Use of dry powder inhalers for the treatment or prevention of pulmonary fungal infections

Abstract

Methods for treating or preventing a pulmonary fungal infection in a recipient of, for example, a lung transplant are disclosed.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 539,424, filed on September 20, 2023. Background Technology

[0002] Over the past 40 years, the incidence of pulmonary fungal infections has increased significantly, coinciding with a rise in the number of immunocompromised patients. Immunocompromised patients account for the largest proportion of the approximately 100,000 serious fungal infections that occur annually in the United States. Aspergillus species (…) Aspergillus ) and Candida species fungi ( Candida Opportunistic fungal infections are the most frequently involved pathogens. Although the incidence of Candida infections has decreased since the introduction of fluconazole, mortality rates attributable to invasive pulmonary aspergillosis (IPA) have been reported to be as high as 85%, and overall mortality as high as 95%, even with aggressive intravenous therapy. Early intervention is crucial for the treatment of IPA, but diagnosis of opportunistic fungal infections remains challenging and may be further delayed in at-risk patients. Therefore, infection prevention (if achievable) would be a significant medical advancement. Patients at particularly high risk of developing IPA include those with hematologic malignancies, solid tumors, AIDS, and organ transplant recipients.

[0003] Amphotericin B remains the most effective agent against Aspergillus fungi. Pulmonary fungal infections are usually acquired through inhalation of fungal conidia. These conidia, with a diameter of 2.5 to 3.5 micrometers, can reach both the large and small airways in the lower respiratory tract. In immunocompromised hosts, impaired host defenses allow them to germinate into hyphal forms and subsequently invade tissues. Maintaining an inhibitory concentration at the same location in the lungs where the conidia have reached should provide protection against infection. Targeting the lungs can reduce or mitigate most of the toxicity associated with systemic AmB products. Prophylactic delivery of AmB via nebulization has been studied clinically. However, nebulization is time-consuming and cumbersome, and can lead to adverse events due to high pharyngeal deposition of aerosols and additives such as sodium deoxycholate. Furthermore, nebulizers are inefficient for targeting the lungs. Therefore, inhalable dry powder formulations of AmB for the prevention of pulmonary fungal infections offer advantages. Given the above, there is an unmet need for the development of new antifungal therapeutics and prophylaxis. Summary of the Invention

[0004] In some respects, this document discloses a method for treating pulmonary fungal infections, the method comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

[0005] In some respects, this article discloses a method for preventing pulmonary fungal infections, the method comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2). Attached Figure Description

[0006] Figure 1A The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with AmB in Fungizone is shown.

[0007] Figure 1B The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with ABCI-003 (containing crystalline AmB and Chol in a 0.4 Chol:AmB mol / mol ratio) is shown.

[0008] Figure 2 The minimum hemolytic concentrations (in percent) of hemolyzed blood cells after treatment with AmB and ABCI with different Chol contents are shown: ABCI-003 (0.4 Chol:AmB mol / mol); CM22001 (0.2 Chol:AmB mol / mol); CM22002 (0.1 Chol: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); Figure 3A The minimum hemolytic concentration of blood cells after treatment with AmB in DMSO is shown as a percentage.

[0009] Figure 3B The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with AmB in Fungizone is shown.

[0010] Figure 3C The minimum hemolysis concentration, expressed as a percentage, of hemolyzed blood cells after treatment with AmBisome is shown.

[0011] Figure 3D The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with powder R21052 (30% AmB prepared with PFOB) is shown.

[0012] Figure 4A The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21008 (AmB:Chol (1:2.5) prepared with DSPC and DSPG) is shown.

[0013] Figure 4B The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21012 (AmB:Chol (1:2.5) prepared with DSPC, CaCl2, and PFOB) is shown.

[0014] Figure 4C The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21013 (AmB:Chol (1:2.5) prepared with DSPC, DSPG, and CaCl2) is shown.

[0015] Figure 4D The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21018 (AmB:Chol (1:2.5) [1.23 mol Chol:mol lipid] prepared with DSPC, DSPG, and CaCl2) is shown.

[0016] Figure 4E The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21022 (20% AmB prepared with DSPC, DSPG, and CaCl2) is shown.

[0017] Figure 4F The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21023 (20% AmB prepared with HSPC, DSPG, and CaCl2) is shown.

[0018] Figure 4G The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21024 (20% AmB prepared with HSPC, DSPG, and CaCl2) is shown.

[0019] Figure 4HThe minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21026 (15% AmB prepared with HSPC, DSPG, and CaCl2) is shown.

[0020] Figure 4I The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21027 (30% AmB prepared with HSPC, DSPG, and CaCl2) is shown.

[0021] Figure 4J The minimum hemolytic concentration of blood cells after placebo treatment is shown as a percentage.

[0022] Figure 5A The minimum hemolysis concentration, expressed as a percentage, of hemolyzed blood cells after treatment with AmBisome is shown.

[0023] Figure 5B The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with powder R21034 (ABCI-002 prepared with 0% v / v ethanol) is shown.

[0024] Figure 5C The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with powder R21038 (ABCI-002 prepared with 6% v / v ethanol) is shown.

[0025] Figure 5D The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with powder R21039 (ABCI-002 prepared with 9% v / v ethanol) is shown.

[0026] Figure 5E The minimum hemolysis concentration, expressed as a percentage, of blood cells after hemolysis following treatment with powder cM21041 (ABCI-002) is shown.

[0027] Figure 5F The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder FP21008 (ABCI-002 prepared with 10% v / v ethanol, highly crystalline AmB).

[0028] Figure 5G The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with powder FP21010 (ABCI-002 prepared with 10% v / v ethanol, AmB with lower crystallinity) is shown.

[0029] Figure 5HThe minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powdered FP21011 (30% AmB prepared with 10% v / v ethanol, highly crystalline AmB).

[0030] Figure 6A The minimum hemolyzed concentration, expressed as a percentage, of blood cells after treatment with powder FP21019 (3% w / w solid prepared with 5% v / v ethanol) is shown.

[0031] Figure 6B The minimum hemolyzed concentration, expressed as a percentage, of blood cells after treatment with powder FP21020 (3% w / w solid prepared with 10% v / v ethanol) is shown.

[0032] Figure 7A The minimum hemolytic concentration, expressed as a percentage, of blood cells after treatment with powder R21034 (placebo) is shown.

[0033] Figure 7B The use of powder R21030 (with Novec 7500 (C9H5F)) was shown. 15 The minimum hemolytic concentration (in %) of hemolyzed blood cells after treatment with ABCI-001 (prepared O).

[0034] Figure 7C The minimum hemolytic concentration, expressed as a percentage, of hemolyzed blood cells after treatment with powder R21052 (ABCI-001 prepared with PFOB) is shown.

[0035] Figure 8 The mean concentration-time curve of AmB ASL concentration of ABCI-003 in rats on day 29 is shown.

[0036] Figure 9A A scanning electron micrograph (SEM) of spray-dried ABCI-003 powder is shown.

[0037] Figure 9B The RS01 dry powder inhaler is shown in digital rendering.

[0038] Figure 10 The concentration of the inhaled dose of the formulation of the present invention relative to time is shown.

[0039] Figure 11 The geometric mean (95% CI) plasma concentration-time curves of amphotericin B (ng / mL) in each treatment group are shown (top plot is semi-logarithmic scale, bottom plot is linear scale) - part B (PK evaluable population). Detailed Implementation

[0040] Over the past 40 years, the incidence of pulmonary fungal infections has increased significantly, coinciding with a rise in the number of immunocompromised patients. Immunocompromised patients account for the largest proportion of the approximately 100,000 serious fungal infections that occur annually in the United States. Aspergillus species (…) Aspergillus ) and Candida species fungi ( Candida Opportunistic fungal infections are the most frequently involved pathogens. Although the incidence of Candida infections has decreased since the introduction of fluconazole, mortality rates attributable to invasive pulmonary aspergillosis (IPA) have been reported to be as high as 85%, and overall mortality as high as 95%, even with aggressive intravenous therapy. Early intervention is crucial for the treatment of IPA, but diagnosis of opportunistic fungal infections remains challenging and may be further delayed in at-risk patients. Therefore, infection prevention (if achievable) would be a significant medical advancement. Patients at particularly high risk of developing IPA include those with hematologic malignancies, solid tumors, AIDS, and organ transplant recipients. The most effective agent against Aspergillus remains amphotericin B. Pulmonary fungal infections are usually acquired through the inhalation of fungal conidia. Fungal conidia, with a diameter of 2.5 to 3.5 micrometers, are able to reach the large and small airways in the lower respiratory tract, where impaired host defenses in immunocompromised hosts allow them to germinate into hyphal forms and subsequently invade tissues. Maintaining an inhibitory concentration at the same location in the lungs where conidia arrive should provide protection against infection. Targeting the lungs can reduce or mitigate most of the toxicity associated with systemic AmB products. Prophylactic delivery of AmB via nebulization has been studied clinically. However, nebulization is time-consuming and cumbersome, and can lead to adverse events due to high pharyngeal deposition of aerosols and additives such as sodium deoxycholate. Furthermore, nebulizers are inefficient for targeting the lungs. Therefore, inhalable dry powder formulations of AmB for the prevention of pulmonary fungal infections offer advantages. In light of the above, there is an unmet need for the development of new antifungal therapeutics and prophylaxis. We have created a novel inhaled dry powder formulation called BCYstetic for Inhalation (ABCI), which consists of wet-milled AmB crystals coated with a porous layer of phospholipids (hydrogenated soybean phosphatidylcholine (HSPC); distearate phosphatidylglycerol (DSPG)), cholesterol (Chol), and calcium chloride, and is administered via oral inhalation to directly target the apical side of airway epithelial cells.

[0041] During the development of ABCI, we studied dozens of formulations and observed their cytotoxicity (minimum hemolytic concentration (MHC)) curves. Figure 1AThe MHC curves of AmB formulations dissolved in bile salt micelles in Fungizone are shown. AmB significantly causes hemolysis in human blood cells because it extracts Chol from the lipid bilayer. Formulations containing amorphous AmB but without Chol still cause hemolysis in human blood cells; this relationship can be simply explained by the formulations with the highest solubility in water (i.e., deoxycholate micelles, amorphous drugs). However, the ABCI-003 formulation (Figure 1C) containing both crystalline AmB and Chol (0.4 Chol:AmB (mol / mol)) is not toxic to human blood cells. The formulation containing crystalline AmB and a Chol:AmB (mol / mol) ratio of as low as 0.05 (CM22003) in ABCI... Figure 2 Preparations containing crystalline AmB but not Chol (CM22004 and CM22006) will not cause hemolysis (see [link to relevant documentation]). Figure 3A – Figure 7C (All MHC curves). It is unclear why some AmB formulations showed toxicity in this assay while others did not.

[0042] During the ABCI development process, we also investigated the minimum inhibitory concentrations (MICs) of formulations with different AmB, Chol, co-solvent, EtOH, or foaming agent contents, and compared them with reference formulations of AmB (water-soluble) and AmBisome (liposome 2.5 Chol:AmB (mol / mol) formulations) in Fungizone. The MICs of all studied formulations, as well as the reference MICs, were determined in biologically grade water. The MIC values ​​of all studied formulations are listed in Tables 12-18. The results showed that powders without co-solvents or containing ethanol appeared to have higher average MIC values, while powders containing fluorinated foaming agents (CF3-CF2-CF2-CF(OCH2CH3)-CF(CF3)-CF3; Novec 7500 or PFOB) had lower MIC values ​​(i.e., more potent antifungal activity), even though the drug was crystalline in those powders and therefore presumably insoluble in water. It is unclear why the different AmB formulations exhibited such differences in efficacy. We speculate that this may be due to the improved drug dissolution from the particles caused by their porous morphology and reduced aggregation, leading to higher drug utilization for fungicide activity. On the other hand, other unknown factors may also play a role. The ABCI-001 (R21052) formulation of the fluorinated foaming agent PFOB is characterized by excellent antifungal activity and no toxicity to human cells was observed. Figure 7A – Figure 7CBased on the data provided, we predict that our final formulation, ABCI-003 (currently being tested in clinical trials and also formulated with PFOB), will exhibit high antifungal activity. Furthermore, ABCI-003 is non-toxic to human blood cells. Figure 2 Furthermore, no serious adverse events have been observed in clinical studies to date, which together make it an ideal targeted IFI therapy for individuals with cancer, acquired immunodeficiency syndrome, those who have undergone transplantation, or those whose immune systems are compromised in other ways.

[0043] Currently available treatments using intravenous nebulizer formulations rely on the redistribution of the drug present in lung tissue to provide protection, with dose reduction from twice weekly to once every two weeks. This can lead to insufficient airway surface fluid (ASL) concentrations and potentially breakthrough infections. In contrast, ABCIs are designed for daily administration and have significantly higher AmB concentrations than the MIC in areas where pathogen deposition is maintained after inhalation of fungal conidia. The nominal dose administered is also much lower, which reduces dose-related adverse events (e.g., bronchospasm, unpleasant taste, cough). This also influences the desired composition and results in lower drug content in the formulation to achieve a lower daily dose. The shorter administration time (i.e., a single inhalation relative to a longer time on the nebulizer) also reduces the likelihood of adverse events, especially in immunocompromised cancer patients who cannot tolerate longer administration times.

[0044] To date, all inhaled AmB formulations have been administered weekly or bi-weekly under steady-state conditions. This may be suboptimal for a prophylactic strategy, as the half-life of inhaled amphotericin B in the airway surface fluid (ASL) deposited by conidia upon inhalation is approximately 10–20 hours. The drug initially deposited in the ASL is cleared via multiple pathways, such as absorption, mucociliary clearance, cough clearance, and macrophage clearance. Most of the inhaled AmB deposits in the lung tissue and is cleared by circulating and tissue-resident macrophages. AmB redistributes from the lung tissue into the ASL with a half-life of approximately 20 days. However, the concentration of the drug in the ASL is very low compared to the initial concentrations achieved after AmB inhalation. Therefore, it may be advantageous to inhale a lower dose of AmB daily to maintain AmB concentrations well above the MIC in the ASL. The formulations described herein are designed for daily administration of a lower nominal dose while still maintaining ASL concentrations well above the MIC.

[0045] Pharmaceutical compositions containing AmB The pharmaceutical compositions used in the currently 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.

[0046] In some respects, the pharmaceutical composition comprises: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

[0047] In some embodiments, the amount of AmB is from about 0.5% to about 30% w / w. In other embodiments, the amount of AmB is from about 3% to about 16% w / w. In still other embodiments, the amount of AmB is about 14% w / w.

[0048] In some embodiments, the amount of Chol is from about 0.1% to about 8% w / w. In other embodiments, the amount of Chol is from about 0.3% to about 6% w / w. In still other embodiments, the amount of Chol is from about 0.5% to about 3% w / w.

[0049] In some embodiments, the amount of CaCl2 is from about 1% to about 10% w / w. In other embodiments, the amount of CaCl2 is from about 4% to about 7% w / w.

[0050] In some embodiments, the amount of phospholipid is from about 60% to about 95% w / w. In other embodiments, the amount of phospholipid is from about 70% to about 90% w / w.

[0051] In some embodiments, the weight ratio of Chol to phospholipid is from about 0.001:1 to about 0.1:1. In other embodiments, the weight ratio of Chol to phospholipid is from about 0.005:1 to about 0.05:1.

[0052] In some embodiments, the weight ratio of soybean phosphatidylcholine (HSPC) to distearyl phosphatidylglycerol (DSPG) is from about 2:1 to about 19:1. In other embodiments, the weight ratio of soybean phosphatidylcholine (HSPC) to distearyl phosphatidylglycerol (DSPG) is from about 7:1 to about 12:1.

[0053] In some embodiments, the molar ratio of Chol to AmB is from about 0.05:1 to about 1.2:1. In other embodiments, the molar ratio of Chol to AmB is from about 0.4:1 to about 1.2:1. In still other embodiments, the molar ratio of Chol to AmB is from about 0.05:1 to about 0.4:1. In still other embodiments, the molar ratio of Chol to AmB is about 0.4:1.

[0054] In some embodiments, the molar ratio of phospholipid to CaCl2 is about 4:1 to about 2:1. In other embodiments, the molar ratio of phospholipid to CaCl2 is about 2:1.

[0055] In some embodiments, AmB has a crystallinity greater than about 75%. In other embodiments, AmB has a crystallinity greater than about 85%. In still other embodiments, AmB has a crystallinity greater than about 95%.

[0056] In some embodiments, the pharmaceutical composition comprises, is substantially composed of, or is composed of the following substances: (i) Approximately 30.0% w / w amphotericin B (AmB); (ii) Approximately 14.6% w / w cholesterol (Chol); (iii-a) Approximately 35.8% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 15.9% w / w distearate (DSPG); and (iv) Approximately 3.7% w / w calcium chloride (CaCl2).

[0057] In some embodiments, the pharmaceutical composition comprises, is substantially composed of, or is composed of the following substances: (i) Approximately 14.0% w / w amphotericin B (AmB); (ii) Approximately 6.81% w / w cholesterol (Chol); (iii-a) Approximately 51.2% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 22.8% w / w distearate (DSPG); and (iv) Approximately 5.2% w / w calcium chloride (CaCl2).

[0058] In some embodiments, the pharmaceutical composition comprises, is substantially composed of, or is composed of the following substances: (i) Approximately 14.0% w / w amphotericin B (AmB); (ii) Approximately 2.3% w / w cholesterol (Chol); (iii-a) Approximately 70.3% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 7.8% w / w distearate (DSPG); and (iv) Approximately 5.52% w / w calcium chloride (CaCl2).

[0059] In some embodiments, the pharmaceutical composition comprises, is substantially composed of, or is composed of the following substances: (i) Approximately 3.4% w / w amphotericin B (AmB); (ii) Approximately 0.57% w / w cholesterol (Chol); (iii-a) Approximately 80.72% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 8.97% w / w distearate (DSPG); and (iv) Approximately 6.34% w / w calcium chloride (CaCl2).

[0060] In some embodiments, the pharmaceutical composition comprises: (i) A Chol / AmB ratio of approximately 0.4 to 1.2 mol / mol; (ii) A Chol / PL ratio of less than approximately 0.05 w / w; (iii) An HSPC / DSPG ratio of approximately 2.3 to approximately 9.0 w / w; and (iv) Approximately 2:1 mol / mol PL / Ca 2+ ratio.

[0061] In some implementations, AmB and Chol are not compounded; and AmB is not encapsulated in liposomes.

[0062] In some implementations, AmB is coated with a porous shell of phospholipids and choline.

[0063] In some embodiments, the pharmaceutical composition is formulated as a dry powder.

[0064] In some implementations, the mass median diameter X of the powder particles 50 The diameter is approximately 1.0 to approximately 4.0 µm. In another embodiment, the mass median diameter X of the powder particles is... 50 It is approximately 1.5 to approximately 3.5 µm.

[0065] In some embodiments, the tap density of the powder particles is from about 0.03 to about 0.4 g / mL. In other embodiments, the tap density of the powder particles is from about 0.06 to about 0.2 g / mL.

[0066] In some implementations, the Carr's index of the powder particles is about 20 to about 32.

[0067] In some implementations, the main transformation temperature (T) of the shell m The temperature should be at least 80°C.

[0068] In some embodiments, the water content of the powder is from about 1.5% to about 6% w / w.

[0069] In some embodiments, the median aerodynamic mass diameter of the powder particles is from about 1.5 µm to about 4.0 μm. In other embodiments, the median aerodynamic mass diameter of the powder particles is from about 2.0 µm to about 3.5 μm.

[0070] In some embodiments, the pharmaceutical composition is formulated for pulmonary or airway administration.

[0071] In some embodiments, the pharmaceutical composition is formulated for aerosol administration.

[0072] In some embodiments, the pharmaceutical composition is formulated for administration as a dry powder inhaler.

[0073] In some embodiments, the nominal dose or dosage of the pharmaceutical composition is from 0.01 mg to 10 mg. In other embodiments, the nominal dose or dosage of the pharmaceutical composition is 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, or 4.0 mg.

[0074] In some implementations, the pharmaceutical composition is administered once daily.

[0075] In some implementations, the absolute bioavailability of AmB is from about 0.1% to about 5%.

[0076] In some embodiments, a dry powder composition of engineered particles is provided, the dry powder composition comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein the Chol / AmB ratio is from about 0.05 to about 1.2 mol / mol, such as from about 0.2 to about 0.6 mol / mol.

[0077] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell containing PL and Chol, wherein the Chol / PL ratio is less than 0.10 w / w or less than 0.05 w / w.

[0078] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell containing PL and Chol. In some embodiments, PL comprises hydrogenated soybean phosphatidylcholine (HSPC), distearyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylglycerol (DSPG), or a combination thereof.

[0079] In some embodiments, a dry powder composition of engineered particles is provided, the dry powder composition comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein PL comprises (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.

[0080] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), 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 be reduced to below about 2.0 mol / mol.

[0081] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), 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 from about 0.5% to about 25% w / w, and the nominal Chol / AmB ratio is from about 0.05 to about 1.2 mol / mol.

[0082] In some embodiments, a dry powder composition of engineered particles (ABCI-003) is provided, comprising spray-dried core-shell particles of fine crystalline AmB particles (about 14.0% w / w) coated with a porous shell of PL and Chol, 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 PC / PG ratio in PL is about 9.0 w / w.

[0083] In some embodiments, a dry powder composition of engineered particles (ABCI-004) is provided, comprising spray-dried core-shell particles of fine crystalline AmB particles (about 3.4% w / w) coated with a porous shell of PL and Chol, wherein 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 PC / PG ratio in PL is about 9.0 w / w.

[0084] In some embodiments, a dry powder composition of engineered particles is provided, comprising spray-dried core-shell particles of fine crystalline AmB particles (14.0% w / w) coated with a porous shell of PL and Chol, wherein the Chol / AmB ratio is from 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 PC / PG ratio in PL is between about 2.3 and about 9.0 w / w.

[0085] In some implementations, the maximum Chol / AmB ratio is approximately 1.2 mol / mol, but this high ratio may only be effective for maintaining lipids in a highly ordered state. A lower drug loading (e.g., not exceeding 10.0% w / w) is acceptable. A Chol / AmB reduction to 0.4 mol / mol is permissible in this phase. Higher drug loading within the phase (e.g., not exceeding 22% w / w).

[0086] In some embodiments, the compositions described herein comprise an HSPC / DSPG ratio of about 2.3 to about 9.0 w / w and a PL / Ca ratio of about 2.0 mol / mol. 2+ ratio.

[0087] In some embodiments, the composition described herein is selected based on maintaining the lipids in a single phase (i.e., the gel phase). ),in (More than 50°C higher than the accelerated storage temperature of 40°C).

[0088] In some embodiments, the composition described herein is selected based on the maximum increase in ASL pH maintained over a wide range of AmB concentrations.

[0089] In some embodiments, the compositions described herein are selected based on the reduction in hygroscopicity relative to compositions with HSPC / DSPG < 9.0.

[0090] In some embodiments, the composition described herein is selected based on increased production yield.

[0091] In some embodiments, the composition described herein is selected based on improved powder flowability.

[0092] In some embodiments, the composition described herein is selected based on its content relative to the amount of The aerosol properties of the phase powder are improved.

[0093] In some embodiments, the compositions described herein are selected based on the absence of erythrocyte hemolysis even at low Chol / AmB molar ratios.

[0094] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), 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.

[0095] In some embodiments, a dry powder composition of engineered particles is provided, the dry powder composition comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the median mass diameter (X) of the particles is [missing information]. 50 The range is from about 1.0 to about 5.0 µm, such as from about 1.5 to about 4.0 µm.

[0096] In some embodiments, a dry powder composition of engineered particles is provided, the dry powder composition comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the X of the particles 90 It ranges from about 3 µm to about 10 µm, such as from about 3.5 µm to about 7 µm.

[0097] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), wherein the tap density of the particles is from about 0.03 to about 0.40 g / mL, such as from about 0.06 to about 0.20 g / mL.

[0098] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), wherein the water content in the powder is from about 1.0% to about 10.0%, preferably from about 2.0% to about 5.0%.

[0099] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), wherein when administered with a portable dry powder inhaler, the median aerodynamic mass diameter (MMAD) is from about 1.0 µm to about 6.0 µm, such as from about 2.0 µm to about 4.0 µm.

[0100] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), wherein when administered with a portable dry powder inhaler, the fraction of fine particles less than 5 µm, expressed as a percentage of the nominal dose, is at least 30% w / w, at least 50% w / w, or at least 60% w / w.

[0101] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of crystalline AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the powder is prepared by spray drying a liquid feedstock comprising fine AmB crystals suspended in an oil-in-water emulsion stabilized by a monolayer of the lipid mixture described herein.

[0102] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of crystalline AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the lipids have a primary transition temperature of at least 80°C, such as at least 90°C. ).

[0103] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of crystalline AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the powder is produced by passing liquid feedstock through a process with a minimum density less than that of lipids. It is prepared by spray drying at an outlet temperature. In some embodiments, the outlet temperature is at least 50°C, at least 60°C, or at least 70°C.

[0104] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of crystalline AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the powder is produced by passing liquid feedstock through a process with a minimum density less than that of lipids. It is prepared by spray drying at an outlet temperature. In some embodiments, the outlet temperature is at least 60°C or at least 70°C.

[0105] In some embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of crystalline AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), wherein the powder is prepared by spray drying a liquid feedstock on a PSD-1 grade spray dryer at a total gas flow rate of about 70 to about 100 scfm.

[0106] In some embodiments, a dry powder composition of engineered particles is filled using a rotary drum filling machine. The dry powder composition comprises AmB drug particles coated with a porous shell containing PL, Chol, and calcium chloride (CaCl2).

[0107] In some embodiments, the powder filling mass in capsule No. 3 or No. 2 is about 1.0 mg to about 40 mg, such as about 3 mg to about 20 mg, or about 10 mg to about 15 mg.

[0108] In some implementations, the powder filling mass has good precision (e.g., RSD < 3%) and accuracy relative to the target filling mass.

[0109] Treatment In some respects, this document discloses a method for treating pulmonary fungal infections, the method comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

[0110] In some respects, this article discloses a method for preventing pulmonary fungal infections, the method comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

[0111] In some implementations, the subjects had cystic fibrosis.

[0112] In some embodiments, the subject has cancer, acquired immunodeficiency syndrome, has undergone transplantation (e.g., lung transplantation), is otherwise immunocompromised, or any combination thereof. In other embodiments, the subject is a person. In still other embodiments, the subject is a person aged at least 6 years.

[0113] In some implementations, application is long-term (e.g., daily application).

[0114] In some implementations, the subject is the recipient of a lung transplant.

[0115] definition As used herein, the term "treat / treating" refers to an intervention that results in (a) the prevention of the development of a symptom or disease in a subject who may be at risk of developing a symptom or disease or who is susceptible to a symptom or disease but has not yet been diagnosed with such a symptom or disease; or (b) the suppression of a symptom or disease, such as slowing or halting its development; or (c) the relief or improvement of a symptom or disease, such as causing the symptom or disease to regress. In one embodiment, the term "treat / treating" refers to an intervention that results in (a) the suppression of a symptom or disease, such as slowing or halting its development; or (b) the relief or improvement of a symptom or disease, such as causing the symptom or disease to regress.

[0116] As used herein, "subject" or "patient" refers to a living mammal. In various embodiments, a patient is a non-human mammal, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, sheep, goats, cats, dogs, pigs, horses, cattle, or non-human primates. In some embodiments, a patient is a human.

[0117] As used in this article, “effective amount” means any amount sufficient to achieve the desired biological effect.

[0118] As used in this article, "therapeutic effective amount" refers to any amount sufficient to achieve the desired therapeutic effect, such as treating CF.

[0119] As used in this article, “active ingredient,” “therapeutic active ingredient,” “active agent,” “drug,” or “medicinal substance” refers to the active ingredient of a drug, also known as an active pharmaceutical ingredient (API).

[0120] As used in this article, "amorphous" refers to a state in which a material lacks long-range order at the molecular level and exhibits physical properties that vary with temperature, resembling either a solid or a liquid. Typically, such materials do not produce distinctive X-ray diffraction patterns and, while exhibiting solid properties, are more formally described as liquids. Upon heating, a change from solid to liquid-like properties occurs during the "glass transition," a change often defined as a second-order phase transition.

[0121] As used herein, “crystallization” refers to a solid phase in which the material has a regular, ordered internal structure at the molecular level and produces a distinctive X-ray diffraction pattern with defined peaks. Such materials will also exhibit liquid properties when sufficiently heated, but the change from solid to liquid is characterized by a phase transition, typically a first-order phase transition (“melting point”). In the context of this disclosure, crystalline active ingredient means an active ingredient with a crystallinity greater than 75%. In some embodiments, the crystallinity is suitably greater than 90%. In other embodiments, the crystallinity is greater than 95%. In still other embodiments, the crystallinity is less than 10% or less than 5%.

[0122] As used herein, “drug loading” refers to the percentage of the active ingredient in the total mass of the composition based on a mass meter.

[0123] As used in this article, "median diameter of mass" or "MMD" or "X" 50 "This refers to the median diameter of a group of particles (i.e., composed of particles of a range of sizes) in a polydisperse particle population. Unless the context otherwise indicates, X is as reported in this paper." 50 The values ​​were determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany).

[0124] As used in this article, “taper density” or “ρ” 振实 "Measured in a manner similar to Method I, such as USP" <616> The bulk density and tapped density of powders are described below. Tapped density represents an approximation of particle density that is closer than the dumping method bulk density, and the measured value is about 20% smaller than the actual particle density.

[0125] As used herein, “aerodynamic mass median diameter” or “MMAD” refers to the aerodynamic median size of multiple particles in a polydisperse population. “Aerodynamic diameter” is the diameter of a sphere of unit density that typically has the same settling velocity as powder in air, and is therefore an effective way to characterize aerosolized powders or other dispersed particles or particle compositions based on settling behavior. Aerodynamic particle size distribution (APSD) and MMAD were determined in this paper using the NEXTGENERATION IMPACTOR™ (Copley Scientific) via cascade impaction. Generally, if the aerodynamic size of the particles is too large, fewer particles will reach specific areas of the lung. If the particles are too small, a larger percentage of the particles may be exhaled. In contrast, d a The aerodynamic diameter of a single particle.

[0126] As used herein, “nominal dose” or “ND” refers to the mass of medication contained in a container (e.g., capsule or blister pack) in a non-reservoir type dry powder inhaler. ND is sometimes also referred to as the dose.

[0127] As used herein, “expelled dose” or “ED” refers to an indication of the amount of dry powder delivered from the inhaler device following the actuation or dispersion event of the powder unit. ED is defined as the ratio of the dose delivered by the inhaler device to the nominal or metered dose. ED is a parameter determined experimentally and can be determined using an in vitro device that simulates patient administration. ED is sometimes also referred to as delivered dose (DD).

[0128] As used herein, “Fine Particle Fraction” (FPF) refers to the percentage of active ingredient in the ejected dose with an aerodynamic size less than 5 µm. Aerodynamic Particle Size Distribution (APSD) was determined in this paper using the NEXT GENERATION IMPACTOR™ via cascade impaction.

[0129] As used herein, “solid content” refers to the concentration of active ingredients and excipients dissolved or dispersed in the liquid solution or dispersion to be spray-dried.

[0130] As used in this article, “the subject’s airway” refers to any or all of the following lung structures: trachea, bronchi, and bronchioles.

[0131] The term “about” refers to numerical variations that are commonly encountered by those skilled in the art of breathable compositions, including variations of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the values ​​described herein.

[0132] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprising” or variations such as “including” or “containing” shall be understood to mean including the said integer or step or group of integers or steps, but not excluding any other integer or step or group of integers or steps.

[0133] Unless otherwise stated or clearly apparent from the context, the range of values ​​includes the endpoints and any values ​​in between.

[0134] Example Various aspects and embodiments of this disclosure will be further illustrated with reference to the following examples. These examples are illustrative in nature and should not be construed as limiting the subject matter of this disclosure.

[0135] Characterization methods Amphotericin B (AmB) content and purity. The content and purity of AmB in prepared bulk powder or aerodynamic particle size distribution (APSD) samples were determined by reversed-phase high-performance liquid chromatography (RP-HPLC) with detection at 383 nm. Samples were analyzed using an Agilent 1260 Infinity II HPLC system (Wilmington, DE, USA). Separation was achieved using a gradient method (solvent A = 10 mM acetate buffer, pH 4.2; solvent B = acetonitrile / methanol, 1.0 v / v) with an Agilent InfinityLab Poroshell 120 EC-C18 (3.0 x 150 mm, 2.7 µm) column. AmB was quantified using a single-point calibration with USP-certified reference standards.

[0136] Cholesterol content. Cholesterol content was determined by RP-HPLC, with detection at 210 nm. Samples were analyzed using an Agilent 1260 Infinity II HPLC system (Wilmington, DE, USA). Separation was achieved using an isocratic method (acetonitrile / isopropanol, 1.0 v / v) with a Haisil Clipeus™ C18 column (5 µm). Cholesterol quantification was performed using high-purity cholesterol (Carbogen Amcis, Beuvry-la-Foret, France) conforming to the European Pharmacopoeia and the United States Pharmacopoeia, with single-point calibration.

[0137] Primary particle size distribution.Primary particle size distribution was determined by laser diffraction (Sympatec GmbH, Clausthal-Zellerfeld, Germany). The Sympatec H3296 unit was equipped with an R2 lens, an ASPIROS micro-dosing unit, and a RODOS / M dry powder dispersion unit. Approximately 2 to 5 mg of powder was filled into the ASPIROS tube and fed at 5 mm / s into the RODOS, which was operating at a dispersion pressure of 4 bar and a vacuum of 65 mbar. The powder was introduced at an optical concentration of approximately 1% to 5%, and data were collected over a measurement duration of up to 15 seconds. The particle size distribution was calculated using the Fraunhofer model via the instrument software. The reported values ​​represent the average of three independent measurements for each collector.

[0138] Tap density. Using a known volume (0.593 cm³) 3 The tap density of the cylindrical cavity was determined. Use a small scraper to fill the sample holder with powder. Then gently tap the sample cell on the work surface. As the sample volume decreases, add more powder to the cell. Repeat the tapping and powder addition steps until the cavity is full and the powder bed is no longer compacted with further tapping. The tapped density is defined as the mass of the tapped powder bed divided by the volume of the cavity.

[0139] Bulk density. Bulk density ( This indicates the mass of powder loaded into the sample holder to the desired volume without being struck.

[0140] Carr's index. Carr index An indication of powder compressibility is provided. It is given by the following formula: It is believed that powder flowability decreases as the Karl Fischer index increases. A value of <10% indicates good free-flowing powder, a value between 11-15% is associated with good free-flowing powder, a value between 16-20% is associated with normal powder flow, a value between 21-25% is associated with acceptable powder flow, a value between 26-31% is associated with poor flowability and cohesiveness, a value between 32-37% is associated with very poor flowability, and a value ≥38% is associated with almost no flowability.

[0141] Water content. The water content was determined by Karl Fischer titration using a Nittoseiko Analytech CA-310 moisture analyzer with a glassless cathode and a VA-300 vaporizer.

[0142] Dynamic vapor adsorption. Moisture adsorption isotherms at 25 °C were measured using a Dynamic Vapor Adsorption (DVS) instrument manufactured by Surface Measurement Systems (UK). This instrument measures the absorption and desorption of water vapor by the material via gravimetric analysis. The DVS system is equipped with a recording microbalance with a resolution of ±0.1 μg and a daily drift of approximately ±1 μg. In the first step of the experimental run, the sample was dried at 25 °C and 0% RH for 24 hours to achieve a constant mass. The instrument program was then set to proceed from 0 to 2% RH, to 5% RH, then to 90% RH in 5% RH increments, and then to 0% RH in 5% RH increments. A balancing criterion of dm / dt = 0.005% / min was selected to allow the system to automatically proceed to the next RH step after each RH step. Sample masses between 10 and 15 mg were used in this study.

[0143] Differential scanning calorimetry. The DSC temperature spectrum of a given sample was measured using a TA Instruments Q2000 differential scanning calorimeter equipped with a refrigerated cooling system (New Castle, Delaware). Dry nitrogen gas was used at a velocity of 50 cm⁻¹. 3 The sample cell was purged at a flow rate of 110 cm / min; the cryogenic control system (RCS) used a flow rate of 110 cm / min. 3 Nitrogen gas was applied at a rate of 5°C / min. A Tzero aluminum disc containing approximately 5 to 10 mg of powder was hermetically sealed using a sample encapsulation press. The sample was equilibrated at -40°C and then heated to 200°C at a rate of 5°C / min.

[0144] Aerodynamic particle size distribution. Aerodynamic particle size distribution (APSD) was measured using an RS01 dry powder inhaler (Mod. 7 Ultra-High Resistance Type 2), a USP induction port (IP), and a Next Generation Impactor™ or NGI™ and conformed to USP standards. <601> And Ph. Eur. 2.9.18 requirements. Adjust the flow control device to operate at a pressure drop of 4 kPa and a total volume through the inhaler of 4 L. The RS01 DPI variant used has 0.143 cm H2O. 0.5 L -1 min (0.045 kPa 0.5 L -1 The resistance at (min) corresponds to 44.2 L / min at a voltage drop of 4 kPa. -1The flow rate was adjusted. Approximately 10 mg was manually filled into #3 inhalation-grade HPLC capsules (VCaps, Qualicaps). The aerosol powder expelled from the inhaler was aspirated through the USP IP and fractionated at the NGI. Each stage of the NGI, the empty capsules, and the device were extracted with a sample dissolving solution containing methanol. Further dilutions were performed to reduce the AmB concentration within the linear range of the detection range. The mass of AmB at each stage was determined using the HPLC method described above, and the fraction less than 5 µm (FPD) was calculated. <5 µm The fine particulate dose and aerodynamic mass median diameter (MMAD) of the particles.

[0145] Cell lines and growth conditions. NuLi, CuFi-1, and CuFi-4 cells (Welsh Laboratory, University of Iowa) were used in Thermo Scientific BioLite 75 cm⁻¹ 2Cell culture was performed on flasks containing cryopreserved cells. These flasks were previously coated with 4 mL of 60 μg / mL human placental type IV collagen (Sigma-Aldrich) at 37°C for at least 1 hour, washed twice with PBS, dried, and then inoculated. Cells were cultured in 12 mL of bronchial epithelial cell growth medium (BEGM) Bullet Kit (Lonza CC-3170), which contained basal medium and eight SingleQuots 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 culture medium was supplemented with 50 μg / mL penicillin-streptomycin (Corning Cellgro), 50 μg / mL gentamicin (Sigma-Aldrich G1397), and 2 μg / mL fluconazole (Sigma-Aldrich). Genotyping of the original CF transplant donor was performed using Integrated Genetics. The cell line was reconfirmed by the ATCC repository to have the correct genotype and be free of mycoplasma contamination. MycoAlert Mycoplasma Detection Kit (Lonza LT07-418) was used to detect any RNA transcripts common to broad-spectrum mycoplasma. The cell line was confirmed to be mycoplasma-free. Cells were cultured at 37°C in 5% CO2 until >90% confluence, with the medium changed every two to three days, followed by trypsinization with 4 mL of 0.25% trypsin containing 1 mM EDTA (Gibco 25200-056). Trypsin was inactivated using 10 mL of HEPES-buffered saline (Lonza CC-5024) containing 1% fetal bovine serum. Cells were centrifuged at 1,500 rpm for 5 minutes at room temperature in an Eppendorf 5430R centrifuge and resuspended in BEGM medium for passage. For culture on membrane supports used for differentiation, cells were resuspended in Ultroser G medium after centrifugation. This medium contained 1:1 DMEM:Ham's F-12, supplemented with 4% v / v Ultroser G (Crescent Chemical) and 50 μg / mL penicillin-streptomycin (Corning Cellgro), 50 μg / mL gentamicin (Sigma-Aldrich G1397), and 2 μg / mL fluconazole (Sigma-Aldrich). The membrane support used for all studies was Corning Costar 0.4-μm 24-well Transwell transparent polyester membrane insert (0.33 cm). 2(Corning 3470). These membranes were coated with collagen in the same manner as the flasks detailed above, except that 100 mL of collagen was used and the membranes were rinsed only once with PBS. Each plug was seeded with 115,000 cells. The membranes were allowed to mature at the air-liquid interface for at least 14 days to achieve complete differentiation, with the Ultroser G medium changed weekly or more as needed. After maturation, the medium was changed every seven days. For covariate control, the membranes used in the experiments were made as similar as possible in terms of culture time and maturity.

[0146] For primary cultured airway epithelial cells, lung tissue was obtained from individuals with CF undergoing lung transplantation or organ donation. The tissue was dissected, and the dissected cells were directly seeded onto a transwell filter and cultured at the air-liquid interface. The culture was used more than three weeks after seeding, at which point the epithelial cells had differentiated into typical airway cell types and the electrical properties of the cells reflected the excised tissue.

[0147] Fluorescence microscopy analysis for measuring the pH of fluids on the airway surface. Fresh ABCI suspensions for each experiment were prepared by dispersing approximately 2 mg of ABCI in approximately 100 μL of perfluorohexane PFH (FC-72, Sigma-Aldrich) to achieve a final concentration of approximately 1 mM AmB. After dissolution in methanol, the AmB concentration in the stock suspensions was measured in triplicate by absorption spectroscopy. Beer's law was used to determine the AmB concentration at 406 nm (ε0). 406 = 164,000 M -1 cm -1 ) Calculate the concentration. Next, dilute the stock suspension with PFH to achieve... AmB concentration in suspensions ranging from 0.5 to 50 μM.

[0148] ASL pH was measured using small-diameter NuLi and CuFi cells. ASL pH was measured using a ratiometric pH indicator, SNARF-dextran conjugate (Molecular Probes). SNARF powder was suspended in PFH by sonication and distributed onto the top surface of the cells. ASL pH was measured after 2 hours. 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 objective for cell line culture at 40x magnification. To generate a standard curve for pH measurement, SNARF was dissolved in a colorless pH standard solution, and the fluorescence ratio was converted to pH. The powder to be tested in this assay was suspended in an appropriate volume of PFH and sonicated for 1 minute to aid resuscitation. AmBisome was resuscitated by vortexing in PFH. Subsequently, 20 µL of the suspension was applied to cultured airway epithelium (A = 0.33 cm⁻¹) at concentrations between 0.5 and 50 μM. 2 On the surface of the epithelium. In all experiments, the ASL pH of the compound-treated epithelium was measured and compared with the results of the solvent-treated epithelium. For top compound application, the cultured airway epithelium was incubated at 37°C for approximately 22 hours before measuring the ASL pH.

[0149] Preparation of red blood cell reserve suspension. One mL of human whole blood (heparin sodium preparation; BioIVT, Westbury, NY) was centrifuged at 10,000 x g for 2 minutes at room temperature. The supernatant was removed, and the pellet was resuspended by gently inverting in 1 mL of 0.9% (m / v) saline (red blood cells will lyse by aspiration and vortexing). The resulting suspension was centrifuged at 10,000 g for 2 minutes. The supernatant was then removed, and the saline wash was repeated twice. After the final wash, the supernatant was removed, and the red blood cell pellet was resuspended in 1 mL of resuspension buffer (10 mM Na2HPO4∙7H2O, 10 mM NaH2PO4∙H2O, 150 mM NaCl, 1 mM MgCl2∙6H2O, pH 7.4) to prepare a red blood cell stock suspension.

[0150] Example 1: Compositions of lipid-coated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004) The nominal anhydrous compositions of four lipid-coated AmB compositions (ABCI-001, ABCI-002, ABCI-003, and ABCI-004) are shown in Table 2. Two controls are also shown in the table: AmBisome. ®(i.e., liposomal amphotericin B, L-AmB); and lyophilized AmB / Chol complexes prepared by Burke et al. (US 2019 / 0083517; US 2020 / 0352970; both incorporated herein by reference).

[0151] Table 2. Theoretical anhydrous inhalation AmB compositions

[0152] a In addition to lipids and AmB, AmBisome also contains 67.8% sucrose, 2.0% disodium succinate hexahydrate, and 0.2% tocopherol. Although commercially available L-AMB contains the same lipid components (i.e., HSPC, DSPG, and Chol) as the ABCI composition, they are present in different proportions. Furthermore, the drug and lipids are organized differently in L-AMB compared to the ABCI composition. In L-AMB, the drug substance is encapsulated in small monolayer vesicles (liposomes). This 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). In the presence of a high proportion of sucrose, the lipid particles are lyophilized into a dry powder, with the sucrose acting as a cryoprotectant to maintain the integrity of the liposomes during lyophilization. In four CFTRs... - / - 1cm in pig 2 Administration of 60 mg of reconstituted L-AMB to the apical side of airway epithelial cells through the tracheal window resulted in an increase in ASL pH. 0.2 pH units, increasing from pH 6.8 to 7.0 (US 2020 / 0352970).

[0153] The lyophilized AmB:Chol complex studied by Burke et al. (US 2019 / 0083517; US 2020 / 0352970) has an even higher Chol / AmB ratio of 5.0 mol / mol, but without added PL. The AmB:Chol complex was formed by rapid nanoprecipitation of a solution of the material in dimethyl sulfoxide / chloroform upon rapid injection into a non-solvent (water). The resulting suspension was lyophilized to form a dry powder. The AmB:Chol complex exhibited bicarbonate secretion and ASL pH ( ) in CuFi-1 cells. A significant increase (0.1-0.2 pH units).

[0154] The compositions of three ABCI compositions (ABCI-001, ABCI-002, and ABCI-003) that have been used in non-clinical toxicology studies are also provided. These powders have a Chol / AmB ratio of 0.4 to 1.2 mol / mol. It is not desirable to be bound by theory, but the high Chol / AmB ratios utilized in L-AMB and AmB:Chol complexes may not be suitable for dry powder compositions containing PL, as the presence of large amounts of Chol may lead to disordered packing of PL acyl chains, resulting in an unacceptable increase in interparticle cohesion and “sticky” powder.

[0155] The DSC temperature spectrum of ABCI-001 (30% w / w AmB, Chol / AmB = 1.2 mol / mol) contains a significant proportion of Chol-rich material. (Example 4). For ABCI-002, the drug content was reduced to 14% w / w while maintaining a constant Chol / AmB molar ratio, resulting in a decrease in the Chol / PL ratio from 0.29 to 0.09 w / w. Nevertheless, a small proportion of Chol-rich material was still retained. Phase. Phase separation exists. Dry powders of this phase can exhibit strong interparticle cohesion and increased hygroscopicity and deliquescence at high relative humidity. These characteristics can adversely affect powder yield during spray drying and may result in a relatively large aerodynamic median diameter (MMAD). 4 µm).

[0156] ABCI-003 maintained the 14% w / w drug loading of ABCI-002, but with a reduced nominal Chol / AmB ratio of 0.4 mol / mol. Overall, the Chol content decreased from 14.6% in ABCI-001 to 2.4% in ABCI-003, and the Chol / PL ratio decreased to 0.03 w / w. At this Chol / PL ratio, Phase dissolves in In the ABCI-003 phase, the HSPC / DSPG ratio also increased from 2.3 w / w to 9.0 w / w, resulting in increased solubility of Chol in the PL phase, reduced NaCl formation, and decreased powder hygroscopicity.

[0157] Many limitations regarding Chol / AmB, Chol / PL, and HSPC / DSPG ratios may apply only to the development of inhaled dry powder compositions and may not be suitable for liquid-based aerosols such as inhaled L-AMB. They also cannot be directly applied to compositions without PL.

[0158] Example 2: Manufacturing ABCI-001 by spray drying Raw material preparation. First, lipids and calcium chloride are dispersed in hot water using a high-shear mixer (UltraTurrax T-50) to form multilayer vesicles (MLVs). The aqueous phase must be above the major transition temperature (T0) of the lipids. m To promote MLV formation (T>65℃), the MLV dispersion was cooled (T<30℃), and perfluorobromooctane (PFOB) was filtered and added using a Watson-Marlow peristaltic pump while mixing to form water-encapsulated PFOB-type crude emulsion droplets stabilized by a lipid monolayer. The emulsion droplets acted as pore-forming agents to form a porous lipid coating layer on the crystalline drug particles. The crude emulsion was then homogenized individually under high pressure using an M-100 microfluidic jet to form nanoemulsion droplets (diameter...). (200-500 nm). Next, the drug substance is added to the nanoemulsion under high-shear mixing. The composite dispersion containing the suspended drug and nanoemulsion droplets is passed through a homogenizer and then subjected to two separate homogenizations. The homogenization method wet-mills the AmB particles to a suitable size for pulmonary delivery. Based on quantity, most wet-milled AmB crystals have a diameter of less than 1000 nm. In one embodiment, the final feedstock composition has 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 feedstock) or 424.2 g (12 liters feedstock).

[0159] Dry powder is produced by spray drying. Spray drying was performed using a pilot-scale spray dryer (Niro Mobile Minor, Copenhagen, Denmark), equipped with a Schlick 970 / 0 dual-fluid atomizer (0.8 mm inner diameter), a DorrClone cyclone separator, and a lower geometry including a straight tube and a Brewer valve. A 1 L Eagle collector connected below the Brewer valve was jacketed and maintained at 50°C.

[0160] The liquid feed was pumped into the spray dryer using a Watson-Marlow peristaltic pump. The atomizer operated at a gas flow rate of 7.0 ± 0.6 scfm and an inlet temperature of 104 ± 5°C. The liquid feed rate was adjusted to maintain the dryer outlet temperature at 55 ± 3°C. The total gas flow rate was approximately 140 Nm³. 3 / h ( 85 scfm).

[0161] In the suspension-based feed, each atomized droplet (median mass diameter ~10 μm) contains dispersed drug crystals and approximately 1000 submicron emulsion droplets. Initially, the more volatile aqueous phase begins to evaporate. The rapidly shrinking atomized droplet interface drives the slowly diffusing drug and emulsion particles to accumulate at the interface. This results in the formation of void spaces at the center of the dried droplets. As the drying process continues, the less volatile oil phase in the emulsion droplets evaporates, leading to the formation of hollow pores in their locations. In summary, the resulting hollow spray-dried composite particles contain drug crystals embedded in an interfacial layer within a porous lipid matrix.

[0162] ABCI-002, ABCI-003, and ABCI-004 were prepared using the same general method, but the composition of the liquid feed differed.

[0163] Example 3: Wet milling of AmB and its effect on the properties of ABCI-002 The physical form of AmB (crystalline versus amorphous) can influence its wet milling behavior. For this study, two batches of AmB were purchased from North China Pharmaceutical Group Corp. (Hebei, China). The two batches had different crystallinities, with batches '202 and '203 having crystallinities of 77% and 96%, respectively (as determined by quantitative XRPD).

[0164] In fact, the low crystallinity of batch '202 adversely affects the wet milling process, significantly increasing the X-ray content of the wet-milled drug. 50 and X 90 For example, measurements of drug suspensions were performed using the Malvern Mastersizer via laser diffraction (Table 3).

[0165] Table 3. Effect of AmB crystallinity on primary particle size distribution during wet milling

[0166] Table 4. Drug loading and crystallinity of the formulations disclosed herein Flocculent powder (prepared with 10% v / v ethanol) - Experimental batch #2

[0167] Using the method described in Example 2, batches of ABCI-002, namely AmB batches '202 (ABCI-002 batch FP21060) and '203 (ABCI-002 batch FP21059), were manufactured on a Niro Mobile Minor. The total solids content and PFOB% in the liquid feedstock were 2% w / w and 10% v / v, respectively. The liquid feed rate was approximately 44.5 g / min. The atomizer was set at 11.5 ± 1.0 N m. 3 Operating at a gas flow rate of 7.0 scfm / h and an inlet temperature of 104 ± 5 ​​°C. This equates to approximately 44.5 g / min. The total gas flow rate is approximately 140 Nm³. 3 / h ( 85 scfm). Target batch size is 40 g.

[0168] The physicochemical properties and aerosol properties of these two batches are detailed in Table 5. No significant differences were observed in powder properties and aerosol properties, indicating that a crystallinity as low as 77% is still suitable for preparing the ABCI-002 composition.

[0169] Table 5. Comparison of physicochemical properties and aerosol performance of ABCI-002 batches prepared from AmB batches with different crystallinities

[0170] Example 4: Effect of lipids on the phase behavior of lipid-coated crystal compositions of AmB The addition of Chol and DSPG to the ABCI composition results in a more complex temperature spectrum, namely, broadened transition peaks and the appearance of multiple peaks.

[0171] At a Chol / PL ratio of 0.29 w / w (ABCI-001), two phase-separated regions were observed: a Chol-rich region at an initial temperature of 62.5 °C. The phase and the wide PL-rich phase with an initial temperature of 83.9℃ Phase. As demonstrated by the hydrated DPPC bilayer, towards Adding Chol to a phase increases disorder and eventually leads to coexistence. Phase separation of phases.

[0172] The phase transition of Chol-rich molecules is very broad and contains overlapping features. X-ray powder diffraction (XRPD) patterns confirm this. The phase contains Chol crystals, and these figures show diffraction peaks of the ABCI variant at approximately 5.2° 2θ. In PLCI-001, the phase separation of cholesterol microcrystals is more easily distinguishable (i.e., fewer diffraction peaks in the same region and no interference from amphotericin B peaks). The PLCI composition is a placebo composition that does not contain AmB but contains other components of the ABCI composition.

[0173] A linear increase in the enthalpy of the Chol-rich phase transition was observed with increasing Chol content in the powder composition. Linear regression was performed on data points where Chol / (Chol+PL) > 10% w / w. Data were extrapolated to the x-axis when the Chol / (Chol+PL) weight ratio was approximately 4.9% w / w. This corresponds to a Chol / PL ratio of 0.05 w / w or 9.4 mol% Chol. Therefore, compositions having less than about 9.4 mol% Chol are expected to have undetectable low-temperature peaks.

[0174] In a dehydrated state The initiation of the two-phase region tends to occur at much higher Chol concentrations. Therefore, the ordering in the ABCI composition... The phase remains at a much higher Chol content (for hydrated DPPC bilayers) 9.4 mol% vs. 6 mol%). Similarly, although exceeding approximately 20 mol% Chol in hydrated DPPC / Chol mixtures... Phase elimination, but in dehydrated ABCI-001, The two-phase region extends beyond 36.5 mol% Chol. Therefore, relative to the phase diagram of the hydrated DPPC-Chol mixture, the phase diagram of ABCI-001 shifts upwards to a higher value. The value shifts to the right towards higher cholesterol levels.

[0175] High in ABCI-001 The peaks are also very broad and contain multiple overlapping features. High The enthalpy of the peak tends to increase with increasing PL content and tends to decrease with increasing Chol content, indicating that below... The peak and It is related.

[0176] Binary mixtures of HSPC / DSPG in 20% AmB compositions (Chol-free) exhibit similar overlapping characteristics, indicating coexisting immiscibility. Phase separation of the PC and PG domains in the presence of calcium ions is consistent with observations in other studies. High levels of calcium ions in ABCI-001... The peak's onset temperature and enthalpy were 83.9℃ and 19.74 J / g, respectively.

[0177] The transition from ABCI-001 to ABCI-002 reduced the drug loading from 30% w / w to 14% w / w while maintaining a Chol / AmB ratio of 1.2 mol / mol. The reduction in AmB and Chol resulted in an increase in PL content and a decrease in the Chol / PL ratio from 0.28 to 0.092 w / w. Although a small amount of low... The peak is still visible in ABCI-002, but in the Chol-rich region The temperature increased from 62.5℃ to 64.6℃, while ΔH decreased from 7.73 to 1.93 J / g. (High) The peaks remain broad and exhibit overlapping characteristics. Compared to ABCI-001, the onset temperature increased from 83.9℃ to 88.5℃, indicating that the acyl chain ordering increases with decreasing Chol content.

[0178] In ABCI-003, the drug loading remained at 14% w / w, but the Chol / AmB ratio decreased from 1.2 to 0.4 mol / mol. This allowed the Chol / PL ratio to decrease from 0.092 w / w to 0.030 w / w relative to ABCI-002. The low temperature spectrum of ABCI-003... Magnified view of the peak region shows that the Chol-rich peaks have been eliminated. The peaks are also sharper, and the onset temperature has increased by 7.2°C compared to ABCI-001, reaching 91.1°C.

[0179] For the long-term stability of amorphous solids, a glass transition temperature may be beneficial. Exceeding storage temperature At least 50°C. Similarly, in PL... This indicates an order-disorder transition. For ABCI-003, 91.1℃ mean Acceleration above 40°C Temperatures exceeding 50°C.

[0180] As previously mentioned, reducing the Chol / PL ratio has a profound impact on powder properties, increasing production yield from 74.5% to 82.4%, reducing the Chol index from 41.4% to 27.0, and significantly reducing interparticle cohesion, as evidenced by a nearly two-fold reduction in MMAD.

[0181] As the Chol content in the powder composition increases, it is observed that... The enthalpy of the phase transition decreases linearly. Regression at these points shows that the x-intercept (i.e., the location where the enthalpy is zero) occurs at a Chol / PL ratio of 0.05 w / w.

[0182] Example 5: Effect of lipid composition on the hygroscopicity of AmB composition under high relative humidity Compared to AmB alone, a significant increase in moisture adsorption was observed in the composition containing Chol and DSPG. Without being bound by theory, it is believed that the increased moisture adsorption is due to the presence of NaCl in the composition resulting from the interaction between DSPG and calcium chloride. Divalent calcium ions can strongly bind to the anion DSPG Na, leading to the displacement of sodium ions, which can then interact with chloride ions from CaCl2 to form NaCl. At high RH, the composition containing DSPG / CaCl2 deliquesces, with the magnitude of moisture adsorption and deliquescence being proportional to the amount of NaCl formed.

[0183] Compared to ABCI-001 and ABCI-002, in which the HSPC / DSPG ratio is 2.3 (i.e. 7 / 3) w / w, ABCI-003, in which the PL component contains an HSPC / DSPG ratio of 9.0 w / w, exhibits significantly reduced hygroscopicity.

[0184] Example 6: Physicochemical properties of ABCI-001 The spray-dried powder of ABCI-001 was manufactured on a Niro Mobile Minor spray dryer as described in Example 2. The physicochemical properties of the small porous particles (e.g., AmB content, AmB purity, Chol content, primary particle size distribution, bulk density, tap density, Karl Fischer index, and water content) are detailed in Table 6.

[0185] Characterized by being a powder spray-dried from a drug suspension, the ABCI-001 powder exhibits an enrichment of AmB of approximately 10% of the nominal drug content. This enrichment results in a decrease in the Chol / AmB molar ratio to 1.0 mol / mol. Despite the enrichment, the AmB and Chol values ​​were consistent across the five batches, with relative standard deviations (RSDs) of 5% and 1%, respectively. The purity of the AmB drug substance fed was 96.7%. The purity of AmB was maintained throughout the manufacturing process, with an average purity of 97.2 ± 0.5% across the five ABCI-001 batches. Furthermore, no new degradation peaks were observed in the RP-HPLC chromatograms.

[0186] Table 6. Physicochemical properties of ABCI-001 bulk powder batches.

[0187] The primary particle size distribution obtained by laser diffraction is typical of spray-dried particles from emulsion-based feedstocks, with an average X from five batches. 50 The value is 1.9 ± 0.1 μm, and the average X 90 The value was 4.7 ± 0.5 μm. In this batch, X was measured before equilibration was established in the spray dryer. 50 The RSD varied between 4.7% and 9.6%, with most of the variation coming from the first collector.

[0188] The observed low bulk density (0.071 ± 0.008 g / cm³) 3 ) and tap density (0.121 ± 0.015 g / cm³) 3 This is also a characteristic of powders manufactured from emulsion-based liquid raw materials (Table 6). The high compressibility of the fine, low-density particles is demonstrated by an average Karl Fischer index value of 41.5 ± 2.0%. This value indicates that the ABCI-001 powder has very little or no flowability. The average residual water content in the powder is 3.2 ± 0.2%.

[0189] Table 7. Characteristics of the ABCI-001 formulation disclosed herein

[0190] Example 7: Physicochemical properties of ABCI-002 The physicochemical properties of ABCI-002 are shown in Table 8. The results are somewhat similar to those presented for ABCI-001. This is not surprising, as 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.

[0191] Table 8. Physicochemical properties of ABCI-002 bulk powder batches

[0192] Example 8: Physicochemical properties of ABCI-003 The physicochemical properties of ABCI-003 are detailed in Table 9. Compared 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.

[0193] Table 9. Physicochemical properties of ABCI-003 bulk powder batches

[0194] Example 9: Comparison of three ABCI compositions The physicochemical properties and aerosol properties of the three ABCI compositions are shown in Table 10.

[0195] Table 10. Comparison of physicochemical properties and aerosol performance of ABCI-001, ABCI-002, and ABCI-003

[0196] As the PL / Chol ratio increased from 3.5 to 10.9 and then to 33.1 w / w (ABCI-001:ABCI-003), several distinct trends emerged. First, the yield increased from 74.5% to 82.4% (+10.6%). Second, the Carr index decreased from 41.4 to 27.0, indicating a significant improvement in powder flowability.

[0197] Significant changes were also observed in aerosol properties. This is reflected in the significant decrease in interparticle cohesion and the increase in powder dispersibility as the Chol content decreased. The percentage of drug in the coarse fraction decreased from 36.0% to 6.1%, while the percentage in the airway fraction increased from 49.3% to 71.8%. FPF <5µm The percentage of ED increased from 56% to 92%, while MMAD decreased from 3.9 μm to 2.1 μm. The significant reduction in MMAD also reduced deposition in the nasal cavity of rodents during nasal aerosol delivery only.

[0198] Example 6: Formulations with ethanol as a co-solvent The formulations in this disclosure, manufactured using ethanol as a co-solvent, are produced without an oil phase. The aqueous phase contains ethanol in various proportions, wherein the lipids are dispersed. Apart from these modifications, the manufacturing procedure for the formulations with an ethanol co-solvent is the same as that previously described for other ABCI formulations.

[0199] Table 11: Ethanol content of formulations prepared using ethanol as a co-solvent Ethanol series

[0200] Example 7: Minimum Hemolytic Concentration Determination (MHC) Preparation of Red Blood Cell Reserve Suspension One mL of human whole blood (heparin sodium preparation; BioIVT, Westbury, NY) was centrifuged at 10,000 x g for 2 minutes at room temperature. The supernatant was removed, and the pellet was resuspended by gently inverting in 1 mL of 0.9% (m / v) saline (red blood cells will lyse by aspiration and vortexing). The resulting suspension was centrifuged at 10,000 g for 2 minutes. The supernatant was then removed, and the saline wash was repeated twice. After the final wash, the supernatant was removed, and the red blood cell pellet was resuspended in 1 mL of resuspension buffer (10 mM Na2HPO4∙7H2O, 10 mM NaH2PO4∙H2O, 150 mM NaCl, 1 mM MgCl2∙6H2O, pH 7.4) to prepare a red blood cell stock suspension.

[0201] MHC assay A series of dilutions of the test compounds were prepared in DMSO (D6-99.9%; Cambridge Isotope Laboratories) solution, each concentration being 25.63 times the final concentration. In 0.2 ml microcentrifuge tubes, the compound dilutions were diluted 1:25 in resuspension buffer to a total of 100 µl, and vortexed to mix the solutions. The negative control (0% lysis) contained only DMSO in the resuspension buffer, while the positive control (100% lysis) contained only DMSO in water, as this caused complete lysis of red blood cells due to osmotic pressure. A 2.52 µl volume of red blood cell suspension was added to each tube (including the control), each tube was mixed by gently inverting, and incubated statically at 37 °C for 2 h. After incubation, each sample was mixed again by gently inverting and centrifuged at 3,214 x g for 6 min. After centrifugation, 60 µl of the supernatant was added to a 96-well plate, and the absorbance was read at 540 nm. The data were normalized to a negative control and processed to represent total hemolysis relative to the positive control.

[0202] Example 8: Determination of the minimum inhibitory concentration (MIC) using the broth microdilution method Candida and Aspergillus Determination of the minimum inhibitory concentration (MIC) of fungi.

[0203] Used to determine Candida The MIC protocol for fungi is adapted from CLSI publications. M27, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts 4th Edition . CandidaThe fungi were subcultured on Difco™ Sabouraud dextrose agar (SDA; Becton, Dickinson, and Co.) at 35°C for 24 hours. Single colonies were selected, suspended in 1 ml of sterile 85% saline, and diluted 1:10 in HyClone™ RPMI-1640 medium (GE Healthcare Life Sciences) containing 165 mM MOPS (FisherScientific), pH 7.0. Cell density was determined using a hemocytometer, and the final inoculum was diluted to 1 x 10⁻⁶ cells / mL using RPMI medium. 3 CFU / ml. Serial dilutions of the test compounds were prepared in DMSO (D6-99.9%; Cambridge Isotope Laboratories) solution, each concentration being 100 times the final concentration, and diluted 1:2.5 in RPMI medium. Positive and negative controls were used with DMSO only for culture growth. Each concentration of each compound (including DMSO-only for the positive control) was then diluted twice in a final inoculum of cell suspension to a final volume of 200 µl in a round-bottom 96-well plate (Corning). Similarly, DMSO-only was diluted in RPMI-only for the negative control. The 96-well plates were then incubated statically at 35°C for 24 hours. Immediately after incubation, the concentrations that appeared identical to the negative control when observed from the bottom of the plate were determined visually, and the average of the two replicates was used to determine the MIC.

[0204] Used to determine Aspergillus The MIC scheme for fungi is based on CLSI publications. M38, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi 3rd Edition It includes several obvious modifications. Aspergillus The fungus was subcultured for 5 days on Difco™ potato dextrose agar (PDA; Becton, Dickinson, and Co.) plates at 35°C. After incubation, 1 mL of 85% saline containing 0.1% Tween® 20 (Sigma-Aldrich) was pipetted onto the plate and used to resuspend the fungal spores in separate test tubes. The spore suspension was then diluted 1:1000 in RPMI medium, and the density was determined using a hemocytometer. The final inoculum was diluted to 1 x 10⁻⁶ in RPMI medium. 4 1 spore / ml. Finally, after diluting the compound in the final inoculum, the resulting 96-well plate was incubated at 35°C for 48 hours.

[0205] Table 12: MIC determination results of AmB, AmBisome and ABCI-001 in Fungizone of representative fungal species.

[0206] Table 13: MIC determination results of AmB, AmBisome and R21052 in Fungizone of representative fungal species.

[0207] Table 14: MIC determination results of AmB, AmBisome, R21008, R210012, R21013, R21018, R21022 and R21023 in representative fungal species in fungizone.

[0208] Table 15: MIC determination results of AmB, AmBisome, R21024, R210026, R21027 (each aqueous dispersion was without ethanol), R21031, R21034 and R21038 in representative fungal species in Fungizone.

[0209] Table 16: MIC determination results of AmB, AmBisome, R21039, R21041, FP21008, FP21010, FP21011 and FP21019 in representative fungal species in fungizone.

[0210] Table 17: MIC determination results of AmB, AmBisome, FP21020, FP21030, FP21052 and FP21034 placebo in representative fungal species.

[0211] Example 9: Mean concentration-time curve of AmB ASL concentration of ABCI-003 in rats on day 29 in study FY22-071.

[0212] In study FY22-071, the mean ASLAMB concentration versus time was also determined for three delivered doses of ABCI-003 administered to rats. Figure 8The measured delivery 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 the ASL was biphasic, with measurable concentrations of AmB present in the ASL 28 days post-administration. The observed biphasic kinetics were consistent with those of IV AmB and were associated with the redistribution of AmB from lung tissue. Assuming single exponential decay, the initial clearance rate of AmB from the ASL was estimated from data within 72 hours post-administration. The half-lives for the measured escalation doses were 17, 15, and 23 hours, respectively. Daily maintenance dosing of the drug is necessary to maintain AmB concentrations above the MIC.

[0213] Example 10: A randomized, double-blind, placebo-controlled, single-dose escalation study of amphotericin B for inhalation (the formulation of the present invention) in healthy volunteers. The formulation of this invention is an investigational medicine-device combination product developed for the treatment of CF patients who are not currently receiving CFTR modulators. The formulation comprises lipid-coated crystals of amphotericin B (AmB), which are administered by oral inhalation using a portable dry powder inhaler. The formulation acts as… "Molecular Repair Agent" It spontaneously forms ion channels in the airway epithelial cell membrane, independent of CFTR. In vitro studies have demonstrated significant improvements in chloride secretion, bicarbonate secretion, and airway surface fluid pH, viscosity, and antimicrobial activity. Significant improvements in nasal potential gradient have also been observed in individuals with CF, comparable to those achieved with ivacaptor. These improvements in biomarkers are independent of CFTR mutations, with substantial improvements in activity observed in class I or other nonsense mutations that produce little or no CFTR protein. Forty-eight healthy subjects participated in a randomized, double-blind, placebo-controlled, single-escalation dose trial (Study CM001001, Part A) at NZCR (Christchurch, NZ). There were six dose groups (0.5, 1.0, 2.0, 4.0, 6.0, 10.0 mg), with eight subjects in each group (six receiving the active ingredient and two receiving placebo).

[0214] Two formulations of the present invention are used: a low-strength powder containing 3.9% w / w AmB (formulation ABCI-004) and a high-strength powder containing 15.8% w / w AmB (formulation ABCI-003). In the context of... After a 13 mg filler mass is encapsulated in a #3 HPMC capsule, the formulation provides nominal dose strengths of approximately 0.5 mg and 2.0 mg, respectively. The powder is administered using the ultra-high resistance variant of the RS01 dry powder inhaler (Plastiape SpA, Osnago, Italy). Figure 1A and Figure 1B Safety parameters were monitored throughout the process, including vital signs, clinical chemistry, lung function, ECG, and adverse events.

[0215] Venous blood samples were collected at 0 (before administration), 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 96 hours after administration for pharmacokinetic characterization. Plasma concentrations were determined using a validated LC-MS / MS method with an LLOQ of 0.1 ng / mL (Resolian Pharma, Malvern, PA).

[0216] Table 18. Baseline Demographics All doses of the inhaled formulation of this invention were well tolerated. No clinically significant abnormalities were observed in vital signs, ECG, and clinical chemistry (including serum electrolytes and serum creatinine). No bronchospasm or dose-limiting pulmonary toxicity were reported. All adverse events were mild and transient, with similar incidences of treatment-related AEs between the active ingredient group and the placebo group (Table 19).

[0217] Table 19. Treatment-related adverse events (AEs) in the single-dose increment portion of the study CM001001. The most common adverse events (AEs) were mild headaches. No systemic AEs observed with intravenous AmB have been reported with a single inhaled dose of the formulation of this invention. Figure 10 Plasma AmB concentration-time curves for the formulation of this invention are presented. Plasma AmB concentrations showed a 1-2 hour delay after administration, peaking at approximately 8 hours. Plasma AmB concentrations increased proportionally with increasing dose, but remained well below the threshold associated with systemic toxicity of intravenously administered AmB (i.e., sustained AmB concentrations >1000 ng / mL). PK parameters determined using WinNonlin are shown in Table 20.

[0218] Table 20. PK parameters calculated using WinNonLin In summary, single doses of up to 10 mg of the formulation of this invention were well tolerated with only mild, transient adverse events. Plasma drug levels of AmB were 2-3 orders of magnitude lower than the systemic effect threshold concentration. No systemic adverse events specific to IV AmB were reported. No evidence of respiratory tolerance problems (bronchospasm, asthma, dyspnea, post-inhalation cough) was observed.

[0219] In the MAD study in healthy volunteers, subjects received a loading dose (LD), followed by 13 doses (low and medium dose groups) or 27 doses daily (high dose group). ) Maintenance dose (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, with no dose-limiting toxicities or bronchospasm observed. All TEAEs were mild to moderate in intensity and not serious; all study drug-related TEAEs were mild in intensity. The incidence of TEAEs in the ABCI groups (range: 50% to 100%) was similar to that in their corresponding placebo groups (75% and 100%). The most common TEAEs overall were headache (20.8% of subjects) and nasal congestion and chest discomfort (12.5% ​​of subjects each). TEAEs of increased bronchial secretions reported in the SAD portion of the study were also reported by 2 subjects (33.3%) (3 events) in the highest ABCI 10.0 mg / 4.0 mg QD dose group in the MAD portion, and were not reported by any subjects in the placebo group. Of the three TEAEs with increased bronchial secretions, all were mild and non-severe, considered possibly or certainly related to the study drug, and had varying durations (1 to 40 days) before remission. One subject in the ABCI 10.0 mg / 4.0 mg QD group experienced a mild AESI with decreased glomerular filtration rate, which investigators considered possibly related to the study drug, lasting 21 days and resolving without changing the study drug dosage. No subjects experienced SAEs, TEAEs leading to early discontinuation of the study drug, or death. During Part B of this study, when any ABCI group was compared to its corresponding placebo group, there were no clinically significant or dose-dependent changes in hematology, chemistry, vital signs (including oximetry), ECG, or vital capacity.

[0220] Geometric mean plasma AmB concentration-time curves following administration of multiple maintenance doses of ABCI 0.5 mg QD and 2.0 mg QD on day 14 in groups G and H, respectively, and after administration of 4.0 mg QD on day 28 in group I, showed that plasma AmB concentrations generally increased with increasing dose. Multiphasic elimination was observed in all groups after discontinuation of dosing, but was most pronounced in subjects in group I (ABCI 10.0 mg / 4.0 mg) during a 56-day sampling period following the last daily dose administration on day 28. Figure 11A comparison of clearance periods after the last dose across all regimens showed that the rate of concentration decrease during the 14-day clearance sampling period in groups G and H did not represent the intrinsic terminal elimination rate of AmB, while the extended sampling shown in group I better characterized these intrinsic terminal elimination rates. Analysis was performed to estimate the terminal t in group I. 1 / 2 value Table 21. Terminal t of Group I 1 / 2 Summary of values ​​– Part B (PK group)

[0221] Abbreviations: CV = Coefficient of Variation; PK = Pharmacokinetics; SD = Standard Deviation When assessing the t-test results of all 6 subjects in group I (ABCI 10.0 mg / 4.0 mg), 1 / 2 At time, the final t 1 / 2 The estimated values ​​ranged from 500.79 to 1347.25 hours—or approximately 21 to 56 days—similar to or slightly longer than those reported after intravenous administration of AmB deoxycholate (Bekersky et al., 2002a; Bellmann, 2007). This study intentionally included a 56-day sampling period following 28 days of once-daily ABCI administration to better characterize the long-term treatment of AmB. The prolonged half-life was comparable to the half-life of AmB in lung tissue observed in animals after ABCI inhalation. Therefore, the terminal half-life is considered to reflect the redistribution of AmB from lung tissue followed by its reabsorption.

[0222] A bronchoalveolar lavage (BAL) study was conducted on six healthy subjects in Group I to determine the trough concentration of AmB in airway surface fluid (ASL). Subjects received a loading dose of 10.0 mg, followed by a maintenance dose of 4.0 mg daily for 28 days. In the six subjects studied, the trough ASL AmB concentration was ≥38.2 mg / mL (Table 33). This is significantly higher than... Aspergillus and Candida Candida The MICs of various strains were determined. Therefore, the concentrations in lung tissue and ASL were much higher than the concentrations required for effective killing.

[0223] Table 22. ASL Amphotericin B Concentrations in Group I – Partial B (PK-evaluable population)

[0224] Incorporate by reference All U.S. patents and published U.S. and PCT patent applications mentioned in the above description are incorporated herein by reference in their entirety.

[0225] Equivalent solution For the purpose of clear understanding, the invention has now been described in considerable detail and in its entirety by way of illustration and example. It will be apparent to those skilled in the art that modifications or alterations may be made to the invention within a broad range of equivalents in terms of conditions, formulations and other parameters without affecting the scope of the invention or any particular embodiment thereof, and such modifications or alterations are intended to be covered within the scope of the appended claims.

Claims

1. A method for treating a fungal infection of the lungs, the method comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

2. A method for preventing pulmonary fungal infection, the method comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising: (i) Amphotericin B (AmB) or its pharmaceutically acceptable salt or hydrate; (ii) Cholesterol; (iii) Phospholipids, including hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

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

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

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

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

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

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

12. The method according to any one of claims 1-11, wherein the amount of CaCl2 is about 1% to about 10% w / w.

13. The method according to any one of claims 1-12, wherein the amount of CaCl2 is about 4% to about 7% w / w.

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

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

16. The method of any one of claims 1-15, wherein the weight ratio of Chol to phospholipid 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 Chol to phospholipid is about 0.005:1 to about 0.05:

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

24. The method according to any one of claims 1-23, wherein the molar ratio of phospholipid to CaCl2 is about 4:1 to about 2:

1.

25. The method of claim 24, wherein the molar ratio of phospholipid to CaCl2 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, is substantially composed of, or is composed of: (i) Approximately 30.0% w / w amphotericin B (AmB); (ii) Approximately 14.6% w / w cholesterol (Chol); (iii-a) Approximately 35.8% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 15.9% w / w distearate (DSPG); and (iv) Approximately 3.7% w / w calcium chloride (CaCl2).

30. The method of claim 1 or 2, wherein the pharmaceutical composition comprises, is substantially composed of, or is composed of: (i) Approximately 14.0% w / w amphotericin B (AmB); (ii) Approximately 6.81% w / w cholesterol (Chol); (iii-a) Approximately 51.2% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 22.8% w / w distearate (DSPG); and (iv) Approximately 5.2% w / w calcium chloride (CaCl2).

31. The method of claim 1 or 2, wherein the pharmaceutical composition comprises, is substantially composed of, or is composed of: (i) Approximately 14.0% w / w amphotericin B (AmB); (ii) Approximately 2.3% w / w cholesterol (Chol); (iii-a) Approximately 70.3% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 7.8% w / w distearate (DSPG); and (iv) Approximately 5.52% w / w calcium chloride (CaCl2).

32. The method of claim 1 or 2, wherein the pharmaceutical composition comprises, is substantially composed of, or is composed of: (i) Approximately 3.4% w / w amphotericin B (AmB); (ii) Approximately 0.57% w / w cholesterol (Chol); (iii-a) Approximately 80.72% w / w hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 8.97% w / w distearate (DSPG); and (iv) Approximately 6.34% w / w calcium chloride (CaCl2).

33. The method of claim 1 or 2, wherein the pharmaceutical composition comprises: (i) A Chol / AmB ratio of approximately 0.4 to 1.2 mol / mol; (ii) A Chol / PL ratio of less than approximately 0.05 w / w; (iii) An HSPC / DSPG ratio of approximately 2.3 to approximately 9.0 w / w; and (iv) Approximately 2:1 mol / mol PL / Ca 2+ ratio.

34. The method of any one of claims 1-33, wherein the AmB and Chol are not compounded; 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 Chol.

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 median mass diameter X of the powder particles 50 It ranges from approximately 1.0 to approximately 4.0 μm.

38. The method of claim 37, wherein the median mass diameter X of the powder particles 50 It is approximately 1.5 to approximately 3.5 μm.

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

40. The method of any one of claims 39, wherein the tap 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 Karl quotient of the powder particles is about 20 to about 32.

42. The method of any one of claims 36-41, wherein the primary transition temperature (T) of the shell is... m The temperature should be 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 aerodynamic mass median diameter of the powder particles is about 1.5 μm to about 4.0 μm.

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

46. ​​The method of any one of claims 1-45, wherein the pharmaceutical composition is formulated for pulmonary 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 dosage 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 said AmB is about 0.1% to about 5%.

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

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

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