm) of at least 80 °C, such as at least 90 °C.

[0167] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the powder is produced by spray drying a liquid feedstock at an outlet temperature that is less than the lowest Tmof the lipids. In some embodiments, the outlet temperature is at least 50 °C, at least 60 °C, or at least 70 °C. In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the powder is produced by spray drying a liquid feedstock at an outlet temperature that is less than the lowest Tmof the lipids. In some embodiments, the outlet temperature is at least 60 °C, or at least 70 °C.

[0168] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the powder is produced by spray drying a liquid feedstock at a total gas flow on a PSD-1 scale spray dryer of about 70 to about 100 scfm.

[0169] In certain embodiments, the dry powder composition of engineered particles comprising drug particles of the compound represented by structural formula I or structural formula II coated with a porous shell of PL, Choi, and calcium chloride (CaCh), is filled with a drum filler.

[0170] In certain embodiments, the powder fill mass is about 1.0 mg to about 40 mg in a size 3 or size 2 capsule, such as about 3 mg to about 20 mg, or about 10 mg to aboutl5 mg.

[0171] In certain embodiments, the powder fill mass has good precision (e.g., RSD < 3%) and accuracy for the target fill mass.

[0172] In some embodiments, the present disclosure relates to 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 the pharmaceutical composition according to any of the embodiments disclosed herein.

[0173] In some embodiments, the compound is the compound represented by structural formula I or a pharmaceutically acceptable salt thereof.

[0174] In some embodiments, the ion channel is an anion channel. For example, in certain embodiments, the ion channel is a chloride channel. Alternatively, the ion channel is a bicarbonate (HCO3 ) channel. In some embodiments, the ion channel is the CFTR channel.

[0175] In some embodiments, the compound is represented by structural formula I or a pharmaceutically acceptable salt thereof; and the ion channel is an anion channel. For example, in certain embodiments, the ion channel is a chloride channel. Alternatively, the ion channel is an HCCh' channel. In some embodiments, the ion channel is the CFTR channel. In some embodiments, the compound is the compound represented by structural formula II or a pharmaceutically acceptable salt thereof.

[0176] In some embodiments, the ion channel is a cation channel. For example, in certain embodiments, the ion channel is a K+channel.

[0177] In some embodiments, the compound is the compound represented by structural formula II or a pharmaceutically acceptable salt thereof; and the ion channel is a cation channel. For example, in certain embodiments, the ion channel is a K+channel.

[0178] In some embodiments, the present disclosure relates to a method of treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, wherein the administration is pulmonary.

[0179] In some 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 ataxia, familial hemiplegic migraine, spinocerebellar ataxia type 13, long QT syndrome, Brugada syndrome, mucolipidosis type IV, and Dravet syndrome. For example, in certain embodiments, the disease or disorder is selected from the group consisting of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis. For example, in certain embodiments, the disease or disorder is cystic fibrosis. In some embodiments, the disease or disorder is cystic fibrosis; and the compound is represented by structural formula I.

[0180] In some embodiments, the present disclosure relates to a method of increasing the pH of airway surface liquid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, thereby increasing the pH of airway surface liquid in the subject; wherein the administration is pulmonary.

[0181] In some embodiments, the present disclosure relates to a method of increasing bicarbonate secretion into airway surface liquid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, thereby increasing bicarbonate secretion into airway surface liquid in the subject; wherein the administration is pulmonary.

[0182] In some embodiments, the present disclosure relates to a method of increasing expiratory volume in one second (FEV1), comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any of the embodiments disclosed herein, thereby increasing the subject’s FEV1; wherein the administration is pulmonary.

[0183] In some embodiments, the subject’s FEV1 is increased by about 3% to about 20%.

[0184] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered systemically.

[0185] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is administered to an airway of the subject. For example, in certain embodiments, the compound or a pharmaceutically acceptable salt thereof is administered as an aerosol to an airway of the subject, such as the bronchial airway of the subject.

[0186] In some embodiments, the subject is a human.

[0187] In some embodiments, the human is an adult. In some embodiments, the human is a juvenile. In some embodiments, the human is less than 12 years old. Alternatively, the human is at least 12 years old. For example, in some embodiments the human is at least 6 years old.

[0188] Examples of diseases and conditions characterized by decreased expression or reduced function of an ion channel (sometimes referred to as channelopathies) include, without limitation, achromatopsia 2 (colorblindness), achromatopsia 3, arrhythmogenic right ventricular dysplasia type 2, autosomal dominant (Thomsen) myotonia, autosomal dominant long-QT syndrome (Romano-Ward syndrome), autosomal dominant nocturnal frontal lobe epilepsy, autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive (Becker) myotonia, autosomal recessive long-QT syndrome with deafness (Jervell-Lange- Nielsen syndrome), autosomal recessive retinitis pigmentosa, Bartter syndrome (renal salt loss, hypokalemic alkalosis), Bartter syndrome type III, Bartter syndrome type IV (associated with sensorineural deafness), BFNC (benign familial neonatal convulsions; epilepsy), BFNC (epilepsy) with myokymia, Brugada syndrome (idiopathic ventricular arrhythmia), calciumopathy, catecholaminergic polymorphic ventricular tachycardia, central core disease, childhood absence epilepsy, CMTX (X-linked Charcot-Marie-Tooth neuropathy), congenital bilateral aplasia of vas deferens, congenital hyperinsulinism, congenital insensitivity to pain, cystic fibrosis, Dent’s disease (X-linked proteinuria and kidney stones), DFNA2 (dominant hearing loss), DFNA3 (autosomal dominant hearing loss), DFNB1 (autosomal recessive hearing loss), epilepsy, episodic ataxia, episodic ataxia with myokymia, erythromelalgia, familial atrial fibrillation, familial hemiplegic migraine, focal segmental glomerulosclerosis, generalized epilepsy with febrile and afebrile seizures, generalized epilepsy with febrile seizures plus (GEFS+), hyperekplexia (startle disease), hyperkalemic periodic paralysis, hypokalemic periodic paralysis, hypokalemic sensory overstimulation, hypomagnesimia with secondary hypocalcemia, juvenile myoclonus epilepsy, Liddle syndrome, Liddle syndrome (dominant hypertension), long-QT syndrome, long-QT syndrome with dysmorphic features (Andersen syndrome), maculopathy, malignant hyperthermia, mucolipidosis type IV, myasthenia congenital, myotonia congenital, nonsyndromic deafness, osteopetrosis (recessive or dominant), paramyotonia congenital, paroxysmal extreme pain syndrome, periodic paralysis, persistent hyperinsulinemic hypoglycemia of infancy (PHHI), potassium- aggravated myotonia, progressive familial heart block type I, pseudohypoaldosteronism type 1 (PHA1), retinitis pigmentosa, Rolandic epilepsy, short-QT syndrome, spinocerebellar ataxia type 6, spinocerebellar ataxia type 13, Timothy syndrome, and X-linked congenital stationary night blindness.

[0189] In certain embodiments, the disease or condition characterized by decreased expression or reduced function of an ion channel is selected from the group consisting of Andersen-Tawil syndrome, autosomal dominant nocturnal frontal lobe epilepsy, autosomal dominant polycystic kidney, Bartter syndrome, benign familial neonatal seizures, Brugada syndrome, calciumopathy, channelome, childhood absence epilepsy, congenital hyperinsulinism, congenital insensitivity to pain, cystic fibrosis, Dent’s disease, episodic ataxia, erythromelalgia, familial atrial fibrillation, familial hemiplegic migraine, focal segmental glomerulosclerosis, generalized epilepsy with febrile seizures plus, hyperkalemic periodic paralysis, hypokalemic sensory overstimulation, hypomagnesemia with secondary hypocalcemia, juvenile myoclonic epilepsy, long QT syndrome, maculopathy, malignant hyperthermia, mucolipidosis type IV, myotonia congenita, nonsyndromic deafness, paramyotonia congenita, paroxysmal extreme pain disorder, periodic paralysis, potassium- aggravated myotonia, pseudohypoaldosteronism, retinitis pigmentosa, Rolandic epilepsy, Romano-Ward syndrome, short QT syndrome, spinocerebellar ataxia type 6, spinocerebellar ataxia type 13, template:channelopathy, Timothy syndrome, and X-linked congenital stationary night blindness.

[0190] In certain embodiments, the disease or condition characterized by decreased expression or reduced function of an ion channel is selected from the group consisting of cystic fibrosis, 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, and mucolipidosis type IV.

[0191] In certain embodiments, the disease or condition characterized by decreased expression or reduced function of an ion channel is cystic fibrosis.

[0192] In certain embodiments, the compound represented by structural formula (I) or (II) or a pharmaceutically acceptable salt thereof is administered systemically.

[0193] In certain embodiments, the compound represented by structural formula (I) or (II) or a pharmaceutically acceptable salt thereof is administered locally.

[0194] In certain embodiments, the compound represented by structural formula (I) or (II) or a pharmaceutically acceptable salt thereof is administered to an airway of the subject. As used herein, “airway” refers to any conducting or respiratory epithelium of the respiratory tract. The term “airway” thus includes upper airways and lower airways, including nasal passages, paranasal sinuses, pharynx, larynx, trachea, bronchi, bronchioles, alveolar ducts, alveolar sacs, and alveoli. In certain embodiments, the compound represented by structural formula (I) or (II) or a pharmaceutically acceptable salt thereof is administered to the bronchial airway of the subject.

[0195] In certain embodiments, the compound represented by structural formula (I) or (II) or a pharmaceutically acceptable salt thereof is administered as an aerosol to an airway of the subject.

[0196] 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.

[0197] 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.

[0198] 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. Multiple doses per day may be contemplated to achieve appropriate systemic levels of 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.

[0199] Generally, daily intravenous doses of API, e.g., the compound represented by structural formula (I) or (II) 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 (II) or a pharmaceutically acceptable salt thereof, will be, for human subjects, similar to or less than usual daily other parenteral doses of AmB.

[0200] 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.

[0201] 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.

[0202] 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 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.

[0203] 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.

[0204] 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.

[0205] For intravenous and other parenteral routes of administration, the API, e.g., the compound represented by structural formula (I) or (II) or a pharmaceutically acceptable salt thereof, generally may be formulated similarly to AmB. For example, the compound represented by structural formula (I) or (II) 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. 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 polyvinylpyrrolidone (PVP). If desired, disintegrating agents 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.

[0206] 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- 1,3 -di oxolane and poly-1, 3, 6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

[0207] 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.

[0208] 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), EudragitL30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

[0209] 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 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.

[0210] 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.

[0211] 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.

[0212] One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-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.

[0213] 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.

[0214] 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.

[0215] 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 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.

[0216] 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.

[0217] 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 sulfosuccinate 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.

[0218] 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 microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0219] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0220] 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. 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.

[0221] 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., Anna! Int Med 3:206-212 (1989) (al -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.

[0222] 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).

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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. 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.

[0229] 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 inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

[0230] 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.

[0231] 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.

[0232] Alternatively, the API may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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 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).

[0237] 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.

[0238] 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).

[0239] 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.

[0240] 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 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.

[0241] 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). 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 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.”

[0242] 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.

[0243] Lipid compositions of AmB-AA or AmB-NAcC

[0244] In certain embodiments, the present disclosure relates to pharmaceutical compositions described comprising highly crystalline AmB-AA or AmB-NAcC particles coated with a porous layer comprising two phospholipids (HSPC and DSPG), cholesterol, and calcium chloride. These materials may exhibit complex phase behavior that directly impacts the physicochemical and aerosol properties of the composition. This in turn may have an impact on the efficacy, safety, and tolerability of the formulated drug product.

[0245] Phospholipids are a class of lipids comprising a glycerol or sphingosine backbone, to which are attached one or more fatty acids and a phosphate group with an alcohol linked to it. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine. Phospholipids are amphiphilic molecules with the two fatty acid acyl chains being lipophilic while the modified phosphate group being hydrophilic. Phospholipids self-assemble to form membranous structures in water. They are ubiquitous in mammalian cell membranes. Hydrated phospholipid bilayers exhibit thermotropic phase behavior. With increases in temperature, the acyl chains undergo a phase transition from a ‘gel phase’ (so) where the acyl chains are present in a highly ordered, solid-like all-trans configuration, to a disordered ‘liquid crystalline phase’ (Zd), where increases in gauche conformer content in the acyl chains leads to disordered, liquid-like packing. The gel to liquid crystal phase transition temperature is sometimes referred to as the main transition temperature, or Tm.

[0246] The Tmof phospholipids depends critically on the length and degree of saturation of the acyl chains, and to a lesser extent, the nature of the headgroup. Table 1 provides a comparison of the Tmvalues of various hydrated phospholipids.

[0247] Table 1. Hydrated Gel to Liquid Crystal Main Transition Temperatures of Various

[0248] Phospholipids

[0249] Increases in the length of the acyl chains for phosphatidylcholines from 12 to 18 leads to increases in the hydrated Tmfrom -2 to 55 °C. Introduction of unsaturation into the acyl chains leads to dramatic reductions in Tmto values less than 0 °C. Changes in the nature of the headgroup also lead to differences in Tm, with the Tmin phosphatidylethanolamine (PE) > phosphatidylserine (PS) > phosphatidylglycerol (PG) ~ phosphatidylcholine (PC). The hydrated Tmvalues for HSPC and DSPG are 53.6 and 55 °C, respectively.

[0250] Cell membranes are inherently multicomponent systems comprising various species of lipids and proteins. The physical properties and biological function of cell membranes are tightly coupled to the arrangement and distribution of lipids within the phospholipid bilayer. Indeed, lipids may phase separate into different domains within a bilayer depending upon their composition. Choi is abundant in cell membranes, playing a critical role in maintaining the structural integrity and regulating membrane fluidity. In certain embodiments, the compositions described herein comprise Choi. Choi may contribute to the safety and ion channel activity of the formulated drug product. The addition of Choi to saturated PL may have a significant impact on PL phase behavior both in cell membranes and in spray-dried particles.

[0251] Figure 6 presents a phase diagram of hydrated DPPC-Chol mixtures (Ipsen JH, Karlstrom G, Mouritsen OG, et al. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim Biophys Acta. 1987; 905: 162-172).

[0252] As depicted in Figure 6, addition of Choi leads to the formation of a new phase referred to as an ordered liquid phase, (Zo). Incorporation of Choi into so below ~6 mol% results in a broadening of the Tmphase transition in DSC thermograms and increases in disorder in the acyl chain packing. In contrast, phospholipid acyl chains present in the disordered Id phase above Tmhave increased order with added Choi. At Choi concentrations between about 6 and 20 mol%, there is coexistence of two phases. Above Tm, the disordered liquid phase and ordered liquid phase coexist (Id — lo). Below Tm, the solid ordered phase coexists with the ordered liquid phase (so — lo). At high Choi concentrations (20-25 mol%), the cooperative Tmphase transition is lost, with only the lo phase present. For HSPC (longer acyl chains), a similar diagram is expected, with the temperatures on the ordinate in Figure 6 shifted upward by about 13 °C.

[0253] In contrast to the fully hydrated PL described above, compositions according to embodiments described herein comprise partially dehydrated PL. For the proposed use as ion channel prosthetics, the lipids in the compositions described herein may be spray dried to form an inhaleable dry powder with low water content.

[0254] In the absence of water, the spacing between phospholipid headgroups decreases, thereby increasing van der Waals interactions between the lipid acyl chains and increasing Tm. For example, the Tmvalue of DPPC increases about 41 °C (fully hydrated) to 105 °C on lyophilization (Ohtake S, Schebor C, Palacek SP, de Pablo JJ: Phase behavior of freeze- dried phospholipid-cholesterol mixtures stabilized with trehalose. Biochim Biophys Acta. 2005, 1713: 57-64).

[0255] For spray-dried DSPC powders, the Tmincreases from 55 °C (fully hydrated) to 71 °C at 75% RH, to 85 °C at 11% RH, to 102 °C at < 3% RH (Pikal -Cleland KA, Zhang J, Lechuga-Ballesteros D, Tarara TE, Weers JG: The impact of Ca2+binding on the packing structure of dry phospholipids. Presented at CRS Annual Meeting, Seoul, Korea, 2002). For current marketed spray-dried compositions comprising PL (e.g., TOBI® Podhaler™, Bevespi® Aerosphere, Breztri® Aerosphere, and Inbrija®), a goal has been to maintain the PL in the so phase during particle formation, in the collector during spraydrying, and on storage over the shelf-life of the product. As a result, these products utilize long-chain saturated phosphatidylcholines, such as DPPC and DSPC, as the principal shellforming excipient.

[0256] During spray drying of an aqueous feedstock, evaporative cooling of the atomized droplets maintains the droplet temperature just above room temperature, and far below the hydrated Tmof the PL (41 to 55 °C). This is true even when the inlet temperature on the spray dryer exceeds Tm. After particle formation, the outlet temperature and collector jacket temperature in the spray dryer are maintained below the Tmin the dehydrated state. Hence, the acyl chains are maintained in the so phase throughout the manufacturing process and on storage.

[0257] For liquid feedstocks comprising DPPC or DSPC, spray drying the so phase enables the formation of discrete spray-dried particles where the surface composition, surface morphology, size, density, porosity of the particles can all be effectively controlled. Moreover, long-chain saturated PLs are believed to be biocompatible in the lungs, due to their presence in endogenous lung surfactant, and natural, rapid clearance pathways from the lungs.

[0258] In contrast, spray drying aqueous feedstocks of unsaturated PL with Tmvalues < 0°C presents a significant challenge. Evaporation of water during spray drying may require an outlet temperature significantly higher than Tm. In this scenario, the PL acyl chains are present in the Id phase during particle formation and the drying process may therefore lead to large agglomerates of fused particles. This may result in low manufacturing yields and aerodynamic particle size distributions that are not suitable for inhalation as dry powder aerosols.

[0259] In certain embodiments, the compositions described herein comprise saturated phospholipids with longer acyl chains (e.g., 16:0 / 16:0 or 18:0 / 18:0). In some embodiments, the compositions described herein comprise DPPC, DSPC, HSPC, DSPG, or a combination thereof.

[0260] Calcium ions may bind to the phosphate group in phospholipids, displacing water molecules and condensing packing between PL molecules. For spray-dried DSPC powders, the addition of Ca ions may improve the environmental robustness (T, RH) of dehydrated powders, particularly at RH values that typically result in significant increases in capillary forces (e.g., 75% RH). In some embodiments, the ratio of PL / Ca is less than 4.0 mol / mol, less than 3.0 mol / mol, or about 2.0 mol / mol (US 8,709,484; US 7,442,388; incorporated by reference in their entirety). In some embodiments, it may be undesirable to decrease the PL / Ca ratio below the stoichiometric 2.0 mol / mol ratio, as excess calcium chloride below this ratio may increase the hygroscopicity of the powder.

[0261] Above Tmin either the Id or Zo phases, the spray-dried powders may become ‘sticky’ viscous liquids. These cohesive powders tend to negatively impact powder flowability and aerosol performance. This observation may be analogous to what occurs for highly disordered amorphous solids above their glass transition temperature, Tg. Spray-dried powders with phase separated so and lo domains tend to have poor powder properties. Hence, it may be desirable to maintain the lipids in the so phase.

[0262] The impact of added Choi on the phase behavior of PL in dry powder formulations is poorly understood. Differential scanning calorimetry thermograms of compositions according to embodiments described herein suggest that the lo phase is fully solubilized into the so phase at a Chol / PL ratio below about 0.05 w / w (< 9.4 mol% Choi). Hence, in the dehydrated state the so phase can solubilize a greater percentage of Choi than was observed for hydrated DPPC-Chol mixtures (Figure 6). Maintenance of the so phase results in Tmvalues that are > 90 °C.

[0263] In some embodiments, decreases in DSPG content in the AmB-AA and AmB-NacC- based powders from a HSPC / DSPG ratio of 2.3 mol / mol (~7 / 3 mol / mol) as utilized in AmBisome® and some lung surfactant preparations, to an HSPC / DSPG ratio of 9.0 w / w or higher may also improve physicochemical and aerosol properties of spray-dried AmB- derivative / Chol powders.

[0264] In some embodiments, increases in the HSPC / DSPG ratio decrease the hygroscopicity of compositions described herein at high relative humidity (RH). DSPG is an anionic phospholipid that may be provided as the sodium salt. Addition of calcium ions may displace the sodium ions, which then may combine with chloride ions from the calcium chloride to form hygroscopic sodium chloride domains in the spray-dried powder. At high RH the NaCl deliquesces (i.e., absorbs moisture from the air and dissolves in it). This may contribute to the stickiness of compositions according to embodiments described herein at high RH.

[0265] Other PLs may provide improvements in ASL pH and acceptable powder and aerosol properties. Their utility may be determined by using the characterization methods described in the Examples section. In certain embodiments, compositions described herein comprise sphingomyelin, phosphatidylserine (PS), or a combination thereof. In some embodiments, the compositions described herein comprise phospholipids with longer saturated acyl chains (e.g., n >16).

[0266] Drug Product Compositions

[0267] In some embodiments, the compositions described herein comprise the drug particles that are phase separated from the lipid excipients in their own crystalline domain. That is, the AmB-derivative (such as AmB-AA or AmB-NacC) and Choi are not complexed in amorphous nanoparticles as in the case of US 2019 / 0083517, US 2020 / 0352970. Nor is the AmB-derivative encapsulated within liposomes as is the case in AmBisome® (US 5,965,156). In some embodiments, the nominal ratio of Chol / AmB -derivative ratio is from 0.4 to 1.2 mol / mol. In some embodiments, due to enrichment of the drug substance in the spray-drying process, the actual range of Chol / AmB-derivative ratios in the final spray-dried powders is about 0.4 mol / mol.

[0268] Maintaining ion channel activity at lower Chol / AmB-derivative ratios in spray-dried powders comprising PL may minimize phase separation of a Chol-rich lo phase, due to its negative effects on powder properties.

[0269] In some embodiments, the nominal daily dose of AmB-derivative for the restoration of ASL pH in patients with CF is about 0.1 to about 10 mg, or about 0.2 mg to about 6 mg.

[0270] Physicochemical Properties

[0271] The physicochemical properties of the compositions described herein may depend on the feedstock composition (e.g., solids content, PFOB content), and drying conditions in the spray dryer. The ranges described below are based on the results for batches of compositions presented in the Examples.

[0272] In some embodiments, the small porous particles have relatively low densities, although not as low as compositions spray-dried from solution-based liquid feedstocks. In some embodiments, the poured bulk density is about 0.04 to about 0.12 g / cm3, and the tapped density is about 0.08 to about 0.20 g / cm3.

[0273] Carr’s Index provides a measure of the flow properties of the bulk powder (Table 2). Fine particles with a geometric size less than 5 pm, such as those according to embodiments described herein, are highly cohesive with very poor powder flow properties. Table 2. Relationship between Carr’s Index and Bulk Powder Flow Properties

[0274] Nonetheless, it is possible to fill fine, cohesive powders accurately and precisely with drum fillers (e.g., machines from Harro Hbfliger) to achieve relative standard deviations less than about 3% on fill masses as low as about 1.0 mg.

[0275] In some embodiments, the powders described herein have a Carr’s Index of about 20 to about 50, such as about 24 to about 30. Powders with lower Carr’s Index values may have noticeably improved powder flow properties, decreased variability in measurements of size and density, and an increased production yield. Compositions that have lo domains in the spray-dried powder have a Carr’s Index of about 40, which tends to lead to low yields and difficulty in handling (e.g., filling and fluidizing) the powders.

[0276] In certain embodiments, a relatively high Carr’s Index value observed (suggesting poor, cohesive flow, i.e., a Carr’s index value of 20-32) does not negatively impact the flow properties of importance for development of a portable dry powder inhaler.

[0277] Primary particle size distributions may be determined by laser diffraction (see, e.g., Characterization Methods, below). In certain embodiments, the powders described herein have an Xio of about 0.60 to about 1.2 pm, an Xso of about 1.5 to about 3.0 pm, and an X90 of about 3.0 to about 5.0 pm.

[0278] Water contents may be determined by coulometric Karl Fischer titrimetry. In certain embodiments, the water content of the powders described herein is about 1.5% to about 6% w / w.

[0279] In certain embodiments, the physicochemical properties of the powders can be modulated throughout the ranges described via variations in manufacturing process parameters. Aerosol Performance

[0280] In certain embodiments, the compositions described herein target the bronchial airways. In this regard, it tends to be beneficial to minimize extrathoracic drug deposition in the mouth and throat and in the alveoli, while maximizing deposition in the large and small airways within the lungs.

[0281] In the context of in vitro aerosol performance metrics, the compositions according to embodiments described herein have an emitted dose from a portable dry powder inhaler at a 4 kPa pressure drop and 4 L inhaled volume of at least 70%, at least 80%, at least 90% or at least 95%. In some embodiments, the compositions described herein are formulated to pass the delivered dose uniformity (DDU) regulatory requirements as delineated in the FDA Draft Guidance on: “Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Products- Quality Considerations (April, 2018) ”, with a standard deviation on mean ED values of 6% or less.

[0282] In certain embodiments, in a Next Generation Impactor (NGI) operated at a 4 kPa pressure drop with a 4 L inhaled volume, the mass median aerodynamic diameter (MMAD) of the compositions described herein is about 1.5 to about 4.0 pm, such as about 2.0 to about 3.5 pm.

[0283] The pattern of deposition within the NGI may be important. In accordance with the desired pattern of regional deposition detailed above, it is beneficial to minimize deposition in the USP throat and impactor stages 1 and 2 (referred to as the ‘coarse fraction’), and in the ‘extrafine fraction’ on stages 6 to filter, while maximizing deposition on stages 3, 4 and 5 (referred to as the ‘airways fraction’). In certain embodiments, deposition in the coarse fraction is less than 30%, less than 20%, or less than 10% w / w of the nominal dose. In some embodiments, deposition in the extrafine fraction is less than 16%, less than 12%, or less than 8% of the nominal dose. In certain embodiments, deposition in the airways fraction is at least 40%, at least 50%, at least 60%, or at least 70% of the nominal dose.

[0284] In certain embodiments, the fine particle fraction less than 5 pm (FPF<s jun) expressed as a percentage of the emitted dose is at least 40%, at least 50%, at least 70%, or at least 90% w / w.

[0285] In certain embodiments, the flow rate dependence of the total lung dose (TLD) in an Alberta Idealized Throat (AIT) model between pressure drops of 1.0 and 6.0 kPa is less than 40% or less than 15%. In certain embodiments, the target fill mass is about 5 to about 40 mg, such as about 10 to about 20 mg. In some embodiments, the compositions described herein are administered to a subject with a portable capsule-based dry powder inhaler (DPI). In certain embodiments, the DPI has a receptacle with a volume of about 0.30 cm3(size 3 capsule) to about 0.37 cm3(size 2 capsule).

[0286] In certain embodiments, the DPI is a variant of the RS01 DPI (Plastiape, Osnago, Italy). Variants of the RS01 differ in the size of capsule they accommodate (size 3 to size 0), and in their resistance to airflow (R = 0.06 to 0.16 cm H2O0 5L'1min). In some embodiments, the DPI is medium to high resistance (e.g., R is about 0.10 to about 0.30 cm H2O0 5L'1min). In certain embodiments, the DPI is a variant of the RS01 DPI with a resistance of about 0.14 cm H2O0 5L'1min. In certain embodiments, medium to high resistance DPIs limit extrathoracic deposition while also limiting the potential for post-inhalation cough.

[0287] In some embodiments, the compositions described herein are administered with a Handihaler (Boehringer Ingelheim), a RS00 (Plastiape) or Aerolizer® (Novartis) (US Patent 3,991,761; incorporated by reference), a Breezhaler® (US 2007 / 0295332; incorporated by reference) (Novartis) In certain embodiments, the compositions described herein are administered with a Turbospin (PH&T) or a variant thereof such as a T-326 (Podhaler™) (Novartis) as described in US Patents 8,069,851 and 7,559,325 (both incorporated by reference), or an AIR inhaler (Acorda Therapeutics).

[0288] In certain embodiments, the compositions described herein are administered with a single-use disposable inhaler. In some embodiments, the single-use disposable inhaler is a TwinCaps (Hovione), a Dosel® (Micro Engineering Solutions), a ICOone™ (Iconovo), or a Cyclops® (Pure IMS).

[0289] In some embodiments, the dry powder compositions of the present disclosure are administered intranasally to increase ASL pH in the nose and increase the nasal potential difference. Devices contemplated include Aptar’s Unidose system.

[0290] Manufacturing

[0291] A mixture of cholesterol and phospholipids may be prepared by dissolution in organic solvents. In certain embodiments, a lipid mixture comprising HSPC / DSPG / Chol in a 54 / 24 / 22 w / w / w ratio was prepared. Depending on the desired composition, the lipid mixture may be supplemented with additional PL and calcium chloride. In some embodiments, a method of preparing the compositions described herein comprises dispersing the lipid mixture in water to form multilamellar liposomes. In certain embodiments, this step involves addition of the lipids to hot water at a temperature greater than the hydrated Tmof the lipids. In some embodiments, the temperature of the water is 65 °C to 90 °C. In some embodiments, the dispersion is accomplished with a high shear mixer, such as an UltraTurrax® T-50.

[0292] In some embodiments, the method further comprises adding a fluorinated (FC) blowing agent, (e.g., perfluorooctyl bromide (PFOB, Perflubron), perfluorooctyl ethane (PFOE), or perfluorodecalin (PFD)) while mixing to form a coarse FC-in-water emulsion comprising micron-sized emulsion droplets. In certain embodiments, the coarse emulsion is homogenized under high pressure with a MicroFluidizer®, or a piston-gap homogenizer (e.g., Avestin Emulsiflex®) to form a submicron emulsion. In some embodiments, the pressure during homogenization is about 10,000 to about 20,000 psi, and homogenization is conducted either for a set period (dependent on batch size), or for a specified number of discrete passes. In some embodiments, a single discrete pass is conducted at the end of a set period process to ensure that all droplets are passed through the homogenizer at least once.

[0293] In some embodiments, the method further comprises adding the drug substance to the submicron emulsion under mixing. In certain embodiments, the addition is performed in a one pot process. In some embodiments, the resulting complex dispersion of emulsion droplets and suspended drug particles is mixed with a high shear mixer or passed through the homogenizer.

[0294] In some embodiments, the emulsion preparation and wet-milling of the drug are conducted in separate tanks in a two pot process. In certain embodiments, this allows for in- process sizing of the micronized drug particles by laser diffraction or dynamic light scattering. In some embodiments, the two pots are then combined into one, while mixing.

[0295] In certain embodiments, the emulsion preparation and wet-milling steps are conducted with concentrated emulsions / suspensions to limit the amount of liquid processed through the homogenizer. In some embodiments, water is added in the final step to achieve the target solids content and PFOB volume fraction in the final liquid feedstock.

[0296] In some embodiments, the feedstock is sprayed into a current of warm filtered air that evaporates the solvents and conveys the dried product to a cyclone separator or baghouse. In certain embodiments, the spent air is then exhausted with the evaporated solvent. In some embodiments, operating conditions of the spray-dryer such as the inlet and outlet temperature, feed rate, atomization pressure, flow rate of the drying air and nozzle configuration can be adjusted to produce the desired particle size, moisture content, and production yield for the resulting dry particles. In certain embodiments, the method further comprises setting a Niro PSD-1 scale spray dryer to have an inlet air temperature between 80 °C and 200 °C, an air outlet temperature between about 40 °C and 120 °C, a liquid feed rate between 30 g / min and 120 g / min, a total air flow between 50 scfm and 230 scfm, and an atomization airflow between 30 and 90 scfm. The solids content in the spray drying feedstock will typically be between 0.5% w / v and 10% w / v, and the blowing agent concentration will typically be between 3% and 30% v / v. In some embodiments, the desired settings depend, at least in part, on the scale and type of equipment used.

[0297] As the water in the atomized droplets is evaporated, the diameter of the aqueous atomized droplet recedes and the slowly diffusing emulsion droplets and the crystals of the compound of structural formula I or structural formula II are concentrated at the air / water interface with a hollow droplet core. When the higher boiling oil phase evaporates, it leaves behind pores in the spray-dried particles. The final particle morphology comprises porous lipid particles with embedded drug particles.

[0298] In certain embodiments, the outlet temperature and the collector jacket temperature (if utilized to control the RH in the collector), must be less than the lowest lipid main transition temperature. In certain embodiments in which the Chol / PL ratio is <0.05 w / w, the lowest lipid main transition temperature is more than 80 °C, providing for outlet and collector jacket temperatures of 70 °C or greater. Decreases in the Chol / PL ratio and increases in the HSPC / DSPG ratio may eliminate the Chol-rich phase, thereby providing for higher outlet temperatures, increases in production rate, and reductions in residual solvents in the spray- dried powder.

[0299] If the atomized droplets are dried too quickly, the benefits of the fine emulsion droplets may be lost. Hence, it may be beneficial to control the drying rate.

[0300] Definitions

[0301] 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.

[0302] 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.

[0303] “Effective amount” as used herein refers to any amount that is sufficient to achieve a desired biological effect.

[0304] “Therapeutically effective amount” as used herein refers to any amount that is sufficient to achieve a desired therapeutic effect, e.g., treating CF.

[0305] A “yeast or fungal infection” as used herein refers to an infection with a yeast or fungus as defined herein.

[0306] 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.

[0307] In one embodiment, the administration is systemically administering.

[0308] In one embodiment, the administration is locally administering.

[0309] 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.

[0310] "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).

[0311] "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. "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%.

[0312] "Drug Loading" as used herein refers to the percentage of active ingredient(s) on a mass basis in the total mass of the composition.

[0313] "Mass median diameter" or "MMD" or “Xso” 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 Xso values as reported herein are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany), unless the context indicates otherwise.

[0314] "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 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.

[0315] "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, da represents the aerodynamic diameter of a single particle.

[0316] "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. "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).

[0317] "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™.

[0318] "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.

[0319] As used herein, an “airway of a subject” refers to any or all of the following pulmonary structures: trachea, bronchi, and bronchioles.

[0320] 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.

[0321] 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.

[0322] Unless otherwise stated, or clear from the context, numerical ranges include both the endpoints and any value between.

[0323] EXAMPLES

[0324] 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.

[0325] General Materials and Methods

[0326] 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. Compound represented by structural formula (I) (AmB-AA) was synthesized as described in WO 2021 / 026520, the teachings of which are incorporated herein by reference in their entirety. Compound represented by structural formula (II) (AmB-NAcC) was synthesized as described in M. Cheron et al., Biochem. Pharmacol. 1988, 37, 827-836.

[0327] 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.

[0328] 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 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 (Coming 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.

[0329] 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 (Corning 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.

[0330] 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 waslOOmM KC1 Yeast Nitrogen Base (YNB) pH 5.8 prepared from 4 g of (NH4)2SO4, 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.

[0331] Small-Molecule Rescue Assay of Potassium-Transporter-Deficient Yeast. All Experiments were done under sterile conditions. Overnight cultures of three to five cultures were grown to saturation over 12-24h, 200XRPM, 30 °C in lOOmM KC1 YNB pH 5.8 media. Afterwards, 50mL of cell suspension was pelleted at 1000XG, 23°C for 5 min. Supernatant 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 ODeoo=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 kmax=406nm, a = 164,000M-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. 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.

[0332] Ussing Chamber Dose-Response and Ion Selectivity Studies: 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. 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- AA, or AmB-NAcC) was performed to determine the ion-dependent flow.

[0333] 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.

[0334] Measurement of ASL pH in cell line and primary cultures of airway epithelia 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 *40 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.

[0335] In all experiments, ASL pH of compound-treated epithelia was measured and compared to the results from vehicle-treated epithelia.

[0336] For apical administration, cultured airway epithelia were incubated for 48 h at 37 °C before measurement of ASL pH.

[0337] Characterization Methods

[0338] 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 (acetonitrile / 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).

[0339] 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.

[0340] 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 further tapping. The tapped density is defined as the mass of this tapped bed of powder divided by the volume of the cavity. Example 1: Ion selectivity and conductance Ussing Chamber studies

[0341] 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 (Figure 2B). 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 (Figure 2B). 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 tolOO pM (Figure 2B). 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 (Figures 3A-3C).

[0342] 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 (Figures 2C-2G).

[0343] 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 [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.

[0344] Despite AmB-NAcC demonstrating the same cation selectivity as AmB in Ussing chamber studies, it performed significantly better in its ability to rescue trklAtrk2A yeast growth (Figures 3A, 3C). In particular, while comparing the max rescue of all three compounds, AmB-NAcC showed a higher significant change compared to AmB (Figure 3D). 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 (Figures 3A, 3C).

[0345] 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 (Figure 3E). 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 (Figure 5B). 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.

[0346] Example 2: AmB-AA increased ASL pH rescue in airway epithelium

[0347] 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, ATP 12 A. 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 capable of rescuing ASL pH to a significant level (Figure 4). AmB-NAcC also did rescue ASL pH, but unlike AmB it only did so at concentrations above 50 pM and as effectively as AmB (Figure 4). Additionally, its extended therapeutic window of efficacy was attributed to its lower conductance. Similar to how cholesterol complexation extended AmB’s doseresponse 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. (Figure 4). 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.

[0348] 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.

[0349] 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 ATP 12 A, 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.

[0350] Example 3. AmB-AA increases primary human airway epithelial defenses

[0351] 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 (Figure 4), AmB-AA restored ASL pH by about 0.4 at both 2 pM and 50 pM.

[0352] 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.

[0353] 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.

[0354] Example 4. Manufacture of lipid-coated particles of amphotericin B derivatives Feedstock Preparation.

[0355] The lipids (HSPC, DSPG, cholesterol) and calcium chloride were first dispersed in hot water with a high-shear mixer (UltraTurrax T-10) to form multilamellar vesicles (MLVs). The aqueous phase must be above the gel-to-liquid-crystal phase transition temperature of the lipids (Tm) to facilitate MLV formation (T > 65 °C). The MLV dispersion was cooled (T < 30°C), and PFOB was added dropwise while mixing to form coarse PFOB-in-water emulsion droplets stabilized by a monolayer of the lipids. The emulsion droplets serve as pore formers to create a porous coating of lipids on the crystalline drug particles. The coarse emulsion was then homogenized continuously under high pressure (15-20 kpsi) using an Avestin Emulsiflex C5 to form nanoemulsion droplets (diameter ~ 200-500 nm). Next, the drug substance was added under high-shear mixing to the nanoemulsion. The complex dispersion comprising suspended drug and nanoemulsion droplets was continuously homogenized to wet mill the drug particles to a suitable size for pulmonary delivery. Finally, a single, discrete pass (17-20 kpsi) was used to ensure that all droplets and drug particles passed through the homogenizer. The final feedstock composition had a solids content of 1.5% w / w, and a PFOB content of 10% v / v. Formulations of AmB-AA and AmB-NAcC were manufactured with and without added cholesterol.

[0356] Production of Dry Powders by Spray Drying.

[0357] Spray drying was conducted with a laboratory-scale spray dryer (Buchi B-191, Flawil, Switzerland) equipped with a custom-built atomizer and cyclone particle collection system. The liquid feed was pumped into the atomizer at 5 ml / min with a Watson-Marlow peristaltic pump. The twin-fluid atomizer was operated at pressure of 70 psi. The dryer aspiration was set at 100%. The dryer inlet and outlet temperatures were 100°C and 62 ± 1 °C, respectively.

[0358] In suspension-based feeds, each atomized droplet (mass median diameter ~ 10 pm) contained dispersed drug crystals and approximately 1000 sub-micron emulsion droplets. During the initial moments of the drying process, the more volatile aqueous phase began to evaporate. The rapidly receding atomized droplet interface drove enrichment of the slowly diffusing drug and emulsion particles at the interface. This led to formation of a void space in the center of the drying droplet. As the drying process continued, the less volatile oil phase in the emulsion droplets evaporated, resulting in formation of hollow pores in their place. Overall, the resulting hollow spray-dried composite particles contained drug crystals embedded in an interfacial layer of a porous lipid matrix.

[0359] Example 5. Physicochemical properties of lipid-coated AmB-AA and AmB-NAcC particles

[0360] The primary particle size distribution and tapped density of the spray-dried particles are detailed in Table 3. The particles are of a size that is suitable for administration as dry powder aerosols with a portable passive dry powder inhaler such as the RS01 DPI. The low density of the particles reflects the hollow porous nature of the spray-dried particulates.

[0361] Table 3. Physicochemical properties of spray-dried formulations comprising AmB-AA and AmB-NAcC.

[0362] Example 6: Designing and Testing Ion Selectivity of AmB-Derivatives

[0363] 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 (Figure 2A). Similarly, the bis-negatively charged derivative, AmB- NAcC, was prepared by acylating the C3’ -amine using succinic anhydride to be used as a control (Figure 2A). Given their net charges, AmB-NAcC was expected to be more cation selective and AmB-AA more anion selective than AmB (Figure 7).

[0364] 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, highly polarized, 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 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 (Figures 8A, 2F, 2G). Similarly, AmB- NAcC, designed to also be cation selective demonstrated a PNa / Pci of 31.7027 (Figures 8B, 2F). Given the high cation selectivity of AmB, we believe this assay was not sensitive enough to discern between two highly cation- selective molecules such as AmB and AmB-NAcC. In contrast, AmB-AA successfully inverted the ion-selective seen by AmB, generating anionselective ion channels, as seen by the average Pci / PNa of 3.0024 (Figures 8C, 2G). Example 7: The Effect of Ion Selectivity on Rescuing Physiology in Channelopathy Models

[0365] 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 (Figure 8D). AmB-NAcC demonstrated a similar ability to rescue yeast growth compared to AmB (Figure 8D). As expected, the anionselective AmB-AA MP showed to be substantially worse at rescuing trkl Atrk2A yeast growth compared to AmB and AmB-NAcC (Figure 3D). These results also tracked with a decreased capacity for the anion selective channels formed by AmB-AA to increase intracellular K+ of yeast cells (Figure 11). 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 (Figure 3D). Unexpectedly, AmB-NAcC lost the ability to rescue ASL pH at 2 pM and at higher concentrations (Figure 8D). Remarkably, AmB-AA resulted in a higher ASL pH rescue compared to AmB (Figure 8D) demonstrating additional evidence towards protein-likeness resulting in improved functional recovery.

[0366] Example 8: Efficacy of AmB-AA in Cystic Fibrosis Models

[0367] 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. 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 at or above 50 pM (Figure 9A). 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 (Figure 9B) Additionally, CF epithelial pretreated with AmB-AA (2 pM) showed a larger reduction of pHint compared to AmB (Figure 9B). These complementary improved results were attributed to the improved efflux of HCCU and CF out of the cell provided by anion selective AmB-AA.

[0368] 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(Figure 9C). Pretreatment of CuFi-4 cells with AmB, at 2 and 50 pM, showed no rescue of ion homeostasis, with nonsignificant changes of 13 and 15 pA / cm2respectively (Figure 9C). 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 (Figure 9C). 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.

[0369] 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 (Figure 9A). 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 (Figure 9D). This conductance dose response study demonstrated that extended window of ASL pH rescue 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+(Figure 9E). 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 (Figure 9F). 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 pM. 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.

[0370] Example 9: Complexation of AmB-AA with Cholesterol Reduces Toxicity

[0371] 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 (Figure 10A and Figure 10B). As a dry powder formulation, AmB, has shown a promising safety profile in ongoing clinical trials in healthy and CF individuals. To reduce the toxicity of 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 (Figure 10A and Figure 10B). The successful AmB- AA:Chol complexation was confirmed by UV-Vis spectroscopy which further supported the binding of cholesterol to AmB-AA (Figure 10C). XPLOR-NH4 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 (Figure 10D). These computational models of AmB-AA further supported this cholesterol dependent toxicity.

[0372] INCORPORATION BY REFERENCE

[0373] All patents and published patent applications mentioned in the description above are incorporated by reference herein in their entirety.

[0374] EQUIVALENTS

[0375] 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.

Claims

CLAIMSWe claim:

1. A method of treating or preventing a disease or condition characterized by decreased expression or reduced function of an ion channel in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is represented by structural formula I or structural formula II:

2. The method of claim 1, wherein the compound is the compound represented by structural formula I or a pharmaceutically acceptable salt or hydrate thereof.

3. The method of claim 2, wherein the ion channel is an anion channel.The method of any one of claims 1-3, wherein the ion channel is a chloride channel.

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

7. The method of claim 1, wherein the compound is the compound represented by structural formula II or a pharmaceutically acceptable salt or hydrate thereof.

8. The method of claim 7, wherein the ion channel is a cation channel.

9. The method of claim 8, wherein the ion channel is a K+channel.

10. The method of any one of claims 1-9, 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.

11. The method of claim 10, wherein the disease or condition is selected from the group consisting of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis.

12. The method of claim 11, wherein the disease or condition is cystic fibrosis.

13. The method of any one of claims 1-12, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered systemically.

14. The method of any one of claims 1-13, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered to an airway of the subject.

15. The method of any one of claims 1-14, wherein the compound or a pharmaceutically acceptable salt or hydrate thereof is administered as an aerosol to an airway of the subject.

16. The method of any one of claims 1-15, wherein the subject is a human.

17. The method of claim 16, wherein the human is less than 12 years old.

18. The method of claim 17, wherein the human is at least 12 years old or at least 6 years old.

19. A pharmaceutical composition, comprising:(i) a compound represented by structural formula I or structural formula II:or a pharmaceutically acceptable salt or hydrate thereof;(ii) calcium chloride (CaCh);(iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and, optionally,(iv) cholesterol (Choi).

20. The pharmaceutical composition of claim 19, wherein the compound represented by structural formula I or structural formula II is present in an amount of about 0.5% to about 25% w / w.

21. The pharmaceutical composition of claim 20, wherein the compound represented by structural formula I or structural formula II is present in an amount of about 2% to about 16% w / w.

22. The pharmaceutical composition of claim 21, wherein the compound represented by structural formula I or structural formula II is present in an amount of about 5% w / w.

23. The pharmaceutical composition of claim 21, wherein the compound represented by structural formula I or structural formula II is present in an amount of about 14% w / w.

24. The pharmaceutical composition of any one of claims 19-23, wherein Choi is present in an amount of about 0.1% to about 8% w / w.

25. The pharmaceutical composition of claim 24, wherein Choi is present in an amount of about 0.3% to about 6% w / w.

26. The pharmaceutical composition of claim 25, wherein Choi is present in an amount of about 0.5% to about 3% w / w.

27. The pharmaceutical composition of any one of claims 19-26, wherein CaCh is present in an amount of about 1% to about 10% w / w.

28. The pharmaceutical composition of claim 27, wherein CaCh is present in an amount of about 4% to about 7% w / w.

29. The pharmaceutical composition of claim 28, wherein CaCh is present in an amount of about 5.5 % w / w.

30. The pharmaceutical composition of any one of claims 19-29, wherein the phospholipids are present in an amount of about 60% to about 95% w / w .

31. The pharmaceutical composition of claim 30, wherein the phospholipids are present in an amount of about 70% to about 90% w / w.

32. The pharmaceutical composition of any one of claims 19-31, wherein Choi and the phospholipids are present in a weight ratio of about 0.001 : 1 to about 0.1 : 1.

33. The pharmaceutical composition of claim 32, wherein Choi and the phospholipids are present in a weight ratio of about 0.05 : 1.

34. The pharmaceutical composition of any one of claims 19-33, wherein HSPC and DSPG are present in a weight ratio of about 2: 1 to about 19: 1.

35. The pharmaceutical composition of claim 34, wherein HSPC and DSPG are present in a weight ratio of about 7: 1 to about 12: 1.

36. The pharmaceutical composition of claim 34, wherein HSPC and DSPG are present in a weight ratio of about 9: 1.

37. The pharmaceutical composition of claim 34, wherein HSPC and DSPG are present in a weight ratio of about 2.25: 1.

38. The pharmaceutical composition of any one of claims 19-37, wherein Choi and the compound represented by structural formula I or structural formula II are present in a molar ratio of about 0.05 : 1 to about 1.2: 1.

39. The pharmaceutical composition of claim 38, wherein Choi and the compound represented by structural formula I or structural formula II are present in a molar ratio of about 0.1 : 1 to about 0.8: 1.

40. The pharmaceutical composition of claim 39, wherein Choi and the compound represented by structural formula I or structural formula II are present in a molar ratio of about 0.4: 1.

41. The pharmaceutical composition of any one of claims 19-40, wherein the phospholipids and CaCh are present in a molar ratio of about 4: 1 to about 1 : 1.

42. The pharmaceutical composition of claim 41, wherein the phospholipids and CaCh are present in a molar ratio of about 2:1.

43. The pharmaceutical composition of any one of claims 19-42, wherein the compound represented by structural formula I or structural formula II has a crystallinity greater than about 75%.

44. The pharmaceutical composition of claim 43, wherein the compound represented by structural formula I or structural formula II has a crystallinity greater than about 85%.

45. The pharmaceutical composition of claim 44, wherein the compound represented by structural formula I or structural formula II has a crystallinity greater than about 95%.

46. The pharmaceutical composition of any one of claims 19-23, wherein the pharmaceutical composition does not comprise Choi.

47. The pharmaceutical composition of claim 19, comprising, consisting essentially of, or consisting of:(i) about 14.0% w / w of the compound represented by structural formula I or structural formula II;(ii) about 2.3% w / w Choi;(iii-a) about 70.3% w / w HSPC;(iii-b) about 7.8% w / wDSPG; and(iv) about 5.52% w / w CaCh.

48. The pharmaceutical composition of claim 19, comprising, consisting essentially of, or consisting of:(i) about 14.0% w / w of the compound represented by structural formula I or structural formula II;(ii) about 6.81% w / w Choi;(iii-a) about 51.2% w / wHSPC;(iii-b) about 22.8% w / DSPG; and(iv) about 5.2% w / w CaCh.

49. The pharmaceutical composition of claim 19, comprising, consisting essentially of, or consisting of:(i) about 3.4% w / w of the compound represented by structural formula I or structural formula II;(ii) about 0.57% w / w Choi;(iii-a) about 80.72% w / wHSPC;(iii-b) about 8.97% w / w DSPG; and(iv) about 6.34% w / w CaCh.

50. The pharmaceutical composition of claim 19, comprising:(i) Choi and the compound represented by structural formula I or structural formulaII in a molar ratio of about 0.2 to about 1.0;(ii) Choi and the phospholipids in a weight ratio of less than about 0.05;(iii) HSPC and DSPG in a weight ratio of about 2.0 to about 10.0; and(iv) the phospholipids and CaCh in a molar ratio of about 2: 1.

51. The pharmaceutical composition of claim 19, comprising:(i) Choi and the compound represented by structural formula I or structural formulaII in a molar ratio of about 0.4;(ii) Choi and the phospholipids in a weight ratio of less than about 0.05;(iii) HSPC and DSPG in a weight ratio of about 9:1; and(iv) the phospholipids and CaCh in a molar ratio of about 2: 1.

52. The pharmaceutical composition of any one of claims 47-51, wherein the compound is represented by structural formula I.

53. The pharmaceutical composition of any one of claims 47-51, wherein the compound is represented by structural formula II.

54. The pharmaceutical composition of any one of claims 19-53, wherein the compound represented by structural formula I or structural formula II and Choi are not complexed; and the compound represented by structural formula I or structural formula II is not encapsulated in liposomes.

55. The pharmaceutical composition of any one of claims 19-54, wherein the compound represented by structural formula I or structural formula II is coated with a porous shell of phospholipids and Choi.

56. The pharmaceutical composition of any one of claims 19-55, wherein the pharmaceutical composition is formulated as a dry powder comprising powder particles.

57. The pharmaceutical composition of claim 56, wherein the mass median diameter, Xso, of the powder particles is about 1.0 to about 4.0 pm.

58. The pharmaceutical composition of claim 57, wherein the mass median diameter, X50, of the powder particles is about 1.5 to about 3.5 pm.

59. The pharmaceutical composition of any one of claims 56-58, wherein the tapped density of the powder particles is about 0.02 to about 0.2 g / mL.

60. The pharmaceutical composition of claim 59, wherein the tapped density of the powder particles is about 0.03 to about 0.1 g / mL.

61. The pharmaceutical composition of any one of claims 19-60, wherein the composition is formulated for pulmonary administration or airway administration.

62. The pharmaceutical composition of claim 56, wherein the composition is formulated for aerosol administration.

63. 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 the pharmaceutical composition of any one of claims 19-62.

64. The method of claim 63, wherein the compound is the compound represented by structural formula I or a pharmaceutically acceptable salt or hydrate thereof.

65. The method of claim 64, wherein the ion channel is an anion channel.66 The method of any one of claims 63-65, wherein the ion channel is a chloride channel.

67. The method of any one of claims 63-66, wherein the ion channel is a CFTR channel.

68. The method of any one of claims 63-65, wherein the ion channel is an HCCh' channel.

69. The method of claim 63, wherein the compound is the compound represented by structural formula II or a pharmaceutically acceptable salt or hydrate thereof.

70. The method of claim 69, wherein the ion channel is a cation channel.

71. The method of claim 70, wherein the ion channel is a K+channel.

72. A method of treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 19-62; wherein the administration is pulmonary.

73. The method of any one of claims 63-72, wherein the disease or condition is cystic fibrosis, chronic obstructive pulmonary disease (COPD), non-cystic fibrosis bronchiectasis (NCFB), hyperkalemic periodic paralysis, paramyotonia congenita,-n -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.

74. The method of claim 73, wherein the disease or disorder is selected from the group consisting of cystic fibrosis, COPD, and non-cystic fibrosis bronchiectasis.

75. The method of claim 74, wherein the disease or condition is cystic fibrosis.

76. A method of increasing the pH of airway surface liquid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of any one of claims 19-62, thereby increasing the pH of airway surface liquid in the subject; wherein the administration is pulmonary.

77. A method of increasing bicarbonate secretion into airway surface liquid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of any one of claims 19-62, thereby increasing bicarbonate secretion into airway surface liquid in the subject; wherein the administration is pulmonary.

78. A method of increasing expiratory volume in one second (FEV1), comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of any one of claims 19-62, thereby increasing the subject’s FEV1; wherein the administration is pulmonary.

79. The method of claim 78, wherein the subject’s FEV1 is increased by about 3% to about 20%.

80. The method of any one of claims 63-79, wherein the composition is administered to an airway of the subject.

81. The method of any one of claims 63-80, wherein the composition is administered as an aerosol.

82. The method of any one of claims 63-81, wherein the subject is a human less than 12 years old.

83. The method of any one of claims 63-81, wherein the subject is a human at least 12 years old or at least 6 years old.

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