WO2025064631A1

WO2025064631A1 - Use of a dry powder inhaler to treat or prevent pulmonary fungal infections

Info

Publication number: WO2025064631A1
Application number: PCT/US2024/047422
Authority: WO
Inventors: Martin D. Burke; Jeffry G. Weers; Agnieszka LEWANDOWSKA; Thomas Tarara; Danforth P. Miller; Corinne SOUTAR
Original assignee: University of Illinois at Urbana Champaign; University of Illinois System; Cystetic Medicines Inc
Current assignee: University of Illinois at Urbana Champaign; University of Illinois System; Cystetic Medicines Inc
Priority date: 2023-09-20
Filing date: 2024-09-19
Publication date: 2025-03-27
Anticipated expiration: 2026-03-20

Abstract

Disclosed are methods for use in treating or preventing pulmonary fungal infections, e.g., in a recipient of a lung transplant.

Description

USE OF A DRY POWDER INHALER TO TREAT OR PREVENT PULMONARY FUNGAL INFECTIONS

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 63/539,424, filed September 20, 2023.

BACKGROUND

The incidence of pulmonary fungal infections has increased significantly over the past 4 decades concurrent with the increase in the number of immunocompromised patients during the same period. Immunocompromised patients comprise the greatest proportion of the approximately 100,000 cases of severe fungal infections that occur in the United States each year. Aspergillus and Candida are the most commonly involved pathogens. While the incidence of Candida infections has reduced since the introduction of fluconazole, attributable mortality from invasive pulmonary aspergillosis (IP A) is reported to be as high as 85% and gross mortality as high as 95%, even with aggressive intravenous therapy. Early intervention is critical in the treatment of IP A, yet diagnosis of opportunistic fungal infections remains difficult, and may be further delayed in patients at risk. Therefore, prevention of the infections, if achievable, would be an important medical advance. Patients particularly at risk for IPA include those with hematological malignancies, solid tumors, AIDS, and organ transplant patients.

The most effective agent against Aspergillus remains amphotericin B. Pulmonary fungal infections are usually acquired through inhalation of fungal conidia. With a diameter of 2.5 to 3.5 micrometers, fungal conidia are able to reach the large and small airways in the lower respiratory tract, where impaired host defense in immunocompromised hosts allows for germination into the hyphal form and subsequent tissue invasion. Maintenance of inhibitory concentrations of inhibitory concentrations in the same locations in the lung that the conidia reach should confer protection against infection. By targeting the lungs, most of the toxicities associated with systemic AmB products can be reduced or mitigated. Prophylactic delivery of AmB via nebulization has been investigated clinically. Nebulization is, however, time consuming and cumbersome, and can result in adverse events from high oropharyngeal deposition of the aerosol, as well as additives like sodium deoxy cholate. In addition, nebulizers are inefficient for targeting the lungs. Hence, an inhalable dry powder formulation of AmB for the prophylaxis of pulmonary fungal infections may provide an advantage. In view of the foregoing, there is an unmet need for the development of new antifungal treatments and prophylactics.

SUMMARY

In certain aspects, disclosed herein is a method of treating a pulmonary fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of 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).

In certain aspects, disclosed herein is a method of preventing a pulmonary fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, comprising:

(ii) cholesterol (Choi);

(iv) calcium chloride (CaCh).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with AmB in Fungizone.

FIG. IB shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with ABCI-003 (containing crystalline AmB and Choi at a 0.4 ChokAmB mol/mol ratio).

FIG. 2 shows the minimum hemolytic concentration as % of blood cells hemolysed after AmB and ABCI with varied Choi content treatments: ABCI-003 (0.4 ChokAmB mol/mol); CM22001 (0.2 Choi: AmB mol/mol); CM22002 (0.1 Choi: AmB mol/mol); CM22003 (0.05 Chol:AmB mol/mol); CM22004 (0 Chol:AmB mol/mol); CM22005 (0.4 Chol:AmB mol/mol); CM22006 (0 Chol:AmB mol/mol);

FIG. 3A shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with AmB in DMSO.

FIG. 3B shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with AmB in Fungizone.

FIG. 3C shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with AmBisome.

FIG. 3D shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21052 (30% AmB formulated with PFOB).

FIG. 4A shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21008 (AmB:Chol (1 :2.5) formulated with DSPC, DSPG).

FIG. 4B shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21012 (AmB: Choi (1 :2.5) formulated with DSPC, CaCh, PFOB).

FIG. 4C shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21013 (AmB:Chol (1 :2.5) formulated with DSPC, DSPG, CaCh).

FIG. 4D shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21018 (AmB:Chol (1 :2.5) formulated with DSPC, DSPG, CaCh [1.23 mol Choi: mol lipid]).

FIG. 4E shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21022 (20% AmB formulated with DSPC, DSPG, CaCh).

FIG. 4F shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21023 (20% AmB formulated with HSPC, DSPG, CaCh).

FIG. 4G shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21024 (20% AmB formulated with HSPC, DSPG, CaCh).

FIG. 4H shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21026 (15% AmB formulated with HSPC, DSPG, CaCh). FIG. 41 shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21027 (30% AmB formulated with HSPC, DSPG, CaCh).

FIG. 4 J shows the minimum hemolytic concentration as % of blood cells hemo lysed after treatment with placebo.

FIG. 5A shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with AmBisome.

FIG. 5B shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21034 (ABCI-002 formulated with 0% v/v ethanol).

FIG. 5C shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21038 (ABCI-002 formulated with 6% v/v ethanol).

FIG. 5D shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21039 (ABCI-002 formulated with 9% v/v ethanol).

FIG. 5E shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder cM21041 (ABCI-002).

FIG. 5F shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder FP21008 (ABCI-002 formulated with 10% v/v ethanol, highly crystalline AmB).

FIG. 5G shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder FP21010 (ABCI-002 formulated with 10% v/v ethanol, less crystalline AmB).

FIG. 5H shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder FP21011 (30% AmB formulated with 10% v/v ethanol, highly crystalline AmB).

FIG. 6A shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder FP21019 (3% w/w solids formulated with 5% v/v ethanol).

FIG. 6B shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder FP21020 (3% w/w solids formulated with 10% v/v ethanol). FIG. 7A shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21034 (placebo).

FIG. 7B shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21030 (ABCI-001 formulated with Novec 7500 (C9H5F15O)).

FIG. 7C shows the minimum hemolytic concentration as % of blood cells hemolysed after treatment with powder R21052 (ABCI-001 formulated with PFOB).

FIG. 8 shows Mean Concentration-Time Profiles on Day 29 of AmB ASL Concentrations for ABCI-003 in Rats.

FIG. 9A shows a scanning-electron micrograph (SEM) of spray-dried ABCI-003 powder.

FIG. 9B shows a digitally rendered RS01 dry powder inhaler.

FIG. 10 shows concentration versus time profiles for inhaled doses of a formulation of the invention.

FIG. 11 shows the geometric mean (95% CI) plasma concentration- time profiles of Amphotericin B (ng/mL) by treatment group (semi-logarithmic in upper panel, linear scale in lower panel) - Part B (PK evaluable population).

DETAILED DESCRIPTION

The incidence of pulmonary fungal infections has increased significantly over the past 4 decades concurrent with the increase in the number of immunocompromised patients during the same period. Immunocompromised patients comprise the greatest proportion of the approximately 100,000 cases of severe fungal infections that occur in the United States each year. Aspergillus and Candida are the most commonly involved pathogens. While the incidence of Candida infections has reduced since the introduction of fluconazole, attributable mortality from invasive pulmonary aspergillosis (IP A) is reported to be as high as 85% and gross mortality as high as 95%, even with aggressive intravenous therapy. Early intervention is critical in the treatment of IP A, yet diagnosis of opportunistic fungal infections remains difficult, and may be further delayed in patients at risk. Therefore, prevention of the infections, if achievable, would be an important medical advance. Patients particularly at risk for IPA include those with hematological malignancies, solid tumors, AIDS, and organ transplant patients. The most effective agent against Aspergillus remains amphotericin B. Pulmonary fungal infections are usually acquired through inhalation of fungal conidia. With a diameter of 2.5 to 3.5 micrometers, fungal conidia are able to reach the large and small airways in the lower respiratory tract, where impaired host defense in immunocompromised hosts allows for germination into the hyphal form and subsequent tissue invasion. Maintenance of inhibitory concentrations of inhibitory concentrations in the same locations in the lung that the conidia reach should confer protection against infection. By targeting the lungs, most of the toxicities associated with systemic AmB products can be reduced or mitigated. Prophylactic delivery of AmB via nebulization has been investigated clinically. Nebulization is, however, time consuming and cumbersome, and can result in adverse events from high oropharyngeal deposition of the aerosol, as well as additives like sodium deoxycholate. In addition, nebulizers are inefficient for targeting the lungs. Hence, an inhalable dry powder formulation of AmB for the prophylaxis of pulmonary fungal infections may provide an advantage. In view of the foregoing, there is an unmet need for the development of new antifungal treatments and prophylactics. We have created a novel inhaled dry powder formulation called Amphoteric B Cystetic for Inhalation (in short ABCI), which is comprised of wet-milled crystals of AmB coated with a porous layer of phospholipids (hydrogenated soy phosphatidylcholine (HSPC); distearoylphosphatidylglycerol (DSPG)), cholesterol (Choi), and calcium chloride and it is administered via oral inhalation to directly target the apical side of airway epithelial cells.

During the process of ABCI’s discovery, we have studied tens of formulations looking at their cytotoxicity (Minimum Hemolytic Concentration (MHC)) profiles. FIG. 1A shows the MHC profile of AmB in Fungizone formulation, solubilized in the bile salt micelles. AmB clearly caused significant hemolysis to the human blood cells due to Choi extraction from the lipid bilayers. Formulation that contained amorphous AmB and did not contain Choi was still causing hemolysis to the human blood cells, a relationship which is explained simply by the formulations with the greatest dissolution in water (i.e., desoxy cholate micelles, amorphous drug). However, ABCI-003 formulation containing both crystalline AmB and Choi (0.4 Choi: AmB (mol/mol)) (FIG. 1C) was not toxic to human blood cells. Formulations containing crystalline AmB and as little as 0.05 Choi: AmB (mol/mol) ratio (CM22003) (FIG. 2) as well as formulations containing crystalline AmB but no Choi (CM22004 and CM22006) also did not cause hemolysis (See all MHC profiles in FIG. 3A - FIG. 7C). in ABCI. It is not clear why some formulations of AmB show toxicity in this assay while others do not. During ABCI development, we have also studied Minimum Inhibitory Concentrations (MICs) of the formulations which varied for example in AmB content, Choi content, co-solvent content, EtOH content or blowing agent content, and we compared them to AmB in Fungizone (water soluble formulation) and AmBisome (liposomal 2.5 Choi: AmB (mol/mol) formulation) as references. MICs for all the studied formulations as well as for references were performed in molecular biology grade water. MIC values for all the formulations studied are listed in Tables 12-18. The results indicate that powders formulated without co-solvent or with ethanol seem to have higher average MIC values, while powders formulated with a fluorinated blowing agent (CF3- CF₂-CF2-CF(OCH2CH3)-CF(CF3)-CF₃; Novec 7500 or PFOB) have lower MIC values (i.e., have more potent antifungal activity) even though the drug is crystalline and thus presumably not water soluble in those powders. It is not clear why different formulations of AmB show such differences in potency. We speculate that this may be due to improved dissolution of the drug from the particles due to their porous morphology and decreased agglomeration, which in turn makes the drug more available to kill fungi. On the other hand, other, not known to us yet factors, may also play a role. ABCI-001 (R21052), formulated with a fluorinated blowing agent PFOB, characterized in an outstanding antifungal activity profile with no toxicity observed to human cells (FIG. 7A - FIG. 7C). Building on the presented data, we predict that our final formulation, ABCI-003, which is currently being tested in clinical trials, and which is also formulated with PFOB, will show high antifungal activity. Moreover, ABCI-003 is not toxic to human blood cells (FIG. 2) and has thus far shown no serious adverse events in clinical studies, which collectively make it an ideal target IFI treatment for people with cancer, acquired immunodeficiency syndrome, having undergone transplantation, or who are otherwise immunocompromised.

Current available treatments with nebulized intravenous formulations decreases dose administration over time from twice weekly to once every two weeks, relying on redistribution of the drug that is present in lung tissue to provide protection. This results in low airway surface liquid (ASL) concentrations that may be insufficient and could lead to breakthrough infections. In contrast, ABCI is intended to be administered daily, and to maintain AmB concentrations significantly above the MIC in the area where the pathogen is deposited following inhalation of fungal conidia. The administered nominal dose is also much lower, which could reduce adverse events associated with dose administration (e.g., bronchospasm, poor taste, cough). It also has an impact on the desired compositions, with a lower drug content in the formulation to enable lower daily doses. The short administration time (i.e., a single inhalation versus extended periods on a nebulizer) also reduces the potential for adverse events, especially in immunocompromised cancer patients who have difficulty with the prolonged administration time.

To date, all inhaled AmB formulations are administered once weekly or once every two weeks at steady state. This may be suboptimal for a prophylaxis strategy as the half-life for inhaled amphotericin in airway surface liquid (ASL) where conidia are deposited on inhalation is about 10-20 hours. Drug initially deposited in ASL is cleared by multiple pathways (e.g., absorption, mucociliary clearance, cough clearance, and macrophages clearance). A large proportion of inhaled AmB is cleared by circulating and tissue resident macrophages, where it is deposited in lung tissue. AmB is redistributed from lung tissue into ASL with a half-life of about 20 days. The concentration of drug in ASL, however, is very low compared to the concentration achieved initially following inhalation of AmB. Hence, it may be advantageous to inhale lower doses of AmB daily to maintain AmB concentrations well above the MIC in ASL. The formulations described herein are designed for daily administration of lower nominal doses that still maintain ASL concentrations well above the MIC.

Pharmaceutical Compositions Comprising AmB

The pharmaceutical compositions 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.

In certain aspects, the pharmaceutical composition comprises:

(ii) cholesterol (Choi);

(iv) calcium chloride (CaCh).

In certain embodiments, the amount of AmB is about 0.5% to about 30% 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.

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. 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.

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.

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.

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.

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 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%.

In certain embodiments, 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).

(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).

(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).

(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).

In certain embodiments, the pharmaceutical composition comprises:

(i) a Choi/ 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.

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

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

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

In certain embodiments, the mass median diameter, X50, of the powder particles is about 1.0 to about 4.0 pm. In further embodiments, the mass median diameter, X50, of the powder particles is about 1.5 to about 3.5 pm. 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.

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

In certain embodiments, the main transition temperature (T_m) of the shell is at least 80 °C.

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

In certain embodiments, the mass median aerodynamic diameter of the powder particles is about 1.5 pm to about 4.0 pm. In further embodiments, the mass median aerodynamic diameter of the powder particles is about 2.0 pm to about 3.5 pm.

In certain embodiments, the pharmaceutical composition is formulated for pulmonary administration or airway administration.

In certain embodiments, the pharmaceutical composition is formulated for aerosol administration.

In certain embodiments, the pharmaceutical composition is formulated for administration as a dry powder inhaler.

In certain embodiments, the nominal dose or metered dose of the pharmaceutical composition is 0.01 mg to 10 mg. In further embodiments, 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.

In certain embodiments, the pharmaceutical composition is administered once daily.

In certain embodiments, the absolute bioavailability of the AmB is about 0.1% to about 5%.

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 Choi/ AmB ratio is about 0.05 to about 1.2 mol/mol, such as about 0.2 to about 0.6 mol/mol.

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.

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 some embodiments, the PL comprises hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylglycerol (DSPG), or a combination thereof.

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.

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.

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 some embodiments, the drug loading is about 0.5% to about 25% w/w, and the nominal Choi/ AmB ratio is about 0.05 to about 1.2 mol/mol.

In certain embodiments, a dry powder composition of engineered particles is provided (ABCI-003) 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.

In certain embodiments, a dry powder composition of engineered particles is provided (ABCI-004) that includes spray-dried core-shell particles of fine crystalline AmB particles (about 3.4% w/w) coated with a porous shell of PL and Choi, where the Chol/AmB ratio is about 0.4 mol/mol, the Chol/PL ratio is about 0.006 w/w, the PL/Ca ratio is about 2 mol/mol, and the ratio of PC/PG in the PL is about 9.0 w/w.

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. 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.

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.

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 T_m that is more than 50°C above an accelerated storage temperature of 40°C).

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.

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.

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

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

In certain embodiments, selection of the compositions described herein is driven by no red blood cell hemolysis even at low Chol/AmB molar ratios.

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 (CaCL), 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.

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 (CaCL), 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.

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 (CaCL), wherein the X90 of the particles is about 3 pm to about 10 pm, such as about 3.5 pm to about 7 pm.

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 (CaCL), 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.

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 (CaCL), wherein the water content in the powder is about 1.0% to about 10.0%, preferably about 2.0% to about 5.0%.

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 (CaCL), 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.

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 (CaCL), 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 (CaCL), 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.

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 (CaCL), wherein the lipids have a main transition temperature (T_m) of at least 80 °C, such as at least 90 °C.

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 T_m of the lipids. In some embodiments, the outlet temperature is at least 50 °C, at least 60 °C, or at least 70 °C.

In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline 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 T_m of the lipids. In some embodiments, the outlet temperature is at least 60 °C, or at least 70 °C.

In certain embodiments, a dry powder composition of engineered particles is provided that includes a plurality of crystalline 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.

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.

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.

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

Methods of Treatment

(ii) cholesterol (Choi);

(iv) calcium chloride (CaCh). In certain aspects, disclosed herein is a method of preventing a pulmonary fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, comprising:

(ii) cholesterol (Choi);

(iv) calcium chloride (CaCh).

In certain embodiments, the subject has cystic fibrosis.

In certain embodiments, the subject has cancer, acquired immunodeficiency syndrome, has undergone transplant (e.g., lung transplant), is otherwise immunocompromised, or any combinations thereof. In further embodiments, the subject is a human. In yet further embodiments, the subject is a human at least 6 years old.

In certain embodiments, the administration is chronic (e.g., daily).

In certain embodiments, the subject is a recipient of a lung transplant.

Definitions

The terms “treat” and “treating” as used herein refer to performing an intervention that results in (a) preventing a condition or disease from occurring in a subject that may be at risk of developing or predisposed to having the condition or disease but has not yet been diagnosed as having it; (b) inhibiting a condition or disease, e.g., slowing or arresting its development; or (c) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease. In one embodiment the terms “treating” and “treat” refer to performing an intervention that results in (a) inhibiting a condition or disease, e.g., slowing or arresting its development; or (b) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease.

A “subject” or “patient” as used herein refers to a living mammal. In various embodiments, a patient is a non-human mammal, including, without limitation, a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, cat, dog, pig, horse, cow, or non-human primate. In certain embodiments, a patient is a human.

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

"Active ingredient", "therapeutically active ingredient", "active agent", "drug" or "drug substance" as used herein means the active ingredient of a pharmaceutical, also known as an active pharmaceutical ingredient (API).

"Amorphous" as used herein refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically, such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid-like properties occurs at a “glass transition”, typically defined as a second-order phase transition.

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

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

"Mass median diameter" or "MMD" or “X50” as used herein means the median diameter of a plurality of particles, typically in a poly disperse 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.

"Tapped densities" or pta_PPed 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.

"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, d_a represents the aerodynamic diameter of a single particle.

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

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

"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™.

"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.

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

The term “about” refers to variations in numerical values typically encountered by one of skill in the art of respirable compositions, including variations of plus or minus 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a numerical value described herein.

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

EXAMPLES

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.

Characterization Methods

Amphotericin B (AmB) Content and Purity. AmB 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). AmB was quantified using a single-point calibration using the USP certified reference standard.

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™ Cl 8 column (5 pm) using an isocratic method (acetonitrile/isopropanol, 1.0 v/v). Cholesterol was quantified using a single-point calibration using Cholesterol HP, Ph. Eur/USP-NF raw material (Carbogen Amcis, Beuvry-la- Foret, France).

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 powderdispersing 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.

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.

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.

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

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.

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.

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. 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 H2O^{0 5} L'¹ min (0.045 kPa^{0 5} L'¹ min). This corresponds to a flow rate of 44.2 L min'¹ at 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 AmB mass on each stage was determined using the HPLC method described above and the fine particle dose less than 5 pm (FPD 5 nm) and mass median aerodynamic diameter (MMAD) were calculated.

Cell Lines and Growth Conditions. NuLi, CuFi-1, and CuFi-4 cells (Welsh Laboratory, University of Iowa) were grown from cryostock on Thermo Scientific BioLite Cell Culture Treated 75 cm² flasks. These flasks were previously coated with 4 mL of 60 pg/mL human placental collagen type IV (Sigma-Aldrich) for a minimum of 1 h at 37 °C, rinsed twice with PBS, and then dried before seeding. The cells were cultured with 12 mL Bronchial Epithelial Cell Growth Medium (BEGM) BulletKit (Lonza CC-3170), which includes the basal medium and eight SingleQuots of supplements (bovine pituitary extract (BPE), 2 mL; hydrocortisone, 0.5 mL; hEGF, 0.5 mL; adrenaline, 0.5 mL; transferrin, 0.5 mL; insulin, 0.5 mL; retinoic acid, 0.5 mL; triiodothyronine, 0.5 mL). The gentamicin-AmB aliquot was discarded, and the medium was instead supplemented with 50 pg/mL penicillin-streptomycin (Corning Cellgro), 50 pg/mL gentamicin (Sigma- Aldrich G1397), and 2 pg/mL fluconazole (Sigma- Aldrich). The original CF transplant donors were genotyped by Integrated Genetics. Cell lines were secondarily confirmed by the ATCC repository to have the correct genotype and were free of mycoplasma contamination. MycoAlert Mycoplasma detection kit (Lonza LT07-418) was used to detect any RNA transcripts common to a broad spectrum of mycoplasma. Cell lines were confirmed to be mycoplasma-free. Cells were grown to >90% confluence at 37 °C in 5% CO2, changing medium every two-three days, and then trypsinized with 4 mL 0.25% trypsin containing 1 mM EDTA (Gibco 25200-056). Trypsin was inactivated with 10 mL HEPES -buffered saline solution (Lonza CC-5024) with 1% fetal bovine serum. Cells were spun down in an Eppendorf Centrifuge 5430R at 1,500 rpm for 5 min at room temperature and resuspended in BEGM medium for passaging. For culturing on membrane supports for differentiation, cells were resuspended after centrifugation in Ultroser G medium. This comprised 1 : 1 DMEM:Ham’s F-12, supplemented with 4% v/v Ultroser G (Crescent Chemical) as well as 50 pg/mL penicillin-streptomycin (Corning Cellgro), 50 pg/mL gentamicin (Sigma-Aldrich G1397), and 2 pg/mL fluconazole (Sigma-Aldrich). The membrane supports used were the Corning Costar 0.4-pm 24-well plate Transwell clear polyester membrane inserts (0.33 cm²) (Corning 3470) for all studies. These membranes were coated with collagen in the same manner as the flasks detailed above, except with 100 mL collagen and only rinsed once with PBS. The inserts were seeded with 115,000 cells each. These membranes were allowed to mature at an air-liquid interface for a minimum of 14 days to reach full differentiation, with the Ultroser G medium changed once or more per week as needed. After maturation, medium was changed every seven days. For covariate control, membranes used in experiments were as close in age and maturation as possible.

For the primary cultured airway epithelial cells, lung tissue was obtained from individuals with CF who were undergoing a lung transplant or organ donation. The tissue was dissociated and the dissociated cells were directly seeded onto transwell filters and cultured at the air-liquid interface. Cultures were used at greater than three weeks post-seeding when epithelial cells were differentiated into the typical cell types of the airways and the electrical properties of the cells reflected excised tissue.

Fluorescence Microscopic Assay for Measurement of Airway Surface Liquid pH. A fresh suspension of ABCI was prepared for each experiment by dispersing approximately 2 mg of ABCI in approximately 100 pL of perfluorohexane, PFH (FC-72, Sigma- Aldrich) to reach a final concentration of approximately 1 mM AmB. Following dissolution in methanol, the AmB concentration in the stock suspension was measured in triplicate by absorbance spectroscopy. Concentrations were calculated from the absorbance at 406 nm (S406 = 164,000 M^_|cm^_| ) using Beer’s law. Next, the stock suspensions were diluted with PFH to achieve suspension AmB concentrations ranging from ~0.5 to 50 pM.

Small-diameter NuLi and CuFi cells were used for measurement of the ASL pH. The ratiometric pH indicator SNARF-conjugated dextran (Molecular Probes) was used to measure ASL pH. SNARF powder was suspended via sonication in PFH and distributed onto the apical surface of the cells. ASL pH was measured 2 h later. SNARF was excited at 514 nm and emission was recorded at 580 nm and 640 nm using a Zeiss LSM 800 microscope equipped with a water immersion lens for cell line cultures at a magnification of 40x. To generate a standard curve for pH determination, SNARF was dissolved in colorless pH standards and fluorescence ratios were converted to pH. Powders tested in this assay were suspended in the appropriate volume of PFH, which were sonicated for 1 min to aid suspension. AmBisome was suspended by vortexing in PFH. Subsequently, 20 pL of suspension was administered onto the surface of cultured airway epithelia (A = 0.33 cm²) at concentrations between 0.5 and 50 pM. In all experiments, ASL pH of compound-treated epithelia was measured and compared to the results from vehicle-treated epithelia. For apical compound administration, cultured airway epithelia were incubated for about 22 hours at 37 °C before measurement of ASL pH.

Preparation of erythrocyte stock suspension. One mL of human whole blood (Na-Heparin preparation; BioIVT, Westbury, NY) was centrifuged at 10,000 x g for 2 min at room temperature. The supernatant was removed, and the pellet was resuspended by gentle inversion in 1 mL 0.9% (m/v) saline (erythrocytes will lyse by pipetting and vortexing). The resulting suspension was centrifuged at 10,000g for 2 min. The supernatant was then removed, and the saline wash repeated twice more. Following the final wash, the supernatant was removed, and the erythrocyte pellet was resuspended in 1 mL resuspension buffer (10 mM Na2HPO4’7H2O, 10 mM NaH2PO4’H2O, 150 mM NaCl, 1 mM MgC12’6H2O, pH 7.4) to make the erythrocyte stock suspension.

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

The nominal anhydrous compositions of four compositions of lipid-coated AmB (ABCI- 001, ABCI-002, ABCI-003, ABCL004) are presented in Table 2. Also shown are two controls: AmBisome® (i.e., liposomal amphotericin B, L-AmB); and the lyophilized AmB/Chol complexes prepared by Burke et al. (US 2019/0083517; US 2020/0352970; incorporated by reference).

Table 2. Theoretical Anhydrous Inhaled AmB Compositions

a In addition to the lipids and AmB, AmBisome contains 67.8% sucrose, 2.0% disodium succinate hexahydrate, and 0.2% tocopherol

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 cm² tracheal window in four CFTR^A pigs, resulted in an increase in ASL pH of ~0.2 pH units, from pH 6.8 to 7.0 (US 2020/0352970).

The lyophilized AmB:Chol 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 AmB: Choi 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 AmB:Chol complexes demonstrated significant increases in bicarbonate secretion and in ASL pH (~0.1-0.2 pH units) in CuFi-1 cells.

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:Chol 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.

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 ABCI-002, 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 still 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).

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.

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.

Example 2: Manufacture of ABCI-001 via Spray Drying

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 (T_m) 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).

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.

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).

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.

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

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

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).

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 3).

Table 3. Impact of AmB Crystallinity on Primary Particle Size Distribution During Wet-Milling

Table 4. Drug loading and Crystallinity of Formulations of this Disclosure

Flurry powders (prepared with 10% v/v ethanol) - Campaign #2

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 ll.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.

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.

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

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

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.

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.

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 Chol/(Chol+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.

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 T_m values, and to the right to higher cholesterol contents.

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

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-T_m peak in ABCI-001 is 83.9°C and 19.74 J/g, respectively.

The transition from ABCI-001 to ABCI-002 decreased the drug loading from 30% w/w to 14% w/w, 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- T_m peak remains visible in ABCI-002, the T_m increases 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- T_m peak 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.

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- T_m peak region of the ABCI-003 thermogram shows that the Chol-rich peak has been eliminated. The high- T_m peak is also much sharper, with an onset temperature increased by 7.2°C relative to ABCI-001, to 91.1°C.

For the long-term stability of amorphous solids, it may be beneficial to have a glass transition temperature, T_g, that exceeds the storage temperature, T_s, by at least 50°C. By analogy, the T_m in PL represents an order-disorder transition. Having a T_m of 91.1 °C for ABCI-003 means that T_m exceeds an accelerated T_s of 40°C by 50°C.

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 two-fold reduction in MMAD.

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.

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

Significant increases in moisture sorption are observed for compositions containing Choi and DSPG relative to AmB alone. 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 CaCh to 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.

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.

Example 6: Physicochemical Properties of ABCI-001

Spray-dried powders of ABCI-001 were manufactured on a Niro Mobile Minor spray-drier as described in Example 2. The physicochemical properties of the small porous particles (e.g., AmB content, AmB purity, Choi content, primary particle size distribution, bulk density, tapped density, Carr’s Index, and water content) are detailed in Table 6.

As is characteristic of powders spray-dried from suspensions of drugs, ABCI-001 powders are enriched in AmB by about 10% of the nominal drug content. The enrichment leads to a decrease in the Chol/AmB molar ratio to 1.0 mol/mol. Despite the enrichment, the AmB and Choi assay values were consistent across the five batches, with relative standard deviations (RSD) of 5% and 1%, respectively. The purity of the incoming AmB drug substance was 96.7%. Purity of AmB was preserved through the manufacturing process with a mean purity for the five ABCI-001 lots of 97.2 ± 0.5%. Moreover, no new degradant peaks were observed in the RP-HPLC chromatograms.

Table 6. Physicochemical properties of ABCI-001 bulk powder batches.

The primary particle size distribution obtained by laser diffraction is typical of spray-dried particulates from emulsion-based feedstocks with a mean X50 value for the five batches of 1.9 ± 0.1 pm, and a mean X90 of 4.7 ± 0.5 pm. Within the batch, the RSD for the X50 varied between 4.7% and 9.6%, with much of the variability coming from the first collector, before equilibrium was established in the spray dryer.

The low bulk density (0.071 ± 0.008 g/cm³) and tapped density (0.121 ± 0.015 g/cm³) observed are also characteristic of powders manufactured from emulsion-based liquid feedstocks (Table 6). The high compressibility of the fine, low-density particles was demonstrated by the mean Carr’s Index value of 41.5 ± 2.0%. This value suggests that the ABCI-001 powders have little to no flowability. The mean residual water content in the powders was 3.2 ± 0.2%.

Table 7. Properties of ABCI-001 Formulations of this Disclosure

Example 7: Physicochemical Properties of ABCI-002

The physicochemical properties of ABCI-002 are presented in Table 8. The results are somewhat similar to those presented for ABCI-001. That is not surprising, given that ABCI-002 (14% w/w AmB) represents ABCI-001 (30% w/w AmB) diluted with additional PL, while maintaining a constant Chol/AmB ratio of 1.16 mol/mol.

Table 8. Physicochemical properties of ABCI-002 bulk powder batches

Example 8: Physicochemical Properties of ABCI-003

The physicochemical properties of ABCI-003 are detailed in Table 9. Relative to ABCI-001, the drug loading has been reduced from 30% w/w to 14% w/w, and the nominal Chol/AmB ratio has been reduced from 1.2 mol/mol to 0.4 mol/mol.

Table 9. Physicochemical properties of ABCI-003 bulk powder batches

Example 9: Comparison of the Three ABCI Compositions

A comparison of the physicochemical properties and aerosol performance of the three

ABCI compositions is presented in Table 10.

Table 10. Comparison of the Physicochemical Properties and Aerosol Performance of ABCI-

001, ABCI-002, and ABCI-003

There are several apparent trends as the PL/Chol increases from 3.5 to 10.9 to 33.1 w/w

(ABCI-001 to ABCI-003). First, there is an increase in yield from 74.5 to 82.4% (+10.6%). Second, there is a decrease in Carr’s Index from 41.4 to 27.0, indicative of marked improvements in powder flowability. There are also significant changes in aerosol performance. This may reflect significant reductions in interparticle cohesive forces and increases in powder dispersibility as the Choi content decreases. The percentage of drug in the coarse fraction decreases from 36.0% to 6.1%, while the percentage in the airways fraction increases from 49.3% to 71.8%. The FPF<5_fun increases from 56% to 92% of the ED, while the MMAD decreases from 3.9 pm to 2.1 pm. The large decrease in MMAD may also decrease deposition in the nose of rodents during nose-only aerosol delivery.

Example 6: Formulations with Ethanol Co-Solvent

Formulations in this disclosure manufactured with ethanol as a co-solvent are manufactured without an oil phase. The aqueous phase comprises various proportions of ethanol, wherein the lipids are dispersed. Excluding these modifications, manufacturing procedures for formulations with ethanol co-solvent are identical to those previously described for other ABCI formulations.

Table 11: Ethanol Content of Formulations Manufactured with Ethanol Co-Solvent

Ethanol Series

Example 7: Minimum hemolytic concentration assay (MHC)

Preparation of erythrocyte stock suspension

One mL of human whole blood (Na-Heparin preparation; BioIVT, Westbury, NY) was centrifuged at 10,000 x g for 2 min at room temperature. The supernatant was removed, and the pellet was resuspended by gentle inversion in 1 mL 0.9% (m/v) saline (erythrocytes will lyse by pipetting and vortexing). The resulting suspension was centrifuged at 10,000g for 2 min. The supernatant was then removed, and the saline wash repeated twice more. Following the final wash, the supernatant was removed, and the erythrocyte pellet was resuspended in 1 mL resuspension buffer (10 mM Na₂HPO₄-7H₂O, 10 mM NaH₂PO₄ H₂O, 150 mM NaCl, 1 mM MgCl₂-6H₂O, pH 7.4) to make the erythrocyte stock suspension.

MHC assay

Compounds to be tested were prepared in a solution of DMSO (D6-99.9%; Cambridge Isotope Laboratories) in a dilution series, with each concentration at 25.63 x final concentration. Compound dilution series were diluted 1:25 in resuspension buffer to a total of 100 pl in a 0.2 ml microcentrifuge tube and vortexed to mix the solution. The negative control (0% lysis) contained DMSO only in the resuspension buffer, while the positive control (100% lysis) contained DMSO only in water, as this causes erythrocytes to lyse completely due to osmotic pressure. Erythrocyte suspension at a volume of 2.52 pl was added to each tube (including controls), each tube was mixed by gentle inversion, and incubated statically at 37 °C for 2 hrs. Following incubation, each sample was mixed again by gentle inversion and centrifuged at 3,214 x g for 6 min. After centrifugation, 60 pl of the supernatant was removed, added to a 96-well plate, and the absorbance was read at 540 nm. The data was normalized to the negative control and processed as a % total hemolysis relative to the positive control.

Example 8: Broth Microdilution Minimum Inhibitory Concentration (MIC) Assay Determination of Minimum Inhibitory Concentration (MIC) for Candida and Aspergillus spp.

The protocol for determining the MIC on Candida spp. was adapted from CLSI publication M27, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts 4^th Ed. Candida spp. were subcultured on Difco™ Sabouraud Dextrose Agar (SDA; Becton, Dickinson, and Co.) plates at 35 °C for 24 hrs. A single colony was selected and suspended in 1 ml sterile 85% saline and diluted 1 : 10 in HyClone™ RPMI-1640 media (GE Healthcare Life Sciences) with 165 mM MOPS (Fisher Scientific), pH 7.0. Cell density was determined using a hemocytometer, and the final inoculum was diluted to 1 x 10³ CFU/ml using RPMI media. Compounds to be tested were prepared in a solution of DMSO (D6-99.9%; Cambridge Isotope Laboratories) in a dilution series, with each concentration at lOOx final concentration and diluted 1:2.5 in RPMI media. DMSO only was used in positive and negative controls for culture growth. Each concentration sample for each compound (including DMSO only for positive control) was then diluted 1:40 in the cell suspension final inoculum to a final volume of 200 pl in a round-bottom, 96-well plate (Corning) in duplicate. In the same manner, DMSO only was diluted in RPMI only for the negative control. The 96- well plates were then incubated statically at 35 °C for 24 hrs. Immediately following incubation, the MIC was determined by visually observing the concentration at which there was no difference from the negative control when looking from the bottom of the plate and averaging the values of both replicates.

The protocol for determining the MIC on Aspergillus spp. contained a few notable changes based on CLSI publication M38, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi 3^rd Ed. Aspergillus spp. were subcultured on Difco™ Potato Dextrose Agar (PDA; Becton, Dickinson, and Co.) plates at 35 °C for 5 days. Following growth, 1 mL of 85% saline with 0.1% Tween® 20 (Sigma-Aldrich) was pipetted onto the plate and used to resuspend fungal spores in a separate tube. This spore suspension was then diluted 1: 1000 in RPMI media and the density determined using a hemocytometer, with the final inoculum diluted to 1 x 10⁴ spores/ml in RPMI media. Finally, following dilution of compounds in the final inoculum, the resulting 96- well plates were incubated at 35 °C for 48 hrs.

Table 12: MIC assay results for AmB in Fungizone, AmBisome, and ABCI-001 in representative fungal species.

Table 13: MIC assay results for AmB in Fungizone, AmBisome, and R21052 in representative fungal species.

Table 14: MIC assay results for AmB in Fungizone, AmBisome, R21008, R210012, R21013, R21018, R21022, and R21023 in representative fungal species.

Table 15: MIC assay results for AmB in Fungizone, AmBisome, R21024, R210026, R21027 (each aqueous dispersions with no added ethanol), R21031, R21034, and R21038 in representative fungal species.

Table 16: MIC assay results for AmB in Fungizone, AmBisome, R21039, R21041, FP21008 FP21010, FP21011, and FP21019 in representative fungal species.

Table 17: MIC assay results for AmB in Fungizone, AmBisome, FP21020, FP21030, FP21052, and FP21034 placebo in representative fungal species.

. Example 9: Mean Concentration-Time Profiles on Day 29 of AmB ASL Concentrations for ABCI-003 in Rats in Study FY22-071

Mean ASL AmB concentration versus time profiles were also determined for three delivered doses of ABCI-003 administered to rats in Study FY22-071 (FIG. 8). The measured delivered doses (loading dose/maintenance dose) were: 1.1/0.2 mg/kg, 2.5/0.4 mg/kg, and 5.7/1.1 mg/kg). Clearance of AmB from ASL is biphasic, with measurable concentrations of AmB present in ASL at 28 days after administration. The biphasic kinetics observed is consistent with results for IV AmB and is associated with re-distribution of AmB from lung tissue. The initial rate of clearance of AmB from ASL was estimated from the data out to 72 hours post-administration assuming a monoexponential decline. The measured half-lives for the ascending doses were 17, 15, and 23 hr, respectively. In order to maintain concentrations of AmB above the MIC it is desired to maintain daily dosing of the drug. Example 10: Amphotericin B Cystetic for Inhalation (a formulation of the invention): A randomized, double-blind, placebo-controlled, single ascending dose study in healthy volunteers

A formulation of the invention is an investigational drug-device combination product being developed for the treatment of people with CF who are currently not receiving CFTR modulators. The formulation comprises lipid-coated crystals of amphotericin B (AmB) that are administered via oral inhalation with a portable dry powder inhaler. The formulation acts as a ‘molecular prosthetic spontaneously forming ion channels in airway epithelial cell membranes independent of CFTR. In vitro studies have demonstrated significant improvements in chloride secretion, bicarbonate secretion, and airway surface liquid pH, viscosity, and antimicrobial activity. Significant improvements in nasal potential difference, comparable to those achieved with ivacaftor, were also observed in people with CF. The improvements in these biomarkers are independent of the CFTR mutation, with large improvements in activity observed for Class I or other nonsense mutations where little or no CFTR protein is produced. Forty-eight healthy subjects were enrolled in a randomized, double-blind, placebo-controlled, single ascending dose trial (Study CM001001, Part A) at NZCR (Christchurch, NZ). There were six dose cohorts (0.5, 1.0, 2.0, 4.0, 6.0, 10.0 mg), with 8 subjects per cohort (6 active, 2 placebo).

Two formulations of the invention were used: a low-strength powder comprising 3.9% w/w AmB (Formulation ABCI-004), and a high-strength powder comprising 15.8% w/w AmB (Formulation ABCI-003). Following encapsulation in size #3 HPMC capsules at a fill mass of ~13 mg, the formulations provided nominal dose strengths of about 0.5 mg and 2.0 mg, respectively. Powder was administered with an ultrahigh resistance variant of the RS01 dry powder inhaler (Plastiape S.p.A., Osnago, Italy) (FIG. 1A and FIG. IB). Safety parameters including vital signs, clinical chemistry, pulmonary function, ECGs, and adverse events were monitored throughout. Venous blood samples were drawn at 0 (pre-dose), 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 96 hours post-dose for pharmacokinetic characterization. Plasma concentrations were determined with a validated LC- MS/MS method with a LLOQ of 0.1 ng/mL (Resolian Pharma, Malvern, PA). Table 18. Baseline demographics

All doses of inhaled formulations of the invention were well tolerated. There were no clinically significant outliers in vital signs, ECGs, and clinical chemistry, including serum electrolytes and serum creatinine. There were no reports of bronchospasm, or dose-limiting pulmonary toxicities. All adverse events were mild and transient with a similar incidence of treatment-related AEs between active and placebo groups (Table 19).

Table 19. Treatment-related AEs in the Single Ascending Dose portion of Study CM001001

The most prevalent AE was a mild headache. None of the systemic AEs observed with intravenous AmB were reported with a single inhaled dose of a formulation of the invention. FIG. 10 presents the plasma AmB concentration-time profiles for a formulation of the invention. Plasma AmB concentrations exhibited a 1 -2 hr delay in appearance post-administration peaking at around 8 hr. Plasma AmB concentrations increased proportionally with increasing dose, yet concentrations were well below the threshold associated with systemic toxicity with intravenous AmB (i.e., sustained AmB concentrations >1000 ng/mL). PK parameters determined using WinNonlin are presented in Table 20.

Table 20. PK parameters calculated using WinNonLin

In conclusion, single doses of a formulation of the invention up to 10 mg were well tolerated with only mild, transient adverse events. Plasma drug levels of AmB were 2-3 orders of magnitude below the threshold concentration for systemic effects. There were no systemic adverse events characteristic of IV AmB reported. No evidence of respiratory tolerability issues (bronchospasm, wheezing, dyspnea, post-inhalation cough) were observed.

In the MAD study in healthy volunteers, subjects received a loading dose (LD) followed by 13 (low and mid-dose groups) or 27 daily (high-dose group)_maintenance doses (MD). Multiple doses of ABCI 1.5 mg LD/0.5 mg MD, 6.0 mg LD/2.0 mg MD, and 10.0 mg LD/4.0 mg MD QD were well tolerated without any dose-limiting toxicities or bronchospasm observed. All TEAEs were mild or moderate in intensity and nonserious; all study drug-related TEAEs were mild in intensity. The incidences of TEAEs in the ABCI cohorts (range: 50% to 100%) were similar to those in their respective Placebo cohorts (75% and 100%). The most common TEAEs overall were headache (20.8% subjects) and nasal congestion and chest discomfort (12.5% subjects each). TEAEs of increased bronchial secretion, which had been reported in the SAD portion of the study, were also reported by 2 (33.3%) subjects (3 events) in the highest ABCI 10.0 mg/4.0 mg QD dose cohort in the MAD portion, and in no subjects in the placebo cohort. Of the 3 TEAEs of increased bronchial secretion, all were mild, non-serious, considered probably or definitely related to study drug, and of varied duration (1 to 40 days) before resolving. One subject in the ABCI 10.0 mg/4.0 mg QD cohort experienced a mild AESI of glomerular filtration rate decreased that was considered possibly related to study drug by the Investigator, lasted 21 days, and resolved without a change in study drug dosing. No subjects experienced an SAE, TEAE leading to early discontinuation of study drug, or death. There were no clinically significant or dose-dependent changes in hematology, chemistry, vital signs (including oximetry), ECGs, or spirometry when comparing any ABCI cohort to its respective placebo cohort during Part B of this study.

Geometric mean plasma AmB concentration-time profiles after administration of multiple maintenance doses of ABCI 0.5 mg QD and 2.0 mg QD on Day 14 for Cohorts G and H, respectively, and 4.0 mg QD on Day 28 for Cohort I showed that plasma AmB concentrations generally increased with increasing dose. Multiphasic elimination was observed following cessation of dosing in all cohorts, but was most pronounced in subjects in Cohort I (ABCI 10.0 mg/4.0 mg) during the 56-day sampling period following administration of the last daily dose on Day 28 (FIG. 11). Comparison of washout phases following administration of the last doses in all regimens shows that the rates of decline in concentrations during the 14-day washout sampling period in Cohorts G and H do not represent intrinsic terminal elimination rates of AmB, which are better characterized by the prolonged sampling shown for Cohort I. An analysis was performed to estimate the terminal ti/2 values in Cohort I

Table 21. Summary of Terminal ti/2 Values for Cohort I - Part B (PK Population)

Abbreviations: CV=coefficient of variation; PK=pharmacokinetic; SD=standard deviation

When evaluating ti/2 in all 6 subjects in Cohort I (ABCI 10.0 mg/4.0 mg), terminal ti/2 estimates ranged from 500.79 to 1347.25 hours — or between approximately 21 to 56 days, similar to, or somewhat longer than, those reported following IV administration of AmB deoxycholate (Bekersky et al, 2002a; Bellmann, 2007). This study intentionally included a 56-day sampling period after administration of ABCI once daily for 28 days in an attempt to better characterize the prolonged disposition of AmB. The prolonged half-life is comparable to that observed for AmB in lung tissue in animals following inhalation of ABCI. As such, it is believed that the terminal half- life reflects redistribution of AmB from lung tissue that is subsequently absorbed.

A bronchoalveolar lavage (BAL) study was conducted in 6 healthy subjects in Cohort I to determine trough concentrations of AmB in airway surface liquid (ASL). Subjects received a 10.0 mg loading dose followed by daily 4.0 mg maintenance doses over 28 days. The trough ASL AmB concentrations were >38.2 mg/mL in the six subjects studied (Table 33). This is well above the MIC for various strains of Aspergillus spp. and Candida spp. Hence the concentrations in both lung tissue and in ASL are well above the concentrations needed for effective killing. Table 22. ASL Amphotericin B Concentrations for Cohort I - Part B (PK Evaluable Population)

INCORPORATION BY REFERENCE All U.S. patents and published U.S. and PCT patent applications mentioned in the description above are incorporated by reference herein in their entirety.

EQUIVALENTS

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

CLAIMS We claim:

1. A method of treating a pulmonary fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, comprising:

(ii) cholesterol (Choi);

(iv) calcium chloride (CaCh).

2. A method of preventing a pulmonary fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, comprising:

(ii) cholesterol (Choi);

(iv) calcium chloride (CaCh).

3. The method of claim 1 or 2, wherein the subject has cancer, acquired immunodeficiency syndrome, has undergone transplant (e.g., lung transplant), is otherwise immunocompromised, or any combinations thereof.

4. The method of any one of claims 1-3, wherein the subject is a human.

5. The method of any one of claims 1-4, wherein the subject is a human at least 6 years old.

6. The method of any one of claims 1-5, wherein the amount of AmB is about 0.5% to about

30% w/w.

7. The method of any one of claims 1-6, wherein the amount of AmB is about 3% to about 16% w/w.

8. The method of claim 7, wherein the amount of AmB is about 14% w/w.

9. The method of any one of claims 1-8, wherein the amount of Choi is about 0.1% to about 8% w/w.

10. The method of any one of claims 1-9, wherein the amount of Choi is about 0.3% to about 6% w/w.

11. The method of any one of claims 1-10, wherein the amount of Choi is about 0.5% to about 3% w/w.

12. The method of any one of claims 1-11, wherein the amount of CaCh is about 1% to about 10% w/w.

13. The method of any one of claims 1-12, wherein the amount of CaCh is about 4% to about 7% w/w.

14. The method of any one of claims 1-13, wherein the amount of phospholipids is about 60% to about 95% w/w.

15. The method of any one of claims 1-14, wherein the amount of phospholipids is about 70% to about 90% w/w.

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

17. The method of any one of claims 1-16, wherein the weight ratio of Choi to phospholipids is about 0.005:1 to about 0.05: 1.

18. The method of any one of claims 1-17, wherein the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 2: 1 to about 19: 1.

19. The method of any one of claims 1-18, wherein the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 7:1 to about 12: 1.

20. The method of any one of claims 1-19, wherein the molar ratio of Choi to AmB is about 0.05:1 to about 1.2:1.

21. The method of any one of claims 1-20, wherein the molar ratio of Choi to AmB is about 0.4:1 to about 1.2: 1.

22. The method of any one of claims 1-21, wherein the molar ratio of Choi to AmB is about 0.05:1 to about 0.4:1.

23. The method of claim 22, wherein the molar ratio of Choi to AmB is about 0.4:1.

24. The method of any one of claims 1-23, wherein the molar ratio of phospholipids to CaCh is about 4: 1 to about 2: 1.

25. The method of claim 24, wherein the molar ratio of phospholipids to CaCh is about 2:1.

26. The method of any one of claims 1-25, wherein the AmB has a crystallinity greater than about 75%.

27. The method of claim 26, wherein the AmB has a crystallinity greater than about 85%.

28. The method of claim 27, wherein the AmB has a crystallinity greater than about 95%.

29. The method of claim 1 or 2, 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).

30. The method of claim 1 or 2, 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).

31. The method of claim 1 or 2, 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).

32. The method of claim 1 or 2, 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 (CaCL).

33. The method of claim 1 or 2, 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.

34. The method of any one of claims 1-33, wherein the AmB and Choi are not complexed; and the AmB is not encapsulated in liposomes.

35. The method of any one of claims 1-34, wherein the AmB is coated with a porous shell of phospholipids and Choi.

36. The method of any one of claims 1-35, wherein the pharmaceutical composition is formulated as a dry powder.

37. The method of claim 36, wherein the mass median diameter, X50, of the powder particles is about 1.0 to about 4.0 pm.

38. The method of claim 37, wherein the mass median diameter, X50, of the powder particles is about 1.5 to about 3.5 pm.

39. The method of any one of claims 36-38, wherein the tapped density of the powder particles is about 0.03 to about 0.4 g/mL.

40. The method of any one of claim 39, wherein the tapped density of the powder particles is about 0.06 to about 0.2 g/mL.

41. The method of any one of claims 36-40, wherein the Carr’s index of the powder particles is about 20 to about 32.

42. The method of any one of claims 36-41, wherein the main transition temperature (T_m) of the shell is at least 80 °C.

43. The method of any one of claims 36-42, wherein the water content of the powder is about 1.5 to about 6% w/w.

44. The method of any one of claims 36-43 wherein the mass median aerodynamic diameter of the powder particles is about 1.5 pm to about 4.0 pm.

45. The method of claim 44, wherein the mass median aerodynamic diameter of the powder particles is about 2.0 pm to about 3.5 pm.

46. The method of any one of claims 1-45, wherein the pharmaceutical composition is formulated for pulmonary administration or airway administration.

47. The method of any one of claims 1-46, wherein the pharmaceutical composition is formulated for aerosol administration.

48. The method of any one of claims 1-47, wherein the pharmaceutical composition is formulated for administration as a dry powder inhaler.

49. The method of any one of claims 46-48, wherein the nominal dose or metered dose of the pharmaceutical composition is about 0.01 mg to about 10 mg.

50. The method of claim 49, wherein the nominal dose or metered dose of the pharmaceutical composition is about 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, or 4.0 mg.

51. The method of any one of claims 46-50, wherein the pharmaceutical composition is administered once daily.

52. The method of any one of claims 46-51, wherein the absolute bioavailability of the AmB is about 0.1% to about 5%.

53. The method of any one of claims 1-52, wherein the administration is chronic (e.g., daily).

54. The method of any one of claims 1-53, wherein the subject is a recipient of a lung transplant.