Additive effects for molecular prosthetcs and CFTR modulators in cf epithelia

Abstract

Provided are methods of using a combination of an ion channel prosthetic and an ion channel modulator for treating pulmonary disorders.

Description

[0001] ADDITIVE EFFECTS FOR MOLECULAR PROSTHETICS AND CFTR MODULATORS IN CF EPITHELIA

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 539,472, filed September 20, 2023.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under R35 GM118185 and P01 HL152960 (subaward S02766-01) awarded by the National Institute of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Anion secretion by airway epithelia into the airway surface liquid (ASL) is essential for mucociliary clearance, antimicrobial peptide function, fluid secretion, and pH homeostasis. The transport of HCCN into the ASL plays an important role in ensuring proper cell physiology. The main transporter of HCOs' is the cystic fibrosis conductance regulator (CFTR), and mutations affecting this anion channel transporter lead to the genetic disease cystic fibrosis (CF). Previous studies show that CFTR and the electroneutral Cl / HCCh’ channel exchanger, pendrin, function in concert to transport HCCU into the ASL under inflammatory conditions and have a maximal increase in ASL pH when both are active. The present disclosure investigates whether a molecular prosthetic (MP), Amphotericin B (AmB) can work together with modulator rescued CFTR to enhance anion transport into the ASL of cultured airway epithelia. In view of the foregoing, there is an unmet need for new treatments for CF.

[0008] SUMMARY

[0009] In certain aspects, provided herein is a method of treating a disease characterized by reduced function or decreased expression of an ion channel, the method comprising administering to a subject in need thereof a therapeutically effective amount of (a) an ion channel prosthetic selected from:

[0010] - 1 -

[0011] SUBSTITUTE SHEET (RULE 26)

[0012] or a pharmaceutically acceptable salt or hydrate of either of them; and

[0013] (b) an ion channel modulator.

[0014] In certain aspects, provided herein is a method of increasing transepithelial current of airway cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0015] (a) an ion channel prosthetic selected from: or a pharmaceutically acceptable salt or hydrate of either of them; and

[0016] (b) an ion channel modulator.

[0017] In certain aspects, provided herein is a method of increasing the pH of airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0018] (a) an ion channel prosthetic selected from: or a pharmaceutically acceptable salt or hydrate of either of them; and

[0019] (b) an ion channel modulator.

[0020] In certain aspects, provided herein is a method of increasing bicarbonate secretion into airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0021] (a) an ion channel prosthetic selected from:

[0022] or a pharmaceutically acceptable salt or hydrate of either of them; and

[0023] (b) an ion channel modulator.

[0024] In certain aspects, provided herein is a method of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of: (a) an ion channel prosthetic selected from: or a pharmaceutically acceptable salt or hydrate of either of them; and (b) an ion channel modulator.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1A displays a non-CF airway with a functional CFTR channel.

[0027] FIG. IB displays loss of function mutations in CFTR result in the internal cell accumulation of HCOs' impairing ASL pH, viscosity, and antibacterial activity. FIG. 1C displays CFTR modulators partially restoring CFTR function dependent of genotype.

[0028] FIG. ID displays the small molecule ion channel utilizing the electrochemical gradient of HCCh to unselectively restore ion movement independent of genotype.

[0029] FIG. IE displays the hypothesis that these two distinct mechanisms can work in tandem to restore CF airway physiology in an additive manner.

[0030] FIG. 2A displays the increase in short-circuit current after the apical addition of 10 pM of Forskolin followed by Ivacaftor to phosphorylate and potentiate mutant CFTR (n=5) in cultures of G551D / AF508 cultured lung epithelia cells.

[0031] FIG. 2B displays the increase in short-circuit current following the addition of 10 pM of the molecular prosthetic in the absence of functional CFTR (n=5) in cultures of G551D / AF508 cultured lung epithelia cells.

[0032] FIG. 2C displays the increase in short-circuit current after the addition of 10 pM of Ivacaftor and forskolin followed by 10 pM of AmB, showing an additive increase in transepithelial current for G551D / AF508 cultured lung epithelia cells (n=5).

[0033] FIG. 2D displays a summary bar graph for the change in short-circuit current for FIG. 2A - FIG. 2C demonstrating an additive increase in electrocurrent when both Ivacaftor and AmB are used together.

[0034] FIG. 3A displays a dose response analysis post-48 hrs of incubation with ETI / f:(pM) = Elexacaftor,Tezacaftor: 1,3,5,10; Ivacaftor, Forskolin: 0.33, 1,1.66, 3.33 in G551D / AF508 cultured lung epithelial cells showing that ASL pH does not linearly increase.

[0035] FIG. 3B displays a dose response analysis post-48 hrs of incubation with ETI / f:(pM) = Elexacaftor,Tezacaftor: 1,3,5,10; Ivacaftor, Forskolin: 0.33, 1, 1.66, 3.33 in AF508 / AF508 cultured lung epithelial cells showing that ASL pH does not linearly increase.

[0036] FIG. 3C displays an Ussing chamber dose response analysis for short-circuit current with Ivacaftor and forskolin on G551D / AF508 cultured lung epithelia cells.

[0037] FIG. 3D displays a dose response analysis of H14CO3‘ efflux in AF508 / AF508 cultured lung epithelia cells post-48 hrs of incubation with ETI / f:(pM) = Elexacaftor,Tezacaftor: 1,3,5,10; Ivacaftor,Forskolin: 0.33, 1, 1.66, 3.33.

[0038] FIG. 3E shows a sub-optimal increase in ASL pH with the unselective molecular prosthetic ion channel AmB (2 pM) post-48 hrs incubation in AF508 / AF508 cultured lung epithelia cells.

[0039] FIG. 3F shows a maximal increase in ASL pH of AF508 / AF508 cultured lung epithelia cells post-48 hrs. incubation with AmB-AA (2 pM). FIG. 4A shows the increase in ASL pH of AF508 / AF508 cultured lung epithelia cells after 48-hrs of incubation with ABCI-003 (2 pM) or ETI / F (1,1,0.33 / 0.33 pM), and coincubation with ABCI-003 and ETEf.

[0040] FIG. 4B shows H14CO3‘ efflux in AF508 / AF508 cultured lung epithelia cells after 48- hrs of incubation with ABCI-003 (2 pM) or ETI / F (1,1,0.33 / 0.33 pM), and co-incubation with ABCI-003 and ETI / f.

[0041] FIG. 4C shows the increase in ASL pH of G551D / AF508 cultured lung epithelial cells after 48-hrs of incubation with ABCI-003 (2 pM) or Ivacaftor / forskolin (0.33 / 0.33 pM), and co-incubation with ABCI-003 and Ivacaftor / forskolin.

[0042] FIG. 4D shows H14CO3‘ efflux in G551D / AF508 cultured lung epithelia cells after 48-hrs of incubation with ABCI-003 (2 pM) or Ivacaftor / forskolin (0.33 / 0.33 pM), and co- incubation with ABCI-003 and Ivacaftor / forskolin.

[0043] FIG. 4E shows the increase in ASL pH of G551D / AF508 cultured lung epithelial cells after 48-hrs of incubation with ABCI-003 (2 pM) or ETI / f (1,1 0.33 / 0.33 pM), and co- incubation with ABCI-003 and ETEf.

[0044] FIG. 4F shows H14CO3‘ efflux in G551D / AF508 cultured lung epithelia after 48-hrs of incubation with ABCI-003 (2 pM) or ETEf (1,1,0.33 / 0.33 pM), and co-incubation with ABCI-003 and ETI.

[0045] FIG. 5A demonstrates that pH measurements suggest an intracellular HCOf accumulation in G551D / AF508 cultured lung epithelial cells that is partially mitigated post- 48 hrs. by treatment with ABCI-003 or CFTR modulators.

[0046] FIG. 5B shows molecular prosthetic mediated H14CO3‘ efflux post-48 hrs of incubation with ABCI-003 (2 pM) in G551D / AF508 cultured lung epithelial cells is eradicated when the Na+ / K+ATPase is inhibited by the basolateral addition of ouabain (10 pM).

[0047] FIG. 5C shows modulator-rescued CFTR mediated H14CO3‘ efflux post-48 hrs of incubation with ETI / f (1,1,0.33 / 0.33 pM) in G551D / AF508 cultured lung epithelial cells is eradicated when the Na+ / K+ATPase is inhibited by the basolateral addition of ouabain (10 pM).

[0048] FIG. 5D shows H14CO3‘ efflux mediated by a molecular prosthetic mechanism and modulator rescued CFTR post-48 hrs of incubation with ABCI-003 (2 pM) and ETEf (1,1,0.33 / 0.33 pM) in G551D / AF508 cultured lung epithelial cells was eradicated when the Na+ / K+ATPase was inhibited by the basolateral addition of ouabain (10 pM). FIG. 6A shows ICPMS analysis demonstrating Na+concentration (mM) in the ASL of AF508 / AF508 cultured lung epithelia is not affected by the combined mechanisms of MP and modulators.

[0049] FIG. 6B shows ICPMS analysis demonstrating K+concentration (mM) in the ASL of AF508 / AF508 cultured lung epithelia is not affected by the combined mechanisms of MP and modulators.

[0050] FIG. 6C shows ICPMS analysis demonstrating Mg2+concentration (mM) in the ASL of AF508 / AF508 cultured lung epithelia is not affected by the combined mechanisms of MP and modulators.

[0051] FIG. 6D shows ICPMS analysis demonstrating Ca2+concentration (mM) in the ASL of AF508 / AF508 cultured lung epithelia is not affected by the combined mechanisms of MP and modulators.

[0052] FIG. 7A shows dose-response behavior for elexacaftor (E), tezacaftor(T), ivacaftor (I), and forskolin (f) on the ASL pH of G551D / AF508 cultured lung epithelia cells.

[0053] FIG. 7B shows dose-response behavior for ABCI-003 on the ASL pH of AF508 / AF508 cultured lung epithelia cells.

[0054] FIG. 7C shows dose-response behavior for elexacaftor, tezacaftor, ivacaftor, and forskolin in combination with ABCI-003 on the ASL pH of AF508 / AF508 cultured lung epithelia cells.

[0055] FIG. 8A shows dose-response behavior for elexacaftor, tezacaftor, ivacaftor, and forskolin on the ASL pH of AF508 / AF508 cultured lung epithelia cells.

[0056] FIG. 8B shows dose-response behavior for ABCI-003 on the ASL pH of AF508 / AF508 cultured lung epithelia cells.

[0057] FIG. 8C shows dose-response behavior for elexacaftor, tezacaftor, ivacaftor, and forskolin in combination with ABCI-003 on the ASL pH of AF508 / AF508 cultured lung epithelia cells.

[0058] FIG. 8D shows composite data and statistical analysis for treatment groups from FIG. 8A - FIG. 8C

[0059] FIG. 9A shows short circuit current change does not increase following a second addition of protein kinase A activators and Ivacaftor (10 micromolar), follow-up addition of AmB (10 micromolar) increases short circuit current.

[0060] FIG. 9B shows post-48 hrs. incubation with increasing concentrations of CFTR modulators (ETI) does not lead to a linear increase of14HCO3‘ efflux into the ASL in CF airway epithelia. FIG. 9C shows post-48 hrs. incubation with increasing concentrations of CFTR modulators (ETI) does not lead to an increase in ASL pH in CF airway epithelia (G551D / AF508 cells).

[0061] FIG. 9D shows post-48 hrs. incubation with increasing concentrations of CFTR modulators (ETI) does not lead to an increase in ASL pH in CF airway epithelia.

[0062] FIG. 9E shows ASL pH of Cufi-1 cells post-48 hrs. incubation with AmB (2 micromolar) shows as increase in airway in ASL pH less than ETI, additive effects are observed for both groups.

[0063] FIG. 9F shows that for the anion- selective chemical biology probe, AmB-AA (2 micromolar) and the ASL pH of Cufi-1 cells is increased with a higher change than ETI, additive effects are only observed for the ETI experimental group. AmB maximizes increase of ASL pH.

[0064] FIG. 10A shows the effect of ABCI-003 and ETEf on ASL pH post-48 hrs. incubation in Cufi-1 epithelia. AF508 / AF508 cells.

[0065] FIG. 10B shows the effect of ABCI-003 and ETI / f on ASL pH post-48 hrs. incubation in Cufi-1 epithelia. G551D / AF508 cells.

[0066] FIG. IOC shows the effect of ABCI-003 and ETEf on14HCO3‘ efflux from basolateral to the ASL post-48 hrs. incubation in Cufi-1 epithelia. AF508 / AF508 cells.

[0067] FIG. 10D shows the effect of ABCI-003 and ETEf on14HCO3‘ efflux from basolateral to the ASL post-48 hrs. incubation in Cufi-1 epithelia. G551D / AF508 cells.

[0068] FIG. 10E shows post-48 hr. incubation with PFC-72 vehicle or AmB-AA (2 micromolar), ABCI-003 (2 micromolar), ETI / f, (1 / 1 / 0.33 / and 0.33 micromolar, respectively) or co-incubation with ABCI-003 (2 micromolar) and ETI / f (1 / 1 / 0.33 / and 0.33 micromolar, respectively) response of cultured lung epithelia to the ENaC channel inhibitor amiloride (100 micromolar) was measured. Cufi-4 epithelia have an increased response relative to Nuli, which is mitigated by addition of molecular prosthetics and / or CFTR-modulators.

[0069] DETAILED DESCRIPTION

[0070] Anion secretion by airway epithelia into the airway surface liquid (ASL) is essential for mucociliary clearance, antimicrobial peptide function, fluid secretion, and pH homeostasis. The transport of HCOs' into the ASL plays an important role in ensuring proper cell physiology. The main transporter of HCOs' is the cystic fibrosis conductance regulator (CFTR), and mutations affecting this anion channel transporter lead to the genetic disease cystic fibrosis (CF). Previous studies show that CFTR and the electroneutral Cl / HCCh’ channel exchanger, pendrin, function in concert to transport HCCU into the ASL under inflammatory conditions and have a maximal increase in ASL pH when both are active. The present disclosure investigates whether a molecular prosthetic (MP), Amphotericin B (AmB) can work together with modulator rescued CFTR to enhance anion transport into the ASL of cultured airway epithelia.

[0071] The MP AmB is a small molecule natural product that forms ion channels in the membrane that are unselective for cations versus anions. AmB functions by utilizing the electrochemical gradient of the deficient ion to move it into the cytoplasm or out to the lumen. Its versatile role in cell physiology restoration led to wildtype growth of yeast missing a K+ channel essential for their growth; it also led to an increase in ASL pH, HCCU transport to the ASL, and restored host defenses in cystic fibrosis airway epithelia. A new formulation of AmB, ABCI-003, has a robust effect in correcting CF related defects in primary cultures of CF airway epithelia and is currently in clinical trials. Despite the promising results of AmB, its unselective nature of monovalent ion transport leads us to believe that the therapeutic effects in the CF airway have not been maximized.

[0072] Another example of a suboptimal therapeutic are ion channel modulators, e.g., CFTR modulators. CFTR modulators are small molecule drugs that partially restore CFTR function and abundance in the membrane of epithelial cells. They are categorized as either correctors (Elexacaftor / Tezacaftor) which increase processing of the misfolded protein, or as potentiators (Ivacaftor) that allow for an increase probability of CFTR channel opening. Since the approval of CFTR modulators there has been a decrease in lung transplants, and improvements in life expectancy and quality of life. However, for some, the effects of the modulators in real-world conditions have led to either dose reductions or complete discontinuation of the modulator therapy altogether due to adverse effects associated with modulator treatment. These include: an increase in transaminases, mental health deterioration, insomnia, and anxiety. Also, inflammation and persistent lung infections remain a concern in people with CF (pwCF) under modulator therapy. The present disclosure is based on the hypothesis that modulator rescued- CFTR stoichiometry leaves room for improvement in CF airway epithelia cells.

[0073] Using CF cultured lung epithelia, two suboptimal therapeutics are shown to work in concert to enhance anion flow into the ASL. To assess ion transport to the apical side of the membrane, ion flow promoted by the distinct mechanisms of either rescued-CFTR, AmB, or both, was measured. To test whether rescued CFTR stoichiometry limits modulator efficacy, the effects of increasing modulator concentration on ASL pH, HCCU efflux, and electrocurrent change were evaluated. The efficacy of AmB in its new formulation, ABCI-003, to that of the modulators was compared to assess whether there is an additive benefit when CF lung epithelia are co-incubated with both MP and CFTR-modulators for ASL pH, bicarbonate efflux, and intracellular pH. To assess whether ion efflux through rescued-CFTR, ABCI-003, or the combined mechanisms interfaced with basolateral channels; the electrochemical gradient that favors intracellular HCCL" accumulation by the Na+ / K+ATPase in CF airway epithelia was eradicated.

[0074] With a voltage clamped Ussing chamber assay, we compared the electrophysiological response of cultured lung epithelial cells heterozygous for the gating and trafficking mutation (G551D / AF508). To establish a concentration gradient, G551D / AF508 cells were bathed in a ringers solution of low chloride concentration on the apical side and a high chloride concentration on the basolateral side. Ivacaftor, AmB, or Ivacaftor followed by AmB with forskolin were acutely added to the apical side of the chamber at a final concentration of lOpM (FIG. 2A - FIG. 2C). We observed an additive increase in current change supporting the notion that AmB and CFTR-modulators have distinct mechanisms of action for ion transport (FIG. 2D)

[0075] To assess whether the potentiation action of Ivacaftor was limited by its bioavailability, we performed a concentration-response analysis with Ivacaftor and observed its potentiating effect to be saturating at ~lpM (FIG. 3C). Similarly, due to the positive effects of HCO3- on airway host defenses, radioactive bicarbonate (H14CO3‘) was used to quantify its transport to the ASL at increasing concentrations of Elexacaftor / Tezacaftor / Ivacaftor / forskolin (ETIf) on cultured lung epithelia cells homozygous for the trafficking mutation (AF508 / AF508) after chronic exposure to ETI / f (FIG. 3D). We observed that like the Ussing chamber concentration-response analysis the change in H14CO3‘ efflux was saturated at an ETI / f micromolar concentrations of 1, 1, 0.33, and 0.33 micromolar. Analogously, ASL pH showed this same trend for G551D / AF508 and AF508 / AF508 cultured lung epithelial cells (FIG. 3A - FIG. 3B) These results support the hypothesis that the rate limiting step of modulator efficacy is the stoichiometry of rescued CFTR protein, and a higher modulator concentration does not lead to a greater physiological benefit. Next, with the role of AmB as a small molecule ion channel we sought to understand if additivity was a result of its indiscrimination for cation versus anion transport and asked whether the newly discovered anion selective derivative, AmB-AA would still leave room for improvement in CF cultured lung epithelia cells (J&D). To do this, we chronically exposed AF508 / AF508 cells to one of five treatments prior to LSM- 880 confocal microscope imaging and analyses: (i) a DMSO and PFC control, (ii) AmB, (iii) ETI / f, (IV) the combination of AmB with ETI / f, (V) AmB-AA, or the combination of AmB- AA with ETI / f. The results demonstrate that additivity still exist when AF508 / AF508 cells are co-incubated with AmB and ETI / f (FIG. 3E). However, the change in ASL pH is maximized when AF508 / AF508 cells are incubated with AmB-AA (FIG. 3F).

[0076] Next, we sought to compare the efficacy of the potential CF therapeutic, ABCI-003 to that of the current CF therapeutics: ETI (i.e., Trikafta) and Ivacaftor. The effect on ASL pH and H14CCL" efflux after chronic exposure of AF508 / AF508 and G551D / AF508 cells with one of six conditions was analyzed: (i) DMSO and PFC control (ii) ABCI-003, (iii) ETI / f, (iv) Ivacaftor with forskolin, (v) the combination of ABCI-003 with ETI / f or the combination of ABCI-003 with Ivacaftor plus forskolin. Our results show that ABCI-003 restores ASL pH and H14COs' efflux to the same capacity as the monotherapy Ivacaftor and ETI. There was also an additive benefit when CF cell cultures were co-incubated with both MP and CFTR-modulators (FIG. 4A - FIG. 4F). To ensure that co-incubating CF cell cultures with CFTR-modulators and ABCI-003 did not disrupt airway homeostasis, the major cation concentrations of the airway were determined by ICPMS. Our data demonstrates that the major cations remained unaffected by the distinct mechanisms that resulted in overall greater ASL pH and H14COs' in the ASL (FIG. 5). We then rationalized that independently the sub-optimal therapeutics must not fully alleviate the intracellular buildup of HCOs' in CF cultures leading to an additive increase in HCOs' secretion when both approaches are used. To test this hypothesis, we first assessed the driving force of HCOs' influx into the cell generated by the Na+ / K+ATPase by using the small molecule inhibitor ouabain. G551D / AF508 cells were chronically incubated with either: ABCI-003, ETI / f, or the combination of ABCI-003 with ETI / f. After the 48-hr incubation period H14CCE' and / or ouabain were simultaneously added to the basolateral side for Ihr prior to ASL H14CO3‘ reading. Our results demonstrate that inhibition of the Na+ / K+ATPase abolishes ABCI-003, rescued-CFTR, and the combination of ABCI-003 with rescued- CFTR mediated H14COs' efflux into the ASL (FIG. 5B - FIG. 5D). Now, to test whether there was residual intracellular HCOs', AF508 / AF508 cells were chronically incubated as before prior to LSM-880 confocal microscopy imaging with the intracellular permeable dye, BCECF- AM. Our results show that the intracellular basicity of DMSO-PFC CF cultures was higher than those with: ABCI-003, ETI / f, or a combination of both (FIG. 5E).

[0077] We have demonstrated that two imperfect mechanisms can come together to additively promote HCOs' efflux into the ASL of CF cell cultures and increase ASL pH. By definition, a CFTR-potentiator is a small molecule that can promote the efflux of ions through an activated CFTR-channel (references). Improvement of CFTR mediated ion transport by potentiators is dependent on the quantity of CFTR present on the surface of the cell. Therefore, as our data suggest, partial rescue of CFTR (quantity, function, stability) results in a residual intracellular HCOf gradient that AmB can harness to allow for efflux of this anion into the ASL. It is notable that AmB also leaves this room for improvement due to it being unselective for cations versus anions. However, incubation of CF epithelia with the more CFTR-like derivative AmB-AA resulted in a significant increase in ASL pH relative to AmB.

[0078] We first measured the intracellular pH (pHi) of CF airway epithelia as a readout of HCCL" to test whether HCCL" accumulated in the absence of CFTR. In the absence of cotreatment with CFTR modulators and small molecule ion channels formed by AmB, CF airway epithelia have a more basic pHi relative to non-CF airway epithelia. An indication of a residual HCCTf gradient is seen when CF airway epithelia are treated alone with CFTR modulators or AmB. However, co-treatment reduces pHi to wildtype levels. We further tested whether the intracellular accumulation of HCCTf in the absence of an apical output resulted from compensatory action of basolateral pumps. For this we utilized H14CO3- to measure efflux mediated by modulator rescued CFTR, small molecule ion channels, and co-treatment with both CFTR modulators and ABCI-003 in the absence and presence of the inhibitor for the Na+ / K+ATPase, ouabain. We found that although H14CO3- effluxes to the ASL by either mechanism alone, co-treatment leads to an additive efflux of H14CO3-. This result emphasizes that a residual HCOf gradient remains untapped with the treatment of CF airway epithelia with CFTR modulators or MP alone. However, when the Na+ / K+ATPase is inhibited with ouabain the electrochemical gradient that favors the movement of Na+into the cell is mitigated. This leads to a loss of HCCL" influx and as a result ceases efflux of H14CO3' to the ASL of CF airway epithelial cells treated with CFTR-modulators, ABCI-003 or both.

[0079] We next chose to investigate why there was room for improvements on top of CFTR modulator-treated CF airway epithelia. We hypothesized that the inherent dependency of CFTR modulators to the quantity and activity of CFTR limits their capacity to restore host defenses. Electrocurrent measurements in an Ussing chamber were recorded for CFTR airway epithelia chronically treated with CFTR modulators (FIG. 9A). Initial activation and potentiation of CFTR with IB MX, forskolin, and Ivacaftor increased electrocurrent. Repeated addition of the respective small molecules did not lead to further changes in ion flow. However, the addition of AmB to CFTR modulator treated airway epithelia did result in an increase in current. Next a dose response analysis with chronic treatment of CFTR modulators on CF airway epithelial was done to assess HCCL" efflux and ASL pH. Notably, there was no further increases in HCCL" efflux, assessed with H14CO3- apical measurements, or ASL pH past the lowest concentration of CFTR modulators (Elxa=lpM, Teza= IpM, Ivacaftor= 0.33 pM, forskolin= 0.33 pM) (FIG. 9B - FIG. 9D).

[0080] Next, we explored why the addition of ABCI-003 alone also left room for improvement. Based on our previous work we predicted that the more cation-selective nature of AmB small molecule ion channels left room for improvement in CF airway epithelia (15*). To test for this, we asked whether additive effects with CFTR modulators and the newly discovered AmB variant, AmB-AA, that forms anion selective small molecule ion channels are observed. We used ASL pH measurements to ask this question and as expected found that AmB did show additive effects when co-incubated with CFTR modulators (FIG. 9E). However, AmB-AA alone showed the maximum increase in ASL pH that is detected for this assay in CF airway epithelia, and did not show additional activity in the presence of CFTR modulators (FIG. 9F).

[0081] We also chronically exposed CF airway epithelia with CFTR modulators, ABCI-003, or both CFTR modulators with ABCI-003 and assessed ASL pH, H14CO3- efflux, and ENaC activity. Notably, ABCI-003 increases ASL pH and promotes H14CO3- efflux to a similar extent as CFTR modulators at both clinically relevant and reduced CFTR modulator concentrations (FIG. 10A - FIG. 10D). Additive effects are also observed highlighting complementarity between these mechanisms. To exemplify the benefits of CFTR modulators with and without ABCI-003 on other membrane channels we asked whether ENaC hyperactivity, which is observed in CF epithelia, could be mitigated. ENaC hyperactivity in the CF airway leads to further airway dehydration and dyshomeostasis of the ASL and cell ion composition. Changes in short current measurement showed that CF airway epithelia were more sensitive to the ENaC inhibitor, amiloride, relative to non-CF airway epithelia cells (FIG. 10E). The addition of CFTR modulators decreased this sensitivity, but not to wildtype levels. Further incubation of CFTR modulator treated epithelia with ABCI-003 did lead to a decrease in ENaC hyperactivity to wildtype levels.

[0082] Thus, the dependency of CFTR modulators on the less than wild type-like quantity and activity of CFTR protein results in a residual intracellular HCOf gradient in CF airway epithelia cells. The residual intracellular HCCL" gradient can be leveraged by small molecule ion channels formed from AmB to achieve an additive increase in CF airway host defenses. The data suggests that the mechanisms of CFTR modulators and MPs work in complement and may lead to an additive benefit in the clinical setting for people with CF currently on modulator therapy. Real-world evidence shows that some individuals currently on CFTR modulator therapy experience adverse effects and require a dose reduction or discontinuation of CFTR modulator therapy. Also, people on highly effective CFTR modulator therapy still experience reoccurring bacterial infections. Here, combination therapy can be of benefit by offering a reduced CFTR modulator concentration with co-administration of ABCI-003 for people with CF who experience adverse effects. We also compare for the first time the efficacy of the clinical formulation ABCI-003 to highly effective CFTR modulators. We show that ABCI-003 restores ASL pH and HCCh' efflux to the same capacity in preclinical models as CFTR modulators. This shows that ABCI-003 offers a complementary mechanism with potentially equivalent clinical impact as CFTR modulators. Collectively, these results also suggest the potential for additive benefit of MPs and CFTR modulators in CF airway host defenses.

[0083] Ion Channel Prosthetic Compositions Comprising AmB

[0084] The ion channel prosthetics used in the presently disclosed methods can be formulated by any suitable method known in the art. Exemplary AmB-containing compositions are disclosed in PCT / US2023 / 015762, which is expressly incorporated herein by reference.

[0085] In certain embodiments, the ion channel prosthetic is formulated as a pharmaceutical composition, comprising:

[0086] (i) amphotericin B (AmB), or a pharmaceutically acceptable salt or hydrate thereof;

[0087] (ii) cholesterol (Choi);

[0088] (iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and

[0089] (iv) calcium chloride (CaCh).

[0090] In certain embodiments, the amount of AmB is about 0.5% to about 25% w / w. In further embodiments, the amount of AmB is about 3% to about 16% w / w. In yet further embodiments, the amount of AmB is about 14% w / w.

[0091] In certain embodiments, the amount of Choi is about 0.1% to about 8% w / w. In further embodiments, the amount of Choi is about 0.3% to about 6% w / w. In yet further embodiments, the amount of Choi is about 0.5% to about 3% w / w.

[0092] In certain embodiments, the amount of CaCh is about 1% to about 10% w / w. In further embodiments, the amount of CaCh is about 4% to about 7% w / w.

[0093] In certain embodiments, the amount of phospholipids is about 60% to about 95% w / w. In further embodiments, the amount of phospholipids is about 70% to about 90% w / w.

[0094] In certain embodiments, the weight ratio of Choi to phospholipids is about 0.001 : 1 to about 0.1 : 1. In further embodiments, the weight ratio of Choi to phospholipids is about 0.005 : 1 to about 0.05: 1. In certain embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 2: 1 to about 19: 1. In further embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 7:1 to about 12: 1.

[0095] In certain embodiments, the molar ratio of Choi to AmB is about 0.05 : 1 to about 1.2: 1. In further embodiments, the molar ratio of Choi to AmB is about 0.4: 1 to about 1.2:1. In yet further embodiments, the molar ratio of Choi to AmB is about 0.05: 1 to about 0.4:1. In still further embodiments, the molar ratio of Choi to AmB is about 0.4: 1.

[0096] In certain embodiments, the molar ratio of phospholipids to CaCh is about 4: 1 to about 2: 1. In further embodiments, the molar ratio of phospholipids to CaCh is about 2: 1.

[0097] In certain embodiments, the AmB has a crystallinity greater than about 75%. In further embodiments, the AmB has a crystallinity greater than about 85%. In yet further embodiments, the AmB has a crystallinity greater than about 95%.

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

[0099] (i) about 30.0% w / w amphotericin B (AmB);

[0100] (ii) about 14.6% w / w cholesterol (Choi);

[0101] (iii-a) about 35.8% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0102] (iii-b) about 15.9% w / w distearoylphosphatidylglycerol (DSPG); and

[0103] (iv) about 3.7% w / w calcium chloride (CaCh).

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

[0105] (i) about 14.0% w / w amphotericin B (AmB);

[0106] (ii) about 2.3% w / w cholesterol (Choi);

[0107] (iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0108] (iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0109] (iv) about 5.52% w / w calcium chloride (CaCh).

[0110] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0111] (i) about 14.0% w / w amphotericin B (AmB);

[0112] (ii) about 6.81% w / w cholesterol (Choi);

[0113] (iii-a) about 51.2% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0114] (iii-b) about 22.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0115] (iv) about 5.2% w / w calcium chloride (CaCh). In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0116] (i) about 3.4% w / w amphotericin B (AmB);

[0117] (ii) about 0.57% w / w cholesterol (Choi);

[0118] (iii-a) about 80.72% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0119] (iii-b) about 8.97% w / w distearoylphosphatidylglycerol (DSPG); and

[0120] (iv) about 6.34% w / w calcium chloride (CaCh).

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

[0122] (i) a Chol / AmB ratio of about 0.4 to 1.2 mol / mol;

[0123] (ii) a Chol / PL ratio of less than about 0.05 w / w;

[0124] (iii) a HSPC / DSPG ratio of about 2.3 to about 9.0 w / w; and

[0125] (iv) a PL / Ca2+ratio of about 2: 1 mol / mol.

[0126] In certain embodiments, the AmB and Choi are not complexed; and the AmB is not encapsulated in liposomes.

[0127] In certain embodiments, the AmB is coated with a porous shell of phospholipids and Choi.

[0128] In certain embodiments, the pharmaceutical composition is formulated as a dry powder.

[0129] In certain embodiments, the mass median diameter, X50, 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.

[0130] In certain embodiments, the tapped density of the powder particles is about 0.03 to about 0.4 g / mL. In further embodiments, the tapped density of the powder particles is about 0.06 to about 0.2 g / mL.

[0131] 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 further 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. In certain embodiments, the composition is formulated for pulmonary administration or airway administration.

[0136] In certain embodiments, the composition is formulated for aerosol administration. In further embodiments, the composition is formulated for aerosol administration as a dry powder.

[0137] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles coated with a porous shell of phospholipids (PL) and Choi, wherein the crystallinity of AmB is at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0138] In certain embodiments, the mass median aerodynamic diameter (MMAD) of the powder particles is about 1.5 pm to about 4.0 pm.

[0139] In certain embodiments, the mass median aerodynamic diameter (MMAD) of the powder particles is about 2.0 pm to about 3.5 pm.

[0140] In certain embodiments, a carrier-free dry powder composition of AmB, Choi, and PL is provided, wherein AmB is not encapsulated in the lipids, and the AmB and lipids are phase separated in their own domains.

[0141] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles coated with a porous shell of PL and Choi, wherein the Chol / AmB ratio is about 0.05 to about 1.2 mol / mol, such as about 0.2 to about 0.6 mol / mol.

[0142] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB 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.

[0143] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles coated with a porous shell comprising PL and Choi. In certain embodiments, the PL comprises hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylglycerol (DSPG), or a combination thereof.

[0144] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB 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.

[0145] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB 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.

[0146] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the percentage of AmB in the composition is less than 60% w / w, such as less than 30% or less than 20% w / w. In certain embodiments, the drug loading is about 0.5% to about 25% w / w, and the nominal Chol / AmB ratio is about 0.05 to about 1.2 mol / mol.

[0147] In certain embodiments, a dry powder composition of engineered particles is provided (ABCL003) that includes spray-dried core-shell particles of fine crystalline AmB particles (about 14.0% w / w) coated with a porous shell of PL and Choi, wherein the Chol / AmB 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.

[0148] In certain embodiments, a dry powder composition of engineered particles is provided that includes spray-dried core-shell particles of fine crystalline AmB particles (14.0% w / w) coated with a porous shell of PL and Choi, where the Chol / AmB 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.

[0149] In certain embodiments, the maximum Chol / AmB 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 / AmB to 0.4 mol / mol may allow higher drug loadings (e.g., no more than 22% w / w) within the so phase.

[0150] 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.

[0151] 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.

[0152] In certain embodiments, selection of the compositions described herein is driven by maximal increases in ASL pH that are maintained across a wide range of AmB concentrations.

[0153] In certain embodiments, selection of the compositions described herein is driven by decreased hygroscopicity relative to compositions with HSPC / DSPG < 9.0. In certain embodiments, selection of the compositions described herein is driven by increased manufacturing yield.

[0154] In certain embodiments, selection of the compositions described herein is driven by improved powder flowability.

[0155] In certain embodiments, selection of the compositions described herein is driven by improved aerosol performance relative to powders containing the lo phase.

[0156] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles 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.

[0157] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles coated with a porous shell of PL, Choi, and calcium chloride (CaCh), wherein the mass median diameter (X50) of the particles is about 1.0 to about 5.0 pm, such as about 1.5 to about 4.0 pm.

[0158] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles 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.

[0159] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles 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%.

[0160] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles 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.

[0161] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of AmB drug particles 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. In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline AmB drug particles 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 AmB crystals suspended in an oil-in-water emulsion stabilized by a monolayer of the mixture of lipids described herein.

[0162] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline AmB drug particles 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.

[0163] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline AmB drug particles 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 certain embodiments, the outlet temperature is at least 50 °C, at least 60 °C, or at least 70 °C.

[0164] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline AmB drug particles 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 certain embodiments, the outlet temperature is at least 60 °C, or at least 70 °C.

[0165] In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline AmB drug particles 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.

[0166] In certain embodiments, the dry powder composition of engineered particles comprising AmB drug particles coated with a porous shell of PL, Choi, and calcium chloride (CaCh), is filled with a drum filler.

[0167] 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 about 15 mg.

[0168] In certain embodiments, the powder fill mass has good precision (e.g., RSD < 3%) and accuracy for the target fill mass. Ion Channel Prosthetic Compositions Comprising AmB-AA

[0169] In certain embodiments, the ion channel prosthetic is formulated as a pharmaceutical composition comprising:

[0170] (i) amphotericin B-AA (AmB-AA), or a pharmaceutically acceptable salt or hydrate thereof;

[0171] (ii) cholesterol (Choi);

[0172] (iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and

[0173] (iv) calcium chloride (CaCh).

[0174] In certain embodiments, the amount of AmB-AA is about 0.5% to about 25% w / w. In further embodiments, the amount of AmB-AA is about 3% to about 16% w / w. In yet further embodiments, the amount of AmB-AA is about 14% w / w.

[0175] In certain embodiments, the amount of Choi is about 0.1% to about 8% w / w. In further embodiments, the amount of Choi is about 0.3% to about 6% w / w. In yet further embodiments, the amount of Choi is about 0.5% to about 3% w / w.

[0176] In certain embodiments, the amount of CaCh is about 1% to about 10% w / w. In further embodiments, the amount of CaCh is about 4% to about 7% w / w.

[0177] In certain embodiments, the amount of phospholipids is about 60% to about 95% w / w. In further embodiments, the amount of phospholipids is about 70% to about 90% w / w.

[0178] In certain embodiments, the weight ratio of Choi to phospholipids is about 0.001 : 1 to about 0.1 : 1. In further embodiments, the weight ratio of Choi to phospholipids is about 0.005 : 1 to about 0.05: 1.

[0179] In certain embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 2: 1 to about 19: 1. In further embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 7:1 to about 12: 1.

[0180] In certain embodiments, the molar ratio of Choi to AmB-AA is about 0.05: 1 to about 1.2:1. In further embodiments, the molar ratio of Choi to AmB-AA is about 0.4: 1 to about 1.2:1.

[0181] In yet further embodiments, the molar ratio of Choi to AmB-AA is about 0.05 : 1 to about 0.4: 1. In still further embodiments, the molar ratio of Choi to AmB-AA is about 0.4: 1.

[0182] In certain embodiments, the molar ratio of phospholipids to CaCh is about 4: 1 to about 2: 1. In further embodiments, the molar ratio of phospholipids to CaCh is about 2: 1. In certain embodiments, the AmB-AA has a crystallinity greater than about 75%. In further embodiments, the AmB-AA has a crystallinity greater than about 85%. In yet further embodiments, the AmB-AA has a crystallinity greater than about 95%.

[0183] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0184] (i) about 5.0% w / w AmB-AA;

[0185] (ii) about 2.3% w / w cholesterol (Choi);

[0186] (iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0187] (iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0188] (iv) about 5.52% w / w calcium chloride (CaCh).

[0189] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0190] (i) about 5.0% w / w AmB-AA;

[0191] (ii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0192] (ii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0193] (iii) about 5.52% w / w calcium chloride (CaCh).

[0194] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0195] (i) about 30.0% w / w AmB-AA;

[0196] (ii) about 14.6% w / w cholesterol (Choi);

[0197] (iii-a) about 35.8% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0198] (iii-b) about 15.9% w / w distearoylphosphatidylglycerol (DSPG); and

[0199] (iv) about 3.7% w / w calcium chloride (CaCh).

[0200] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0201] (i) about 14.0% w / w AmB-AA;

[0202] (ii) about 6.81% w / w cholesterol (Choi);

[0203] (iii-a) about 51.2% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0204] (iii-b) about 22.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0205] (iv) about 5.2% w / w calcium chloride (CaCh).

[0206] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0207] (i) about 14.0% w / w AmB-AA;

[0208] (ii) about 2.3% w / w cholesterol (Choi);

[0209] - 1 - (iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0210] (iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0211] (iv) about 5.52% w / w calcium chloride (CaCh).

[0212] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0213] (i) about 3.4% w / w AmB-AA;

[0214] (ii) about 0.57% w / w cholesterol (Choi);

[0215] (iii-a) about 80.72% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0216] (iii-b) about 8.97% w / w distearoylphosphatidylglycerol (DSPG); and

[0217] (iv) about 6.34% w / w calcium chloride (CaCh).

[0218] In certain embodiments, the pharmaceutical composition comprises:

[0219] (i) a Chol / AmB-AA ratio of about 0.2 to about 1.0 mol / mol;

[0220] (ii) a Chol / PL ratio of less than about 0.05 w / w;

[0221] (iii) a HSPC / DSPG ratio from about 2.0 to about 10 w / w; and

[0222] (iv) a PL / Ca2+ratio of about 2: 1 mol / mol.

[0223] In certain embodiments, the AmB-AA and Choi are not complexed; and the AmB-AA is not encapsulated in liposomes.

[0224] In certain embodiments, the AmB-AA is coated with a porous shell of phospholipids and Choi.

[0225] In certain embodiments, the present disclosure relates to pharmaceutical compositions described comprising highly crystalline AmB-AA 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.

[0226] 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.

[0227] 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’ (Id), 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.

[0228] 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.

[0229] Table 1. Hydrated Gel to Liquid Crystal Main Transition Temperatures of Various

[0230] Phospholipids

[0231] 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) > phosphatidyl serine (PS) > phosphatidylglycerol (PG) ~ phosphatidylcholine (PC). The hydrated Tmvalues for HSPC and DSPG are 53.6 and 55 °C, respectively.

[0232] 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.

[0233] 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).

[0234] 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.

[0235] 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.

[0236] 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 from 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).

[0237] 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 spray-drying, 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 shell-forming excipient.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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).

[0248] Methods of Treatment

[0249] In certain aspects, provided herein is a method of treating a disease characterized by reduced function or decreased expression of an ion channel, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0250] (a) an ion channel prosthetic selected from: or a pharmaceutically acceptable salt or hydrate of either of them; and

[0251] (b) an ion channel modulator.

[0252] In certain embodiments, the disease is cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease. In further embodiments, the disease is cystic fibrosis.

[0253] In certain aspects, provided herein is a method of increasing transepithelial current of airway cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0254] (a) an ion channel prosthetic selected from:

[0255] or a pharmaceutically acceptable salt or hydrate of either of them; and

[0256] (b) an ion channel modulator.

[0257] In certain aspects, provided herein is a method of increasing the pH of airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0258] (a) an ion channel prosthetic selected from: or a pharmaceutically acceptable salt or hydrate of either of them; and

[0259] (b) an ion channel modulator.

[0260] In certain aspects, provided herein is a method of increasing bicarbonate secretion into airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:

[0261] (a) an ion channel prosthetic selected from: or a pharmaceutically acceptable salt or hydrate of either of them; and

[0262] (b) an ion channel modulator.

[0263] In certain embodiments, the subject has cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease. In further embodiments, the subject has cystic fibrosis.

[0264] In certain aspects, provided is a method of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of: (a) an ion channel prosthetic selected from:

[0265] or a pharmaceutically acceptable salt or hydrate of either of them; and

[0266] (b) an ion channel modulator.

[0267] In certain embodiments, the ion channel modulator is a CFTR ion channel modulator. Generally, the therapeutic combination of elexacaftor, tezacaftor, and ivacaftor may be referred to by the name Trikafta. Accordingly, in further embodiments, the CFTR ion channel modulator is selected from the group consisting of elexacaftor, tezacaftor, ivacaftor, lumacaftor, and forskolin, and any combinations thereof.

[0268] In certain embodiments, the cystic fibrosis is refractory to treatment with ivacaftor; or the combination of elexacaftor, tezacaftor, and ivacaftor.

[0269] In certain embodiments, a subject with cystic fibrosis has Class 1 or other mutations that are not treated effectively with current modulators, including combination therapies. This includes nonsense mutations or other mutations where no functional cystic fibrosis transmembrane conductance regulator (CFTR) protein is produced.

[0270] In certain embodiments, the subject has two mutations in the CFTR anion channel, each copy of the CFTR gene contains one of the two mutations, and wherein the two mutations are each independently selected from the mutations listed in Table 2.

[0271] Table 2. Exemplary CTFR Mutations

[0272]

[0273] In certain embodiments, the two mutations are each independently selected from 2184delA, F508del, V520F, 1717-1G->A, E60X, G551D, R553X, and D259G.

[0274] In certain embodiments, the two mutations are a pair of CFTR mutations selected from F508del / F508del, G551D / F508del, R553X / E60X, F508del / 1717-lG->A, F508del / 2184delA, and D259G / V520F.

[0275] In certain embodiments, the two mutations are a pair of CFTR mutations selected from F508del / F508del, R553X / E60X, F508del / 1717-lG->A, F508del / 2184delA, and

[0276] D259G / V520F.

[0277] In certain embodiments, the nominal dose or metered dose of the ion channel prosthetic is 0.01 mg to 10 mg.

[0278] In certain embodiments, the nominal dose or metered dose of the ion channel prosthetic is about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.0 mg, or about 4.0 mg.

[0279] In certain embodiments, the ion channel prosthetic is administered to an airway of the subject.

[0280] In certain embodiments, the ion channel prosthetic is administered to the bronchial airways of the subject.

[0281] In certain embodiments, the ion channel prosthetic is administered as an aerosol.

[0282] In certain embodiments, the ion channel prosthetic is administered as a dry powder aerosol.

[0283] In certain embodiments, the ion channel prosthetic is administered at least once daily, at least twice daily, or at least three times daily.

[0284] In certain embodiments, the ion channel prosthetic is administered once daily.

[0285] In certain embodiments, the ion channel prosthetic is administered at least once weekly, at least twice weekly, or at least three times weekly. In certain embodiments, the administering comprises initially delivering a loading dose of the ion channel prosthetic; and subsequently delivering a maintenance dose of the ion channel prosthetic; wherein the mass ratio of the loading dose to the maintenance dose is about 2: 1 to about 5 : 1. In further embodiments, the mass ratio of the loading dose to the maintenance dose is about 2.5: 1 to about 3: 1. In yet further embodiments, the loading dose is about 1.0 mg to about 20 mg, about 3 mg to about 15 mg, or about 6 mg to about 10 mg. In sill further embodiments, the maintenance dose is about 0.1 mg to about 5 mg, about 0.5 mg to about 5 mg, about 1 mg to about 4 mg, or about 2 mg. In certain embodiments, the loading dose is about 1.5 mg and the maintenance dose is about 0.5 mg. In further embodiments, the loading dose is about 6 mg and the maintenance dose is about 2 mg. In yet further embodiments, the loading dose is about 10 mg and the maintenance dose is about 4 mg.

[0286] All of the masses described above for the loading dose and the maintenance dose refer to the mass of the nominal dose or the metered dose (i.e., not the emitted dose) of the ion channel prosthetic and the ion channel modulator.

[0287] In certain embodiments, provided herein is a method of treating a subject with cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease, comprising administering to a subject in need thereof an effective amount of a lipid-coated crystal composition comprising an ion channel prosthetic and Choi as provided in this disclosure, wherein the lipid-coated crystal composition is administered to the subject via oral inhalation to the bronchial airways in the lungs.

[0288] In certain embodiments, provided herein is a method of treating a subject with cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease, comprising administering to a subject in need thereof an effective amount of a lipid-coated crystal composition comprising an ion channel prosthetic and Choi as provided in this disclosure, wherein the composition provides an increase in ASL pH of about 0.2 to about 1.0 pH units.

[0289] In certain embodiments, provided herein is a method of increasing lung function in individuals with cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease, comprising administering to a subject in need thereof an effective amount of an ion channel prosthetic and an ion channel modulator according to any one of the embodiments described herein, thereby increasing lung function in the subject; wherein the administration is pulmonary.

[0290] In certain embodiments provided herein is a method of increasing health related quality of life (HRQoL) in individuals with cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease, comprising administering to a subject in need thereof an effective amount of an ion channel prosthetic and an ion channel modulator according to any one of the embodiments described herein, thereby increasing HRQoL in the subject; wherein the administration is pulmonary.

[0291] In certain embodiments, provided herein is a method of treating a subject with cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease, comprising administering to a subject in need thereof an effective amount of a lipid-coated crystal composition comprising an ion channel prosthetic and Choi as provided in this disclosure, wherein the lipid-coated crystal composition provides a clinically significant increase in health-related quality of life relative to placebo.

[0292] In certain embodiments, provided herein is a method of improving lung function, comprising administering to a subject in need thereof an effective amount of the ion channel prosthetic and the ion channel modulator of the present disclosure, thereby improving lung function in the subject; wherein the administration is pulmonary.

[0293] In certain embodiments, the ion channel prosthetic is formulated as a dry powder.

[0294] In certain embodiments, the dry powder is administered with a portable dry powder inhaler.

[0295] In certain embodiments, the dry powder inhaler utilizes the subjects inspiratory flow to fluidize and disperse the powder (z.e., the device is a passive dry powder inhaler)

[0296] In certain embodiments, the dry powder inhaler has a medium to high resistance to airflow are preferred (i.e., about 0.10 to 0.30 cm H2O0 5L'1min).

[0297] In certain embodiments, the portable dry powder inhaler includes capsule-based unit dose DPIs, single-use disposable DPIs, or blister-based multi-dose dry powder inhalers.

[0298] Over 1900 different CFTR mutations are found in cystic fibrosis patients, hundreds of which are confirmed to be disease causing through at least five different mechanisms of functional loss. There have been important recent advances in the development of genotypespecific small molecule drugs that bind to certain mutant forms of CFTR and thereby increase its activity. However, about 10% of cystic fibrosis patients have CFTR genotypes that do not respond to current small molecule treatments. These include major truncations that yield a complete lack of functional CFTR protein and very rare mutations for which the mechanistic underpinnings of functional deficiency are unknown.

[0299] In certain embodiments, any of the methods disclosed herein treat a mutation class of cystic fibrosis selected from the group consisting of I, II, III, IV, V, VI, U, and combinations thereof. In certain embodiments, the mutation class is selected from the group consisting of I / I, II / II, II / U, and U / III.

[0300] In certain embodiments, the methods disclosed herein are genotype-independent treatments.

[0301] As used herein, the phrases “genotype-independent” or “genotype-agnostic” refer to any treatment that is independent of the exact nature of the genetic mutation underlying the reduced CFTR expression or reduced CFTR function in cystic fibrosis.

[0302] In certain embodiments, the methods described herein are useful in the treatment of various cystic fibrosis genotypes that are typically non-responsive or minimally responsive to treatment with conventional cystic fibrosis therapeutics. For example, the V520F allele in patients having the D259G / V520F pair of CTFR mutations is refractory to treatment with ivacaftor; or the combination of elexacaftor, tezacaftor, and ivacaftor.

[0303] Accordingly, in certain embodiments, the methods of treating cystic fibrosis described herein are agnostic to cystic fibrosis genotype.

[0304] In certain embodiments, the methods disclosed herein treat refractory or resistant cystic fibrosis. In certain embodiments, the cystic fibrosis is refractory or resistant to one or more cystic fibrosis treatments.

[0305] The pH of airway surface liquid (ASL) in a subject can be measured using any technique known to those of skill in the art. For example, airway pH can be measured by placing a planar pH-sensitive probe on the tracheal surface. Pezzulo AA et al. (2012) Nature. 487: 109-113.

[0306] The pH of ASL in a subject is said to be increased when it is measurably greater than the pH of ASL of an untreated subject. In one embodiment, the pH of ASL in a subject is said to be increased when it is measurably greater than the pH of ASL of the same subject measured prior to or distant in time from treatment according to a method of the present disclosure.

[0307] Remarkably, rescue of ASL pH is observed over a wide range of ion channel prosthetic: sterol concentrations. Accordingly, the method of increasing ASL pH described herein has significant implications for clinical applications.

[0308] In certain embodiments, the increase in pH can be 0.1 pH units to 2.0 pH units. In certain embodiments, the increase in pH can be 0.1 pH units to 1.0 pH units. In certain embodiments, the increase in pH can be 0.3 pH units to 1.0 pH units. In certain embodiments, the increase in pH can be 0.3 pH units to 1.0 pH units. In certain embodiments, the increase in pH can be 0.5 pH units to 0.9 pH units. In certain embodiments, the increase in pH can be 0.5 pH units to 0.8 pH units. In certain embodiments, the increase in pH can be 0.6 pH units to 0.8 pH units. In certain embodiments, the increase in pH can be about 0.7 pH units. In certain embodiments, the increase in pH can be 0.1 pH units to 0.5 pH units. In certain embodiments, the increase in pH can be 0.1 pH units to 0.4 pH units. In certain embodiments, the increase in pH can be 0.2 pH units to 0.4 pH units. In certain embodiments, the increase in pH can be 0.1 pH units to 0.3 pH units. In certain embodiments, the increase in pH can be 0.2 pH units to 0.3 pH units. In certain embodiments, the increase in pH can be 0.1 pH units to 0.2 pH units.

[0309] In certain embodiments, the increase in pH is by apical addition of any one of the methods disclosed herein.

[0310] In certain embodiments, the methods described herein are useful for increasing the pH of airway surface liquid in a patient regardless of their genotype, including a patient having any one of various cystic fibrosis genotypes that are typically non-responsive or minimally responsive to treatment with conventional cystic fibrosis modulator / corrector therapeutics.

[0311] Accordingly, in certain embodiments, the methods of increasing ASL pH described herein are agnostic to the patient’s cystic fibrosis genotype.

[0312] In certain embodiments, any of the methods disclosed herein increase ASL pH in patients having refractory or resistant cystic fibrosis. In certain embodiments, the cystic fibrosis is refractory or resistant to one or more cystic fibrosis treatments, such as ivacaftor; or the combination of elexacaftor, tezacaftor, and ivacaftor.

[0313] In certain aspects, provided herein is a method of restoring anion transport, increasing airway surface liquid hydration and height, thereby decreasing the viscosity of airway surface liquid in a patient having cystic fibrosis, comprising administering to a patient having cystic fibrosis a therapeutically effective amount of the ion channel prosthetic and the ion channel modulator of the present disclosure.

[0314] Conventional understanding of diseases characterized by reduced function or decreased expression of an ion channel (e.g., cystic fibrosis) held that treatment with both an ion channel modulator and an ion channel prosthetic would not lead to an additive therapeutic effect due to the ionic gradient across the relevant cell membrane. However, the studies disclosed herein demonstrate that treatment with both an ion channel prosthetic and an ion channel modulator unexpectedly affords an additive increase in ion transport. Accordingly, in certain embodiments, the administration of the ion channel prosthetic and the ion channel modulator results in an additive effect.

[0315] In certain embodiments, restoration of anion secretion (e.g., bicarbonate secretion) leads to increases in airway surface liquid pH, increased airway hydration, increased airway surface liquid height, increases in the antimicrobial activity of airway surface liquid, or a combination thereof. These improvements in the properties of airway surface liquid may result in clinical improvements in mucociliary clearance, measures of lung function, patient health related quality of life (HRQoL), or a combination thereof.

[0316] In certain embodiments, HRQoL is measured using the Cystic Fibrosis Questionnaire (CFQ). Henry, B. et al., Qua Life Res. 2003 Feb; 12(1): 63-76. In certain embodiments, HRQoL is measured using the Cystic Fibrosis Questionnaire Revised (CFQ-R). Wenninger, K. et al., Qual Life Res. 2003 Feb; 12(1): 77-85. In certain embodiments, administering the present compositions to a patient provides an increase in HRQoL domain score of at least 0.5, at least 0.8, at least 1, or at least 2 as measured by the CFQ or the CFQ-R. In certain embodiments, a subject’s HRQoL domain score is said to be increased when it is measurably greater than the same subject’s HRQoL domain score measured prior to or distant in time from treatment according to a method of the present disclosure. In certain embodiments, a subject’s HRQoL domain score is said to be increased when it is measurably greater than the HRQoL domain score of an untreated subject.

[0317] Subjects with cystic fibrosis may have a reduced ASL height compared to a healthy subject. Muraglia, Katrina A. et al., Nature 2019 Mar; 567(7748):405-408. In certain embodiments, a subject with cystic fibrosis has an ASL height of about 3 pm to about 4 pm. In certain embodiments, the methods described herein increase the ASL height of a subject with cystic fibrosis to the ASL height of a healthy subject (e.g., to an ASL height of about 9 pm to about 10 pm).

[0318] In certain embodiments, the methods described herein increase a subject’s ASL height by about 1 pm to about 8 pm, about 1 pm to about 5 pm, about 1 pm to about 3 pm, about 2 pm to about 8 pm, about 2 pm to about 5 pm, about 3 pm to about 8 pm, about 3 pm to about 5 pm, about 5 pm to about 7 pm, or about 6 pm. In certain embodiments, a subject’s ASL height is said to be increased when it is measurably greater than the same subject’s ASL height measured prior to or distant in time from treatment according to a method of the present disclosure. In certain embodiments, a subject’s ASL height is said to be increased when it is measurably greater than the ASL height of an untreated subject.

[0319] In certain aspects, provided herein is a method of decreasing the viscosity of airway surface liquid, comprising administering to a subject in need thereof an effective amount of the ion channel prosthetic and the ion channel modulator of the present disclosure, thereby decreasing the viscosity of airway surface liquid in the subject; wherein the administration is pulmonary. In certain embodiments, the ion channel prosthetic is administered to an airway of the subject by a dry powder aerosol.

[0320] In certain embodiments, the ion channel prosthetic is administered to the bronchial airways of the subject by a dry powder aerosol.

[0321] In certain embodiments, an airway of a subject refers to the so-called conducting or bronchial airways, i.e., any or all of the following pulmonary structures: trachea, bronchi, and bronchioles in the lower respiratory tract.

[0322] In certain embodiments, the methods described herein are useful for decreasing the viscosity of airway surface liquid in a patient having any one of various cystic fibrosis genotypes that are typically non-responsive or minimally responsive to treatment with conventional cystic fibrosis therapeutics.

[0323] Accordingly, in certain embodiments, the methods of decreasing ASL viscosity described herein are agnostic to the patient’s cystic fibrosis genotype.

[0324] In certain embodiments, any of the methods disclosed herein decrease ASL viscosity in patients having refractory or resistant cystic fibrosis. In certain embodiments, the cystic fibrosis is refractory or resistant to one or more cystic fibrosis treatments, such as ivacaftor; or the combination of elexacaftor, tezacaftor, and ivacaftor.

[0325] In accordance with each of the foregoing embodiments, in certain embodiments, the patient is a human.

[0326] In certain embodiments, the range of ages that are amenable to treatment with the methods described herein is driven by an ability to effectively utilize a passive dry powder inhaler (DPI). Subjects below the age of 6 may not have the cognitive ability to properly perform the steps involved in use of a passive DPI. Moreover, they may lack the respiratory muscle strength needed to generate the inspiratory pressures required. Nevertheless, certain passive DPIs have been approved for subjects down to age 4. Similarly, certain geriatric patients may also have cognitive or atrophied muscles that prevent proper use of passive DPIs. Inspiratory pressures tend to increase with age, maximizing at about age 25, plateauing until about age 40 after which they steadily decrease. Males tend to achieve higher inspiratory pressures than females. Increases in airways disease may also negatively impact the ability for subjects to achieve the pressure drop (~1.0 kPa) needed to effectively operate the passive DPI. Subjects with poor inspiratory muscle strength can be identified based on their ability to achieve a 1.0 kPa inspiratory pressure with an In-Check dial. In accordance with each of the foregoing embodiments, in certain embodiments, the patient is between the ages of 4 and 80 years old.

[0327] In certain embodiments, the patient is at least 4 years old. In certain embodiments, the patient is at least 6 to about 18 years old. In certain embodiments, the patient is under the age of about 80 years old with acceptable inspiratory muscle strength. In certain embodiments, the patient is about 60 to about 70 years old. In certain embodiments, the patient is about 70 to about 80 years old.

[0328] In certain embodiments, any of the methods disclosed herein permeabilize the apical membrane.

[0329] In certain embodiments, any of the methods disclosed herein permeabilize the apical membrane to protons.

[0330] In certain embodiments, any of the methods disclosed herein permeabilize the apical membrane to bicarbonate anions.

[0331] Definitions

[0332] 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.

[0333] 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.

[0334] “Effective amount” as used herein refers to any amount that is sufficient to achieve a desired biological effect.

[0335] “Therapeutically effective amount” as used herein refers to any amount that is sufficient to achieve a desired therapeutic effect, e.g., treating cystic fibrosis. "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).

[0336] "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.

[0337] "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%.

[0338] "Drug Loading" as used herein refers to the percentage of active ingredient(s) on a mass basis in the total mass of the composition.

[0339] "Mass median diameter" or "MMD" or “X50” as used herein means the median diameter of a plurality of particles, typically in a polydisperse particle population, i.e., consisting of a range of particle sizes. The X50 values as reported herein are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany), unless the context indicates otherwise.

[0340] "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.

[0341] "Mass median aerodynamic diameter" or "MMAD" as used herein refers to the median aerodynamic size of a plurality of particles, typically in a polydisperse population. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity, generally in air, as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle composition in terms of its settling behavior. The aerodynamic particle size distributions (APSD) and MMAD are determined herein by cascade impaction, using a NEXT GENERATION IMPACTOR™ (Copley Scientific). In general, if the particles are aerodynamically too large, fewer particles will reach specific regions of the lungs. If the particles are too small, a larger percentage of the particles may be exhaled. In contrast, darepresents the aerodynamic diameter of a single particle.

[0342] "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.

[0343] "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).

[0344] "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™.

[0345] "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.

[0346] As used herein, an “airway of a subject” refers to any or all of the following pulmonary structures: trachea, bronchi, and bronchioles.

[0347] 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.

[0348] 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.

[0349] Unless otherwise stated, or clear from the context, numerical ranges include both the endpoints and any value between. EXAMPLES

[0350] The various aspects and embodiments of the present disclosure will be further clarified with reference to the following examples. The examples are illustrative in nature and should not be understood to limit the subject matter of the present disclosure.

[0351] Characterization Methods

[0352] Ion Channel Prosthetic Content and Purity. Ion channel prosthetic content and purity in formulated bulk powder or aerodynamic particle size distribution (APSD) samples were determined by reverse phase high performance liquid chromatography (RP-HPLC) with detection at 383 nm. Samples were analyzed with an Agilent 1260 Infinity II HPLC system (Wilmington, DE, USA). Separation was achieved with Agilent InfinityLab Poroshell 120 EC- C18, 3.0 x 150 mm, (2.7pm) column using a gradient method (solvent A = 10 mM acetate buffer, pH 4.2; solvent B = acetonitrile / methanol, 1.0 v / v). Ion channel prosthetic was quantified using a single-point calibration using a USP certified reference standard.

[0353] 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 singlepoint calibration using Cholesterol HP, Ph. Eur / USP-NF raw material (Carbogen Amcis, Beuvry-la-Foret, France).

[0354] 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.

[0355] 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.

[0356] Bulk Density. The bulk density (Pbuik) represents the mass of the powder that is loaded into the sample holder to the requisite volume without tapping.

[0357] Carr ’s Index. Carr’s index, C, provides an indication of powder compressibility. It is given by:

[0358] „ > Ptapped Pbulk .nnC — X 100

[0359] Ptapped

[0360] As Carr’s index increases, the flowability of the powder is thought to decrease. Values of <10% are indicative of excellent free-flowing powders, values between 11-15% are associated with good free-flowing powders, between 16-20% with fair powder flow, between 21-25% passable powder flow, between 26-31% poor flow cohesive, between 32-37% very poor flow, and > 38% approximately no flow.

[0361] Water Content. Water content was determined by Karl Fischer titrimetry using a Nittoseiko Analytech Moisture Meter CA-310 with fritless cathode and Vaporizer Model VA- 300.

[0362] Dynamic Vapor Sorption. The moisture sorption isotherm at 25°C was measured using a dynamic vapor sorption (DVS) instrument made by Surface Measurement Systems, UK. This instrument gravimetrically measures uptake and loss of water vapor by a material. The DVS system is equipped with a recording microbalance with a resolution of ±0.1 pg and a daily drift of approximately ±1 pg. In the first step of the experimental run, the sample was dried at 25°C and 0% RH for 24 hours to bring the sample to a constant mass. Then, the instrument was programmed from 0% to 2% RH, to 5% RH, and then RH was increased in steps of 5% RH to 90% RH and decreased in steps of 5% RH from 90% to 0% RH. An equilibration criterion of dm / dt =0.005% / min was chosen for the system to achieve at each RH step before automatically proceeding to the next RH step. Sample masses between 10 and 15 mg were used in this study.

[0363] Differential Scanning Calorimetry. The DSC thermogram of a given sample was measured using a TA Instruments Model Q2000 differential scanning calorimeter equipped with a Refrigerated Cooling System (New Castle, Delaware). The sample cell was purged with dry nitrogen at a flow rate of 50 cm3 / min; the Refrigerated Control System (RCS) used nitrogen at a flow rate of 110 cm3 / minute. Tzero aluminum pans that contained between about 5 and 10 mg of powder were hermetically sealed using a sample encapsulation press. Samples were equilibrated at -40 °C, then heated at 5 °C / min to 200 °C. Aerodynamic Particle Size Distributions. Aerodynamic particle size distributions (APSD) were determined utilizing the RS01 dry powder inhaler (Mod. 7 Ultra High Resistance 2 Model), USP induction port (IP), and Next Generation Impactor™ or NGI™ and conforms with USP <601> and Ph. Eur. 2.9.18. The flow control apparatus was adjusted to operate at a 4 kPa pressure drop and for a total volume of 4 L through the inhaler. The RS01 DPI variant utilized has a resistance of 0.143 cm H2O0 5L'1min (0.045 kPa0 5L'1min). This corresponds to a flow rate of 44.2 L min'1at a 4 kPa pressure drop. Approximately 10 mg was hand filled into size #3 inhalation grade HPLC capsules (VCaps, Qualicaps). The aerosol powder emitted from the inhaler was drawn through the USP IP and sized in the NGI. Each stage of the NGI, emptied capsule and device were extracted with the sample dissolution solution comprising methanol. Further dilutions were made to reduce the AmB concentration within the linearity of the detection range. The ion channel prosthetic mass on each stage was determined using the HPLC method described above and the fine particle dose less than 5 pm (FPD 5 gm) and mass median aerodynamic diameter (MMAD) were calculated.

[0364] Example 1: Compositions of Lipid-Coated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004)

[0365] The nominal anhydrous compositions of four compositions of lipid-coated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004) are presented in Table 3. Also shown are three controls: AmBisome® (i.e., liposomal amphotericin B, L-AmB); the lyophilized AmB / Chol complexes prepared by Burke et al. (US 2019 / 0083517; US 2020 / 0352970); and amphotericin B inhalation powder (ABIP), a dry powder composition of AmB developed by Nektar Therapeutics for the treatment of invasive pulmonary aspergillosis (US 7,326,691; US 8,404,217; US 2012 / 0128728).

[0366] Table 3. Theoretical Anhydrous Inhaled AmB Compositions

[0367] a In addition to the lipids and AmB, AmBisome contains 67.8% sucrose, 2.0% disodium succinate hexahydrate, and 0.2% tocopherol b AB IP utilizes synthetic DSPC instead of HSPC

[0368] Although commercial L-AMB contains the same lipid components as the ABCI compositions (i.e., HSPC, DSPG, Choi), they are present in different proportions. Further, the drug and lipids are organized differently in L-AMB in comparison to the ABCI compositions. The drug substance in L-AMB is encapsulated in small unilamellar vesicles (liposomes). The composition has a Chol / AmB ratio of 2.5 mol / mol and an HSPC / DSPG ratio of 2.3 (i.e., 7 / 3 w / w). The lipid particles are lyophilized into a dry powder in the presence of a large percentage of sucrose, which serves as a cryoprotectant to maintain liposome integrity during lyophilization. Administration of 60 mg of reconstituted L-AMB to the apical side of airway epithelial cells through a 1 cm2tracheal window in four CFTR’ ’ pigs, resulted in an increase in ASL pH of -0.2 pH units, from pH 6.8 to 7.0 (US 2020 / 0352970).

[0369] The lyophilized AmB: Choi complexes studied by Burke et al. (US 2019 / 0083517; US 2020 / 0352970) have an even higher Chol / AmB ratio of 5.0 mol / mol, but contain no added PL. The AmBUhol complexes are formed by flash nanoprecipitation from a solution of the materials in dimethylsulfoxide / chloroform when rapidly injected into a nonsolvent (water). The resulting suspension is lyophilized to form a dry powder. The AmBUhol complexes demonstrated significant increases in bicarbonate secretion and in ASL pH (~0.1-0.2 pH units) in CuFi-1 cells.

[0370] AB IP is a spray-dried composition comprising wet-milled AmB crystals coated with a 2: 1 molar ratio of DSPC to calcium chloride. This composition contains no cholesterol (i.e., Chol / AmB = 0 mol / mol). It also contains only saturated phosphatidylcholine (DSPC) with no added DSPG. While AB IP shows improvements in ASL pH in CuFi-1 cells at low doses (2 pM in FC-70), it does not at high doses (50 pM). This result is consistent with previous studies with neat AmB and suggests that some amount of Choi is necessary for maintaining ion channel activity across a wide range of AmB concentrations. ABIP compositions comprising highly crystalline drug substance have demonstrated good tolerability in early clinical development.

[0371] Compositions are also presented for the three ABCI compositions (ABCI-001, ABCI- 002, and ABCI-003), which have been used in nonclinical toxicology studies. These powders have Chol / AmB ratios of from 0.4 to 1.2 mol / mol. Without intending to be bound by theory, the high Chol / AmB ratios utilized in L-AMB and AmB: Choi complexes may be unsuitable for dry powder compositions comprising PL, as the presence of large amounts of Choi may lead to disorder in the packing of the PL acyl chains, resulting in unacceptable increases in interparticle cohesive forces and ‘sticky’ powders.

[0372] The DSC thermogram for ABCI-001 (30% w / w AmB, Chol / AmB = 1.2 mol / mol) contains a significant proportion of the Chol-rich lo phase (Example 4). For ABCL002, the drug content was decreased to 14% w / w while holding the Chol / AmB molar ratio constant, which leads to a decrease in the Chol / PL ratio of from 0.29 to 0.09 w / w. Nonetheless, a small proportion of the Chol-rich lo phase remained. Dry powders having the phase-separated lo phase present may exhibit strong interparticle cohesive forces and increased hygroscopicity and deliquescence at high relative humidity. These features may negatively impact powder yield during spray drying and may result in relatively large mass median aerodynamic diameters (MMAD ~ 4 pm).

[0373] ABCI-003 retains the 14% w / w drug loading in ABCI-002, but with a decreased nominal Chol / AmB ratio of 0.4 mol / mol. Overall, the Choi content is reduced from 14.6% in ABCI-001 to 2.4% in ABCI-003, and the Chol / PL ratio is reduced to 0.03 w / w. At this Chol / PL ratio the lo phase is solubilized in the so phase. In ABCI-003, the HSPC / DSPG ratio is also increased from 2.3 w / w to 9.0 w / w, leading to improved solubility of Choi in the PL phase, decreases in NaCl formation, and decreases in powder hygroscopicity. Unlike ABIP, all ABCI compositions containing added Choi exhibit significant improvements in ASL pH at both low and high AmB concentrations. Moreover, the presence of DSPG has been shown to bolster the magnitude of the ASL pH improvement observed.

[0374] Many of the constraints regarding Chol / AmB, Chol / PL, and HSPC / DSPG ratios may be specific to the development of dry powder compositions for inhalation and may not be applicable for liquid-based aerosols such as inhaled L-AMB. Nor may they be directly applicable to compositions that contain no PL.

[0375] Example 2: Manufacture of ABCI-001 via Spray Drying

[0376] Feedstock Preparation. The lipids and calcium chloride were first dispersed in hot water with a high-shear mixer (UltraTurrax T-50) to form multilamellar vesicles (MLVs). The aqueous phase must be above the main transition temperature of the lipids (Tm) to facilitate MLV formation (T > 65 °C). The MLV dispersion was cooled (T < 30°C), and perfluorooctyl bromide (PFOB) was filtered and added using a Watson-Marlow peristaltic pump 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 in a single, discrete pass under high pressure with a Model M-100 Microfluidizer 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 passed through the homogenizer for two additional discrete passes. The homogenization process wet mills the AmB particles to a suitable size for pulmonary delivery. On a number basis, most of the wet-milled AmB crystals have a diameter less than 1000 nm. In one embodiment, the final feedstock composition had a solids content of 3.0% w / w, a PFOB content of 20% v / v, and a theoretical batch size of 318 g (9 liters of feedstock) or 424.2 g (12 liters of feedstock).

[0377] Production of Dry Powders by Spray Drying. Spray drying was conducted with a pilotscale spray dryer (Niro Mobile Minor, Copenhagen, Denmark), equipped with a Schlick 970 / 0 twin-fluid atomizer (0.8 mm i.d.), a DorrClone cyclone, and a lower end geometry comprising a straight tube and Brewer valve. The 1 L Eagle collector attached beneath the Brewer valve was jacketed and maintained at 50°C.

[0378] The liquid feed was pumped into the spray dryer with a Watson-Marlow peristaltic pump. The atomizer was operated at a gas flow of 7.0 ± 0.6 scfm and an inlet temperature of 104 ± 5 °C. The liquid feed rate was adjusted to maintain a dryer outlet temperature of 55 ± 3 °C. The total gas flow was about 140 N m3 / h (~85 scfm).

[0379] In suspension-based feeds, each atomized droplet (mass median diameter ~ 10 pm) contains dispersed drug crystals and approximately 1000 sub-micron emulsion droplets. During the initial moments of the drying process, the more volatile aqueous phase begins to evaporate. The rapidly receding atomized droplet interface drives enrichment of the slowly diffusing drug and emulsion particles at the interface. This leads to formation of a void space in the center of the drying droplet. As the drying process continues, the less volatile oil phase in the emulsion droplets evaporates, resulting in formation of hollow pores in their place. Overall, the resulting hollow spray-dried composite particles contain drug crystals embedded in an interfacial layer of a porous lipid matrix.

[0380] ABCI-002, ABCI-003, and ABCI-004 were manufactured using the same general process, although the compositions of the liquid feeds differed.

[0381] Example 3: Wet-Milling of AmB and Impact on Properties of ABCI-002

[0382] The physical form of AmB (crystalline vs. amorphous) can have an impact on its wetmilling behavior. For this study two batches of AmB were obtained from North China Pharmaceutical Group Corp. (Hebei, China). The two batches differed in their crystallinity, with batches ‘202 and ‘203 having crystallinities of 77 and 96%, respectively (as determined by quantitative XRPD).

[0383] Indeed, the decreased crystallinity of batch ‘202 negatively impacted the wet-milling process, significantly increasing the X50 and X90 of the wet-milled drug as determined for suspensions of drug by laser diffraction with a Malvern Mastersizer (Table 4).

[0384] Table 4. Impact of AmB Crystallinity on Primary Particle Size Distribution During Wet- Milling Batches of ABCI-002 were manufactured on a Niro Mobile Minor with AmB batches ‘202 (ABCI-002 Batch FP21060) and ‘203 (ABCI-002 Batch FP21059), using the process described in Example 2. The total solids content and %PFOB in the liquid feedstocks were 2% w / w and 10% v / v, respectively. The liquid feed rate was about 44.5 g / min. The atomizer was operated at a gas flow of 11.5 ± 1.0 N m3 / h (7.0 scfm) and an inlet temperature of 104 ± 5 °C. This equated to about 44.5 g / min. The total gas flow was about 140 N m3 / h (~85 scfm). The target batch size was 40 g.

[0385] The physicochemical properties and aerosol performance of the two batches are detailed in Table 5. No significant differences in powder properties and aerosol performance are observed, suggesting that crystallinities as low as 77% remain suitable for preparing ABCI- 002 compositions.

[0386] Table 5. Comparison of the Physicochemical Properties and Aerosol Performance of ABCI- 002 Batches Prepared from AmB Batches with Different Crystallinities

[0387] Example 4: Impact of Lipids on Phase Behavior of Lipid-Coated Crystal Compositions of AmB

[0388] The addition of Choi and DSPG in the ABCI compositions leads to more complex thermograms with a broadening of the transitions and the presence of multiple peaks. At a Chol / PL ratio of 0.29 w / w (ABCI-001), two phase-separated domains are observed, a Chol-rich lo phase with an onset temperature of 62.5 °C, and a broad PL-rich so phase with an onset temperature of 83.9 °C. As demonstrated for hydrated DPPC bilayers, addition of Choi to the so phase leads to increased disorder and eventually phase separation of a coexisting lo phase.

[0389] The Chol-rich phase transition is very broad and contains overlapping features. The presence of Choi crystallites in the lo phase is supported by X-Ray Powder Diffraction (XRPD) patterns, which show a diffraction peak at about 5.2° 20 for ABCI variants. Phase separation of cholesterol crystallites is more easily distinguished (i.e., fewer diffraction peaks in the same region and no interference from Amphotericin B peaks) in PLCI-001. The PLCI compositions are placebo compositions that do not comprise AmB but comprise the other components of the ABCI compositions.

[0390] A linear increase in the enthalpy of the Chol-rich phase transition is observed with increases in Choi content in the powder composition. A linear regression was performed on the data points with Chol / (Chol+PL) > 10% w / w. The data extrapolate to the x-axis at a Choi / (Choi +PL) weight ratio of about 4.9% w / w. This is equivalent to a Chol / PL ratio of 0.05 w / w or ~9.4 mol% Choi. Thus, compositions with less than about 9.4 mol% Choi may be expected to have an undetectable low-temperature peak.

[0391] In the dehydrated state, the onset of the so — lo two-phase region tends to occur at a much higher Choi concentration. Hence, the ordered so phase in ABCI compositions is maintained at a much higher Choi content (~9.4 mol% vs. 6 mol% for the hydrated DPPC bilayer). As well, while the so phase is eliminated in the hydrated DPPC / Chol mixtures above about 20 mol% Choi, the so — lo two phase region extends beyond 36.5 mol% Choi in dehydrated ABCI-001. Hence, relative to hydrated DPPC-Chol mixtures phase diagram in, the phase diagram for ABCI-001 is shifted upward to higher Tmvalues, and to the right to higher cholesterol contents.

[0392] The high-Tmpeak in ABCI-001 is also very broad and comprises multiple overlapping features. The enthalpy of the high-Tmpeak tends to increase with increasing PL content and tends to decrease with increasing Choi content, suggesting that this peak below Tmis associated with the so phase.

[0393] Binary mixtures of HSPC / DSPG in 20% AmB compositions (no Choi) exhibit similar overlapping features, which is suggestive of coexisting immiscible so phases. The phase separation of PC and PG domains in the presence of calcium ions is consistent with observations in other studies. The onset temperature and enthalpy for the high-Tmpeak in ABCI-001 is 83.9°C and 19.74 J / g, respectively.

[0394] The transition from ABCI-001 to ABCI-002 decreased the drug loading from 30% w / w to 14% vi / vi, while maintaining a Chol / AmB ratio of 1.2 mol / mol. The decrease in AmB and Choi leads to an increase in PL content, and a decrease in the Chol / PL ratio from 0.28 to 0.092 w / w. While a small amount of the low-Tmpeak remains visible in ABCI-002, the Tmincreases for the Chol-rich domain from 62.5 to 64.6°C while AH decreases from 7.73 to 1.93 J / g. The high-Tmpeak remains broad with overlapping features. Relative to ABCI-001, the onset temperature is increased from 83.9 to 88.5°C, suggesting that the acyl chains have increased order as Choi content is decreased.

[0395] In ABCI-003, the drug loading remains 14% w / w, but the Chol / AmB ratio is decreased from 1.2 to 0.4 mol / mol. This enables reductions in the Chol / PL ratio relative to ABCI-002 from 0.092 to 0.030 w / w. Enlargement of the low- Tmpeak region of the ABCI-003 thermogram shows that the Chol-rich peak has been eliminated. The high-Tmpeak is also much sharper, with an onset temperature increased by 7.2°C relative to ABCI-001, to 91.1°C.

[0396] For the long-term stability of amorphous solids, it may be beneficial to have a glass transition temperature, Tg, that exceeds the storage temperature, Ts, by at least 50°C. By analogy, the Tmin PL represents an order-disorder transition. Having a Tmof 91.1 °C for ABCI- 003 means that Tmexceeds an accelerated Tsof 40°C by 50°C.

[0397] As discussed previously, the decrease in Chol / PL ratio has a profound effect on powder properties increasing manufacturing yield from 74.5 to 82.4%, reducing Carr’s Index from 41.4 to 27.0, and dramatically reducing interparticle cohesive forces as evidenced by a nearly twofold reduction in MMAD.

[0398] A linear decrease in the enthalpy of the lo phase transition is observed with increases in Choi content in the powder composition. The regression to the points suggests that the x- intercept (i.e., where the enthalpy goes to zero) occurs at a Chol / PL ratio of 0.05 w / w.

[0399] Example 5: Impact of Lipid Composition on the Hygroscopicity of AmB Compositions at High Relative Humidity

[0400] Without intending to be bound by theory, it is believed that the increases in moisture sorption are due to the presence of NaCl in the composition, which results from the interaction of DSPG and calcium chloride. The divalent calcium ions may bind strongly to anionic DSPG Na, resulting in displacement of the sodium ions which may then interact with the chloride ions from the CaCl2to form NaCl. At high RH, compositions comprising DSPG / CaCh deliquesce, with the magnitude of moisture sorption and deliquescence directly proportional to the amount of NaCl formed.

[0401] ABCI-003, where the PL component contains an HSPC / DSPG ratio of 9.0 w / w, exhibits significantly reduced hygroscopicity compared to ABCI-001 and ABCI-002, where the HSPC / DSPG ratio is 2.3 (i.e., 7 / 3) w / w.

[0402] Example 6: Ussing chamber studies same day electrophysiology measurements

[0403] Apical and basolateral ringer solutions consisting of: 120 mM NaCl / 120mM sodium gluconate, 25 mM NaHCO3, 5 mM KC1, 2 mM CaCl2, 1.2 mM MgCl2, 10mM C6HI2O6and 13.75 mM of NaH2PO4 at a pH of 7.4 were added to each side of the chamber and maintained at 37 °C while gassed with compressed air. The epithelial Na+channel (ENaC) was inhibited by a lOOpM addition of Amiloride to the apical side to achieve a baseline for permeabilization / CFTR activity. Forskolin was then added (10 pM) on the apical side to activate CFTR, followed by the addition of Ivacaftor (lOpM) and / or lOpM of AmB. The transepithelial electrical resistance was measured using the Acquire and Analyze 2.3 software.

[0404] Example 7: Compound preparation and incubation methods

[0405] ABCI-003 sample preparation:

[0406] An arbitrary amount of ABCI-003 was added into a 1.5 mL HPLC vial and suspended in lOOpL of perfluorocarbon-72 (PFC). The absorbance of the stock was measured using the cuvette function of a NanoDrop OneC at 406 nm. The extinction coefficient of AmB (164000) was further used to calculate the concentration of the stock solution. Then, the desired concentrations of ABCI-003 were prepared at 2pM in a final sample volume of 200 pL of PFC. The samples were vortexed and sonicated for 1 minute prior to the apical addition of 20pL of sample per cultured lung epithelia cell.

[0407] Modulator(s) sample preparation:

[0408] Elexacaftor (VX-445), Tezacaftor (VX-661), and Ivacaftor (VX-770) were purchased from Selleckchem as a 5mg powder form and solubilized in DMSO to prepare lOmM stock concentrations. Forskolin was purchased from Sigma-Aldrich as a 10 mg solid and solubilized to prepare a lOmM stock solution in DMSO. 1 mM stock solutions were prepared for each compound listed by obtaining 100 pL from the lOmM stocks and added into 900 pL of DMSO. 550 pL of fresh 1 : 1 DMEM:Ham’s F-12, supplemented with 2% v / v Ultroser G (Crescent Chemical) plate media was added to 24 well plates followed by ETIf addition at the desired volume for the experimental modulator concentrations (Elexacaftor / Tezacaftor range: IpM- lOpM, Ivacaftor / forskolin range: 0.33pM-3.67pM). After the addition of ABCI-003, modulator(s), or both the cells were incubated at 37°C in 5% CO2 for 48-hours.

[0409] Example 8: ASL pH determination - 48 hours post compound addition

[0410] At least one-hour prior to imaging the Zeiss LSM880 confocal microscope was turned on and set to 5% CO2 at 37°C. The excitation wavelength used was 514 nm, emission wavelengths were set at a range of: 578-608 nm (channel 1) and 633-663 nm (channel 2). A standard curve to read the fluorescence ratio was generated by preparing known pH solutions of SNARF-dextran dye in phosphate buffered saline (PBS) at the range of 4-10. One-hour prior to imaging, 2.5 pL of SNARF-dextran in Dulbecco’s PBS was added onto the apical side of the cultured lung epithelia. The cell membranes from each experimental group were then imaged at 40x objective using water immersion for cell line cultures on the objective lens.

[0411] Example 9: H14CC>3_efflux - 48 hours post compound addition

[0412] 14C -labelled sodium bicarbonate was obtained as a sterile 35.7 mM aqueous solution pH 9.5 from PerkinElmer. 5 pL of a 2.8 mM H14CO3‘ stock solution in USG medium was added to the basolateral medium. The lung epithelial cells were then incubated in 5% CO2 at 37 °C for Ihr. After Ihr, the apical membrane was immediately washed with 200 pL of PBS, 150 pL of the wash was collected and added into 3 mL of optiflur. Additionally, 150 pL of PBS and 150 pL of basolateral media were also added into 3 mL of optiflur and analyzed by scintillation liquid counting.

[0413] Example 10: ICPMS - 48 hours post compound addition

[0414] 24 hrs before compound addition, the apical membrane of the cultured airway epithelia cells were washed three times with 200 pL of warm PBS to remove excess mucus. Post-48 hrs after compound addition, 0.1 microcapillary tubes were used to gently touch around the edge of the apical membrane of each epithelial culture insert until they were filled with ASL via capillary action. After collecting 0.1 pL ASL, a p200 pipette was used to push the entire sample into 15 pL of molecular biology grade water. The sample was then quantitatively transferred into a 15-mL conical vial by washing three times with 50 pL of molecular biology grade water. Quantification of sodium, magnesium, potassium, and calcium was accomplished using inductively coupled plasma mass spectrometry (ICP-MS) of acidified samples. Each sample was diluted to a final volume of 5 mL with 1.0% HNO3 (v / v) in double-distilled water. Quantitative standards were made using mixed Na, Mg, K and Ca standards containing 100 pg / mL of each element (Inorganic Ventures), which were combined to create a 100 ng / ml mixed-element standard in 1.0% nitric acid (v / v). ICP-MS was performed on a computer- controlled (QTEGRA software) Thermo iCapQ ICP-MS (Thermo Fisher Scientific) operating in KED mode and equipped with a ESI SC-2DX PrepF AST autosampler. Internal standard was added inline using the prepFAST system and consisted of 1 ng / mL of a mixed element solution containing Bi, In, 6 Li, Sc, Tb and Y (IV-ICPMS-71D from Inorganic Ventures). Online dilution was also carried out using the prepFAST system and used to generate calibration curves with 5,000, 1,000, 500, 100 and 50 ng / mL Na, Mg, K and Ca. Each sample was acquired using one survey run (ten sweeps) and three main (peak-jumping) runs (40 sweeps). The isotopes selected for analysis were23Na,24Mg,39K,44Ca, and89Y (chosen as internal standards for data interpolation and machine stability). Instrument performance was optimized daily through autotuning followed by verification via a performance report (passing manufacturer’s specifications).

[0415] Example 11: Intracellular pH measurement post-48 hours compound addition

[0416] BCECF-AM is a dual excitation (440nm, 490nm), single emission (535 nm) cell permeable ratiometric dye. BCECF-AM was purchased from Millipore-Sigma as a Img powder. A 5 mM solution was prepared by adding 247.31 pL of anhydrous DMSO to 1 mg of the dye. A 5 pM final concentration solution was prepared from the stock in DPBS. Growth medium was removed from CF cell cultures and 2.5 pL from the 5 pM solution was apically added to each membrane. The cell cultures were incubated at 37°C in 5% CO2 for 30 mins. Briefly, the cells were washed with 100 pL of warm DPBS before imaging with the confocal LSM-880 microscope. To obtain intracellular pH values from fluorescence emission ratios a standard curve using epithelia monolayers was generated. Epithelia inserts were kept in known pH buffer solution standards purchased from AAT Bioquest with a pH range of 4.5-8.0 containing 150 mM K+for 10 mins at at 37°C in 5% CO2 in the presence of lOpM Nigericin. The epithelia were then briefly washed with lOOpL of warm DPBS prior to imaging.

[0417] Example 12. Manufacture of lipid-coated particles of amphotericin B derivatives Feedstock Preparation.

[0418] 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.

[0419] Production of Dry Powders by Spray Drying.

[0420] 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.

[0421] 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.

[0422] Example 13. Physicochemical properties of lipid-coated AmB-AA and AmB-NAcC particles

[0423] The primary particle size distribution and tapped density of the spray-dried particles are detailed in Table 6. 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.

[0424] Table 6. Physicochemical properties of spray-dried formulations comprising AmB-AA and AmB-NAcC.

[0425] INCORPORATION BY REFERENCE

[0426] All US patents and published US and PCT patent applications mentioned in the description above are incorporated by reference in their entirety.

[0427] EQUIVALENTS

[0428] Having now fully 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 a disease characterized by reduced function or decreased expression of an ion channel, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic selected from:or a pharmaceutically acceptable salt or hydrate of either of them; and (b) an ion channel modulator.

2. The method of claim 1, wherein the disease is cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease.

3. The method of claim 2, wherein the disease is cystic fibrosis.

4. A method of increasing transepithelial current of airway cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic selected from:or a pharmaceutically acceptable salt or hydrate of either of them; and (b) an ion channel modulator.

5. A method of increasing the pH of airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic selected from:or a pharmaceutically acceptable salt or hydrate of either of them; and (b) an ion channel modulator.

6. A method of increasing bicarbonate secretion into airway surface liquid, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic selected from:or a pharmaceutically acceptable salt or hydrate of either of them; and (b) an ion channel modulator.

7. The method of any one of claims 4-6, wherein the subject has cystic fibrosis, non- cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease.

8. The method of claim 7, wherein the subject has cystic fibrosis.

9. A method of treating cystic fibrosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of:(a) an ion channel prosthetic selected from:or a pharmaceutically acceptable salt or hydrate of either of them; and(b) an ion channel modulator.

10. The method of any one of claims 1-9, wherein the ion channel modulator is a CFTR modulator.

11. The method of any one of claims 1-10, wherein the ion channel modulator is selected from the group consisting of elexacaftor, tezacaftor, ivacaftor, lumacaftor, and forskolin, and any combination thereof.

12. The method of any one of claims 3, 8, or 9, wherein the subject has two mutations in the CF transmembrane conductance regulator (CFTR) anion channel, each copy of the CFTR gene contains one of the two mutations, and wherein the two mutations are each independently selected from the group consisting of:

13. The method of claim 9 or 12, wherein the cystic fibrosis is refractory to treatment with ivacaftor.

14. The method of any one of claims 9, 12, or 13, wherein the cystic fibrosis is refractory to treatment with the combination of elexacaftor, tezacaftor, and ivacaftor.

15. The method of any one of claims 1-14, wherein the ion channel prosthetic is formulated as a pharmaceutical composition comprising:(i) amphotericin B (AmB), or a pharmaceutically acceptable salt or hydrate thereof;(ii) cholesterol (Choi);(iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and(iv) calcium chloride (CaCh).

16. The method of claim 15, wherein the amount of AmB is about 0.5% to about 25% w / w.

17. The method of claim 15 or 16, wherein the amount of AmB is about 3% to about 16% w / w.

18. The method of claim 17, wherein the amount of AmB is about 14% w / w.

19. The method of any one of claims 15-18, wherein the amount of Choi is about 0.1% to about 8% w / w.

20. The method of any one of claims 15-19, wherein the amount of Choi is about 0.3% to about 6% w / w.

21. The method of any one of claims 15-20, wherein the amount of Choi is about 0.5% to about 3% w / w.

22. The method of any one of claims 15-21, wherein the amount of CaCh is about 1% to about 10% w / w.

23. The method of any one of claims 15-22, wherein the amount of CaCh is about 4% to about 7% w / w.

24. The method of any one of claims 15-23, wherein the amount of phospholipids is about 60% to about 95% w / w.

25. The method of any one of claims 15-24, wherein the amount of phospholipids is about 70% to about 90% w / w.

26. The method of any one of claims 15-25, wherein the weight ratio of Choi to phospholipids is about 0.001 : 1 to about 0.1 : 1.

27. The method of any one of claims 15-26, wherein the weight ratio of Choi to phospholipids is about 0.005: 1 to about 0.05: 1.

28. The method of any one of claims 15-27, wherein the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 2: 1 to about 19: 1.

29. The method of any one of claims 15-28, wherein the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 7: 1 to about 12: 1.

30. The method of any one of claims 15-29, wherein the molar ratio of Choi to AmB is about 0.05: 1 to about 1.2: 1.

31. The method of any one of claims 15-30, wherein the molar ratio of Choi to AmB is about 0.4: 1 to about 1.2: 1.

32. The method of any one of claims 15-31, wherein the molar ratio of Choi to AmB is about 0.05: 1 to about 0.4: 1.

33. The method of claim 32, wherein the molar ratio of Choi to AmB is about 0.4: 1.

34. The method of any one of claims 15-33, wherein the molar ratio of phospholipids to CaCh is about 4: 1 to about 2: 1.

35. The method of claim 34, wherein the molar ratio of phospholipids to CaCh is about 2: 1.

36. The method of any one of claims 15-35, wherein the AmB has a crystallinity greater than about 75%.

37. The method of claim 36, wherein the AmB has a crystallinity greater than about 85%.

38. The method of claim 37, wherein the AmB has a crystallinity greater than about 95%.

39. The method of claim 1, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 30.0% w / w amphotericin B (AmB);(ii) about 14.6% w / w cholesterol (Choi);(iii-a) about 35.8% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 15.9% w / w distearoylphosphatidylglycerol (DSPG); and (iv) about 3.7% w / w calcium chloride (CaCh).

40. The method of claim 1, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 14.0% w / w amphotericin B (AmB);(ii) about 6.81% w / w cholesterol (Choi);(iii-a) about 51.2% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 22.8% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 5.2% w / w calcium chloride (CaCh).

41. The method of claim 1, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 14.0% w / w amphotericin B (AmB);(ii) about 2.3% w / w cholesterol (Choi);(iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 5.52% w / w calcium chloride (CaCh).

42. The method of claim 1, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 3.4% w / w amphotericin B (AmB);(ii) about 0.57% w / w cholesterol (Choi);(iii-a) about 80.72% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 8.97% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 6.34% w / w calcium chloride (CaCh).

43. The method of claim 15, wherein the pharmaceutical composition comprises:(i) a Chol / AmB ratio of about 0.4 to 1.2 mol / mol;(ii) a Chol / PL ratio of less than about 0.05 w / w;(iii) a HSPC / DSPG ratio of about 2.3 to about 9.0 w / w; and(iv) a PL / Ca2+ratio of about 2: 1 mol / mol.

44. The method of any one of claims 15-43, wherein the AmB and Choi are not complexed; and the AmB is not encapsulated in liposomes.

45. The method of any one of claims 15-44, wherein the AmB is coated with a porous shell of phospholipids and Choi.

46. The method of any one of claims 1-45, wherein the absolute bioavailability of the AmB is about 0.1% to about 5%.

47. The method of any one of claims 1-14, wherein the ion channel prosthetic is formulated as a pharmaceutical composition comprising:(i) amphotericin B-AA (AmB-AA), or a pharmaceutically acceptable salt or hydrate thereof;(ii) cholesterol (Choi);(iii) phospholipids, comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and(iv) calcium chloride (CaCh).

48. The method of any one of claims 11-14 or 47, wherein the amount of AmB-AA is about 0.5% to about 25% w / w.

49. The method of any one of claims 11-14 or 47-48, wherein the amount of AmB-AA is about 3% to about 16% w / w.

50. The method of any one of claims 11-14 or 47-49, wherein the amount of AmB-AA is about 14% w / w.

51. The method of any one of claims 11-14 or 47-50, wherein the amount of Choi is about 0.1% to about 8% w / w.

52. The method of any one of claims 11-14 or 47-51, wherein the amount of Choi is about 0.3% to about 6% w / w.

53. The method of any one of claims 11-14 or 47-52, wherein the amount of Choi is about 0.5% to about 3% w / w.

54. The method of any one of claims 11-14 or 47-53, wherein the amount of CaCh is about 1% to about 10% w / w.

55. The method of any one of claims 11-14 or 47-54, wherein the amount of CaCh is about 4% to about 7% w / w.

56. The method of any one of claims 11-14 or 47-55, wherein the amount of phospholipids is about 60% to about 95% w / w.

57. The method of any one of claims 11-14 or 47-56, wherein the amount of phospholipids is about 70% to about 90% w / w.

58. The method of any one of claims 11-14 or 47-57, wherein the weight ratio of Choi to phospholipids is about 0.001 : 1 to about 0.1 : 1.

59. The method of any one of claims 11-14 or 47-58, wherein the weight ratio of Choi to phospholipids is about 0.005: 1 to about 0.05: 1.

60. The method of any one of claims 11-14 or 47-59, wherein the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 2: 1 to about 19: 1.

61. The method of any one of claims 11-14 or 47-60, wherein the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 7: 1 to about 12: 1.

62. The method of any one of claims 11-14 or 47-61, wherein the molar ratio of Choi to AmB-AA is about 0.05: 1 to about 1.2: 1.

63. The method of any one of claims 11-14 or 47-62, wherein the molar ratio of Choi to AmB-AA is about 0.4: 1 to about 1.2: 1.

64. The method of any one of claims 11-14 or 47-63, wherein the molar ratio of Choi to AmB-AA is about 0.05: 1 to about 0.4: 1.

65. The method of claim 64, wherein the molar ratio of Choi to AmB-AA is about 0.4: 1.

66. The method of any one of claims 11-14 or 47-65, wherein the molar ratio of phospholipids to CaCh is about 4: 1 to about 2:1.

67. The method of claim 66, wherein the molar ratio of phospholipids to CaCh is about 2: 1.

68. The method of any one of claims 11-14 or 47-67, wherein the AmB-AA has a crystallinity greater than about 75%.

69. The method of claim 68, wherein the AmB-AA has a crystallinity greater than about 85%.

70. The method of claim 69, wherein the AmB-AA has a crystallinity greater than about 95%.

71. The method of claim 47, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 5.0% w / w AmB-AA;(ii) about 2.3% w / w cholesterol (Choi);(iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 5.52% w / w calcium chloride (CaCh).

72. The method of claim 47, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 5.0% w / w AmB-AA;(ii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);(ii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and(iii) about 5.52% w / w calcium chloride (CaCh).

73. The method of claim 47, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 30.0% w / w AmB-AA;(ii) about 14.6% w / w cholesterol (Choi);(iii-a) about 35.8% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 15.9% w / w distearoylphosphatidylglycerol (DSPG); and (iv) about 3.7% w / w calcium chloride (CaCh).

74. The method of claim 47, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 14.0% w / w AmB-AA;(ii) about 6.81% w / w cholesterol (Choi);(iii-a) about 51.2% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 22.8% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 5.2% w / w calcium chloride (CaCh).

75. The method of claim 47, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 14.0% w / w AmB-AA;(ii) about 2.3% w / w cholesterol (Choi);(iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 5.52% w / w calcium chloride (CaCh).

76. The method of claim 47, wherein the pharmaceutical composition comprises, consists essentially of, or consists of:(i) about 3.4% w / w AmB-AA;(ii) about 0.57% w / w cholesterol (Choi);(iii-a) about 80.72% w / w hydrogenated soy phosphatidylcholine (HSPC);(iii-b) about 8.97% w / w distearoylphosphatidylglycerol (DSPG); and(iv) about 6.34% w / w calcium chloride (CaCh).

77. The method of claim 47, wherein the pharmaceutical composition comprises:(i) a Chol / AmB-AA ratio of about 0.2 to about 1.0 mol / mol;(ii) a Chol / PL ratio of less than about 0.05 w / w;(iii) a HSPC / DSPG ratio from about 2.0 to about 10 w / w; and(iv) a PL / Ca2+ratio of about 2: 1 mol / mol.

78. The method of any one of claims 1-14 or 47-77, wherein the AmB-AA and Choi are not complexed; and the AmB-AA is not encapsulated in liposomes.

79. The method of any one of claims 1-14 or 47-78, wherein the AmB-AA is coated with a porous shell of phospholipids and Choi.

80. The method of any one of claims 15-79, wherein the pharmaceutical composition is formulated as a dry powder.

81. The method of claim 80, wherein the mass median diameter, X50, of the powder particles is about 1.0 to about 4.0 pm.

82. The method of claim 81, wherein the mass median diameter, X50, of the powder particles is about 1.5 to about 3.5 pm.

83. The method of any one of claims 80-82, wherein the tapped density of the powder particles is about 0.03 to about 0.4 g / mL.

84. The method of any one of claims 80-83, wherein the tapped density of the powder particles is about 0.06 to about 0.2 g / mL.

85. The method of any one of claims 80-84, wherein the Carr’s index of the powder particles is about 20 to about 32.

86. The method of any one of claims 80-85, wherein the main transition temperature (Tm) of the shell is at least 80 °C.

87. The method of any one of claims 80-86, wherein the water content of the powder is about 1.5 to about 6% w / w.

88. The method of any one of claims 80-87, wherein the pharmaceutical composition is formulated for pulmonary administration or airway administration.

89. The method of claim 88, wherein the pharmaceutical composition is formulated for aerosol administration.

90. The method of any one of claims 88-89, wherein the mass median aerodynamic diameter of the powder particles is about 1.5 pm to about 4.0 pm.

91. The method of any one of claims 88-90, wherein the mass median aerodynamic diameter of the powder particles is about 2.0 pm to about 3.5 pm.

92. The method of any one of claims 88-91, wherein the subject is a human.

93. The method of any one of claims 88-92, wherein the subject is a human less than 12 years old.

94. The method of any one of claims 88-93, wherein the subject is a human at least 12 years old.

95. The method of any one of claims 88-94, wherein the nominal dose or metered dose of the pharmaceutical composition is 0.01 mg to 10 mg.

96. The method of claim 95, wherein the nominal dose or metered dose of the pharmaceutical composition is 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, or 4.0 mg.

97. The method of any one of claims 88-96, wherein the composition is administered once daily.

98. The method of any one of claims 88-97, wherein the administration comprises initially delivering a loading dose of the pharmaceutical composition; and subsequently delivering a maintenance dose of the pharmaceutical composition; wherein the mass ratio of the loading dose to the maintenance dose is about 2: 1 to about 5: 1.

99. The method of claim 98, wherein the mass ratio of the loading dose to the maintenance dose is about 2.5: 1 to about 3: 1.

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