Ion channel prosthetic compositions comprising lipid-coated amphotericin b crystals

Abstract

Disclosed are pharmaceutical compositions comprising: (i) amphotericin B or a pharmaceutically acceptable salt or hydrate thereof; (ii) cholesterol; (iii) a phospholipid comprising hydrogenated soybean phosphatidylcholine and distearoyl phosphatidylglycerol; and (iv) calcium chloride (CaCl2). Also disclosed are methods of treating a disease or disorder (e.g., cystic fibrosis), increasing pH of airway surface liquid, increasing bicarbonate secretion into airway surface liquid, and increasing one-second forced expiratory volume in a subject using the pharmaceutical compositions.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 321,965, filed on March 21, 2022; the contents of which are incorporated herein by reference.

[0003] background

[0004] Cystic fibrosis (CF) is a progressive genetic disease that causes persistent lung infections and limits the ability to breathe over time. The symptoms of CF are caused by a defective protein called the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Researchers are studying potential therapies to restore the normal function of the CFTR protein or correct its production process to produce a normal protein. Although current CFTR modulators and correctors improve lung function in about 90% of CF patients, about 10% of patients have class I mutations or other mutations for which there are no effective treatments.

[0005] Burke and colleagues recently proposed an alternative strategy: the development of small molecule prosthetics for the CFTR protein. To this end, they repurposed the antifungal drug amphotericin B (AmB), which self-assembles with cholesterol (Chol) to form a monovalent ion channel. Complexes of AmB and Chol were made by rapid nanoprecipitation in water from a solution in an organic solvent (US 2019 / 0083517; US 2020 / 0352970). The resulting nanoparticles were then lyophilized. Burke et al. claim that the ion channel formed by Chol and AmB (Chol / AmB = 1.0 to 50.0 mol / mol) is able to restore bicarbonate secretion, increase airway surface liquid (ASL) pH, and improve host defense in CF cell lines (e.g., CuFi-1, CuFi-4 cell lines from U.Iowa) independently of genetic mutations. They also demonstrated that the range of concentrations at which AmB effectively restores ASL pH can be increased if AmB is pre-complexed with cholesterol. That is, pure AmB is only effective at low AmB concentrations, while the AmB:Chol complex remains active over a wide range of AmB concentrations.

[0006] The benefit of AmB was further established in differentiated primary cultures of human airway epithelium from CF patients with different CFTR mutations, some of which do not produce CFTR. Increases in ASL pH were also observed in a CFTR-null pig model.

[0007] at last, Intranasal administration of AmB, a commercial parenteral composition of AmB, was able to alter nasal potential difference in people with CF, including those who are untreatable with current therapies. The improvements in nasal potential difference observed were comparable to those seen with modulators such as ivacaftor.

[0008] Establishing AmB / Chol ion channels in CF patients requires delivery of these materials to the apical side of the airway epithelium, i.e., administration by oral inhalation. Similar challenges are observed in subjects with non-cystic fibrosis bronchiectasis (NCFBE) and chronic obstructive pulmonary disease (COPD).

[0009] Overview

[0010] In certain aspects, provided herein is a pharmaceutical composition comprising: (i) amphotericin B (AmB) or a pharmaceutically acceptable salt or hydrate thereof; (ii) cholesterol (Chol); (iii) phospholipids (PL) comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and (iv) calcium chloride (CaCl2).

[0011] In a further aspect, provided herein is a method of treating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition; wherein the administration is pulmonary administration.

[0012] In a further aspect, provided herein is a method for increasing the pH of airway surface fluid, comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof, thereby increasing the pH of the subject's airway surface fluid; wherein the administration is pulmonary administration.

[0013] In a further aspect, provided herein is a method of increasing the secretion of bicarbonate into airway surface fluid, comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof, thereby increasing the secretion of bicarbonate into the airway surface fluid of the subject; wherein the administration is pulmonary administration.

[0014] In a further aspect, provided herein is a method of increasing a subject's forced expiratory volume in one second (FEV1), comprising administering to the subject an effective amount of the pharmaceutical composition, thereby increasing the subject's FEV1; wherein the administration is pulmonary administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Phase diagram showing a fully hydrated dipalmitoylphosphatidylcholine-cholesterol mixture.

[0017] Figure 2 A ternary phase diagram showing various AmB / lipid compositions is presented. The shaded triangles represent the optimized lipid-coated amphotericin B crystal compositions.

[0018] Figure 3 Shown are differential scanning calorimetry thermograms of Amphotericin B Inhalation Powder (ABIP), ABCI-001, ABCI-002, and ABCI-003.

[0019] Figure 4 X-ray powder diffraction (XRPD) patterns are shown showing evidence of cholesterol crystallization in PLCI-001 and PLCI-002.

[0020] Figure 5 A graph showing that the enthalpy of the Chol-rich phase is proportional to the Chol content is presented.

[0021] Figure 6 Shown are the moisture sorption isotherms (25°C) for various amphotericin B (AmB) and placebo compositions.

[0022] Figure 7A presents a graph showing the increase in airway surface liquid (ASL) pH in CuFi-1 cells for various compositions comprising HSPCs at a Chol / AmB ratio between 0.4 and 1.2 mol / mol and between 70% and 90% w / w of PL.

[0023] 7B presents a graph showing the increase in ASL pH in CuFi-1 cells for various compositions comprising Chol / AmB ratios between 0 and 0.4 mol / mol and compositions without Chol or DSPG.

[0024] FIG. 7C presents a graph showing the effect of changes in the Chol / AmB ratio on ASL pH improvement in CuFi-1 cells.

[0025] Figure 8 Graphs showing increases in ASL pH following administration of ABCI-003 to primary cultured airway epithelial cells from individuals with CF, including those with nonsense mutations, are presented.

[0026] Fig. 9 Graphs showing ASL pH increase in CuFi-1 cells in the presence of ABCI-003 vs. increasing concentrations of AmB suspended in a perfluorohexane (FC-72) vehicle are presented.

[0027] Fig.10 Graphs showing reduction in ASL viscosity following administration of ABCI-003 to primary cultured airway epithelial cells from individuals with CF, including those with nonsense mutations, are presented.

[0028] Fig.11Graphs showing increased ASL antimicrobial activity following administration of ABCI-003 to primary cultured airway epithelial cells from individuals with CF, including those with nonsense mutations, are presented.

[0029] 12A presents a graph showing the effect of nominal and tapped density of a spray-dried powder composition on the calculated drug load of bulk powders for a nominal dose range of 0 to 10 mg.

[0030] Figure 12B presents a graph showing the effect of nominal and tapped density of spray-dried powder compositions on calculated drug load of bulk powders for nominal doses ranging from 0 to 3 mg.

[0031] Fig.13 Shown is an overlay of the X-ray powder diffraction patterns of GMP and GLP batches of ABCI-003, ABCI-004 and PLCI-002.

[0032] Fig.14 Shown is an overlay of DSC thermograms of GMP and GLP batches of ABCI-003, ABCI-004, and PLCI-002.

[0033] Fig.15 The aerodynamic particle size distribution (APSD) of ABCI-003 is shown.

[0034] Details

[0035] Surprisingly, it was found that AmB and Chol do not need to be complexed to achieve the benefits outlined above. In fact, the two components can phase separate into separate domains in the core-shell particles.

[0036] It was also surprisingly found that Chol / AmB ratios much less than 1.0 mol / mol were able to maintain improvements in host defense, including improvements in bicarbonate secretion, ASL pH, ASL viscosity, and / or ASL antimicrobial activity.

[0037] Composition

[0038] In some aspects, a pharmaceutical composition is provided, comprising:

[0039] (i) amphotericin B (AmB) or a pharmaceutically acceptable salt or hydrate thereof;

[0040] (ii) Cholesterol (Chol);

[0041] (iii) phospholipids; and

[0042] (iv) Calcium chloride (CaCl2).

[0043] In a further aspect, there is provided a pharmaceutical composition comprising:

[0044] (i) amphotericin B (AmB) or a pharmaceutically acceptable salt or hydrate thereof;

[0045] (ii) Cholesterol (Chol);

[0046] (iii) phospholipids comprising hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC); and distearoylphosphatidylglycerol (DSPG); and

[0047] (iv) Calcium chloride (CaCl2).

[0048] In a further aspect, there is provided a pharmaceutical composition comprising:

[0049] (i) amphotericin B (AmB) or a pharmaceutically acceptable salt or hydrate thereof;

[0050] (ii) Cholesterol (Chol);

[0051] (iii) phospholipids comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and

[0052] (iv) Calcium chloride (CaCl2).

[0053] In certain embodiments, the pharmaceutical composition comprises about 0.5% to about 25% w / w AmB.

[0054] In certain embodiments, the pharmaceutical composition comprises about 1% to about 22% w / w AmB.

[0055] In certain embodiments, the pharmaceutical composition comprises about 2% to about 16% w / w AmB.

[0056] In certain embodiments, the pharmaceutical composition comprises about 14% w / w AmB.

[0057] In certain embodiments, the pharmaceutical composition comprises about 0.1% to about 8% w / w Chol.

[0058] In certain embodiments, the pharmaceutical composition comprises about 0.5% to about 3% w / w Chol.

[0059] In certain embodiments, the pharmaceutical composition comprises about 0.3% to about 6% w / w Chol.

[0060] In certain embodiments, the pharmaceutical composition comprises about 0.5% to about 3% w / w Chol.

[0061] In certain embodiments, the pharmaceutical composition comprises about 1% to about 10% w / w CaCl2.

[0062] In certain embodiments, the pharmaceutical composition comprises about 4% to about 7% w / w CaCl2.

[0063] In certain embodiments, the pharmaceutical composition comprises about 60% to about 95% w / w phospholipids.

[0064] In certain embodiments, the pharmaceutical composition comprises about 70% to about 90% w / w phospholipids.

[0065] In certain embodiments, the pharmaceutical composition comprises about 75% to about 90% w / w phospholipids.

[0066] In certain embodiments, the weight ratio of Chol to phospholipid is from about 0.001:1 to about 0.1:1.

[0067] In certain embodiments, the weight ratio of Chol to phospholipid is from about 0.005:1 to about 0.05:1.

[0068] In certain embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is from about 2:1 to about 19:1.

[0069] In certain embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is from about 7:1 to about 12:1.

[0070] In certain embodiments, the weight ratio of soy phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is from about 1:1 to about 3:1.

[0071] In certain embodiments, the molar ratio of Chol to AmB is from about 0.05:1 to about 1.2:1.

[0072] In certain embodiments, the molar ratio of Chol to AmB is from about 0.4:1 to about 1.2:1.

[0073] In certain embodiments, the molar ratio of Chol to AmB is from about 0.05:1 to about 0.4:1.

[0074] In certain embodiments, the molar ratio of Chol to AmB is about 0.4:1.

[0075] In certain embodiments, the molar ratio of phospholipid to CaCl2 is from about 4:1 to about 2:1.

[0076] In certain embodiments, the molar ratio of phospholipid to CaCl2 is about 2:1.

[0077] In certain embodiments, the crystallinity of AmB is greater than about 75%.

[0078] In certain embodiments, the crystallinity of AmB is greater than about 85%.

[0079] In certain embodiments, the crystallinity of AmB is greater than about 95%.

[0080] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0081] (i) about 14.0% w / w amphotericin B (AmB);

[0082] (ii) about 2.3% w / w cholesterol (Chol);

[0083] (iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0084] (iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0085] (iv) about 5.52% w / w calcium chloride (CaCl2).

[0086] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0087] (i) about 14.0% w / w amphotericin B (AmB);

[0088] (ii) about 6.81% w / w cholesterol (Chol);

[0089] (iii-a) about 51.2% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0090] (iii-b) about 22.8% w / w distearoylphosphatidylglycerol (DSPG); and

[0091] (iv) about 5.2% w / w calcium chloride (CaCl2).

[0092] In certain embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of:

[0093] (i) about 3.4% w / w amphotericin B (AmB);

[0094] (ii) about 0.57% w / w cholesterol (Chol);

[0095] (iii-a) about 80.72% w / w hydrogenated soy phosphatidylcholine (HSPC);

[0096] (iii-b) about 8.97% w / w distearoylphosphatidylglycerol (DSPG); and

[0097] (iv) about 6.34% w / w calcium chloride (CaCl2).

[0098] In certain embodiments, the pharmaceutical composition comprises:

[0099] (i) a Chol / AmB ratio of about 0.4 to 1.2 mol / mol;

[0100] (ii) a Chol / PL ratio of less than about 0.05 w / w;

[0101] (iii) a HSPC / DSPG ratio of about 2.3 to about 9.0 w / w; and

[0102] (iv) PL / Ca of about 2:1 mol / mol 2+ Compare.

[0103] In certain embodiments, AmB and Chol are not complexed; and AmB is not encapsulated in liposomes.

[0104] In certain embodiments, AmB is coated with a porous shell of phospholipids and Chol.

[0105] In certain embodiments, the pharmaceutical composition is formulated as a dry powder.

[0106] In certain embodiments, the powder particles have a tap density of about 0.03 to about 0.4 g / mL.

[0107] In certain embodiments, the powder particles have a tap density of about 0.06 to about 0.2 g / mL.

[0108] In certain embodiments, the powder particles have a Carr's Index of about 20 to about 32.

[0109] In certain embodiments, the main transition temperature (T m ) is at least 70°C, at least 80°C, or at least 90°C. In certain embodiments, the main transition temperature (T m ) is from about 50°C to about 100°C, from about 70°C to about 100°C, from about 80°C to about 100°C, or from about 85°C to about 95°C.

[0110] In certain embodiments, the powder has a water content of about 1.5 to about 6% w / w.

[0111] In certain embodiments, the composition is formulated for pulmonary or airway administration.

[0112] In certain embodiments, the composition is formulated for aerosol administration.

[0113] In certain embodiments, the composition is formulated for aerosol administration as a dry powder.

[0114] In certain embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of phospholipids (PL) and Chol, wherein the crystallinity of AmB is at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0115] In certain embodiments, the mass median diameter X of the powder particles is 50 From about 0.5 μm to about 5.0 μm, from about 1.0 μm to about 4.0 μm, or from about 1.5 μm to about 3.5 μm.

[0116] In certain embodiments, the powder particles have an X 90 From about 1.5 μm to about 10.0 μm, such as from about 2.0 μm to about 8.0 μm.

[0117] In certain embodiments, the powder particles have a mass median aerodynamic diameter (MMAD) of about 1.5 μm to about 4.0 μm.

[0118] In certain embodiments, the powder particles have a mass median aerodynamic diameter (MMAD) of about 2.0 μm to about 3.5 μm.

[0119] In certain embodiments, a carrier-free dry powder composition of AmB, Chol, and PL is provided, wherein AmB is not encapsulated in lipids, and AmB and lipids are phase separated in their own domains.

[0120] In certain embodiments, dry powder compositions of engineered particles are provided, comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein the Chol / AmB ratio is about 0.05 to about 1.2 mol / mol, such as about 0.2 to about 0.6 mol / mol.

[0121] In certain embodiments, dry powder compositions of engineered particles are provided, comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein Chol / PL is less than 0.10 w / w or less than 0.05 w / w.

[0122] In certain embodiments, a dry powder composition of an engineered particle is provided, comprising a plurality of AmB drug particles coated by a porous shell comprising PL and Chol. In some embodiments, PL comprises hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylglycerol (DSPG), or a combination thereof.

[0123] In certain embodiments, a dry powder composition of engineered particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein PL comprises a mixture of (1) HSPC or DSPC, and (2) DSPG in a w / w ratio of about 2.3 w / w (i.e., 7 / 3 w / w) and about 19.0 w / w (i.e., 95 / 5 w / w), such as about 8 w / w to about 18 w / w.

[0124] In certain 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 below about 2.0 mol / mol.

[0125] In certain 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 load is about 0.5% to about 25% w / w, and the nominal Chol / AmB ratio is about 0.05 to about 1.2 mol / mol.

[0126] In certain embodiments, a dry powder composition of engineered particles (ABCI-003) is provided, which includes 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.

[0127] In certain embodiments, a dry powder composition of engineered particles (ABCI-004) is provided, which includes 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.

[0128] In certain embodiments, dry powder compositions of engineered particles are provided ( Figure 2 ), which includes 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 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 the PL is between about 2.3 and about 9.0 w / w.

[0129] In certain embodiments, the maximum Chol / AmB ratio is about 1.2 mol / mol, but this high ratio may be acceptable only for lower drug loadings (e.g., no greater than 10.0% w / w), where the lipids are maintained in the highly ordered SO phase. Reducing Chol / AmB to 0.4 mol / mol may allow for higher drug loadings (e.g., no greater than 22% w / w) within the SO phase.

[0130] In some embodiments, the compositions described herein occupy Figure 2 Two sides of the triangle represent the lines for nominal Chol / AmB ratios of 0.4 and 1.2 mol / mol, while the third side represents the Chol / PL ratio (Chol / PL = 0.05 w / w) that may lead to the elimination of the Chol-rich phase in the DSC thermogram (Example 4).

[0131] In certain embodiments, the compositions described herein include a 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+ Compare.

[0132] In certain embodiments, the selection of the compositions described herein depends on maintaining the lipids in a single phase (ie, T mIn some embodiments, the selection of the composition described herein depends on the maximum increase in the ASL pH maintained over a wide range of AmB concentrations (Example 11). In some embodiments, the selection of the composition described herein depends on the reduced hygroscopicity of the composition relative to HSPC / DSPG<9.0 (Example 5). In some embodiments, the selection of the composition described herein depends on the increased production yield (Example 9). In some embodiments, the selection of the composition described herein depends on the improved powder flowability (Example 9). In some embodiments, the selection of the composition described herein depends on the improved aerosol performance relative to the powder containing the lo phase (Example 9). In some embodiments, the selection of the composition described herein depends on the excellent performance of restoring host defense in cell-based assays in primary cultures of CuFi-1 cells and from CF patients (even those with nonsense mutations) (Example 10). In some embodiments, the selection of the composition described herein depends on the absence of erythrocyte hemolysis even at low Chol / AmB molar ratios (Example 15).

[0133] In certain 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.

[0134] In certain 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 mass median diameter (X 50 ) is about 1.0 to about 5.0 μm, such as about 1.5 to about 4.0 μm.

[0135] In certain 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 X 90 From about 3 μm to about 10 μm, such as from about 3.5 μm to about 7 μm.

[0136] In certain 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 about 0.03 to about 0.40 g / mL, such as about 0.06 to about 0.20 g / mL.

[0137] In certain 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 about 1.0% to about 10.0%, preferably about 2.0% to about 5.0%.

[0138] In certain 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 from a portable dry powder inhaler, the mass median aerodynamic diameter (MMAD) is about 1.0 μm to about 6.0 μm, such as about 2.0 μm to about 4.0 μm.

[0139] In certain 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 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% or at least 60% w / w when administered with a portable dry powder inhaler.

[0140] In certain 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 spray drying of a liquid feedstock comprising fine AmB crystals suspended in an oil-in-water emulsion stabilized by a monolayer of a lipid mixture described herein.

[0141] In certain embodiments, a dry powder composition of an engineered particle is provided, comprising a plurality of crystalline AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl2), wherein the lipid has a main transition temperature (T m ).

[0142] In certain 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 a liquid feedstock at a temperature below the lowest T of the lipid. m In some embodiments, the outlet temperature is at least 50°C, at least 60°C, or at least 70°C.

[0143] In certain 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 a liquid feedstock at a temperature below the lowest T of the lipid. mIn some embodiments, the outlet temperature is at least 60°C or at least 70°C.

[0144] In certain 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 spray drying of a liquid feedstock at a total gas flow rate of about 70 to about 100 scfm on a PSD-1 scale spray dryer.

[0145] In certain embodiments, a dry powder composition comprising engineered particles of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2) is filled using a drum loader.

[0146] In certain embodiments, the powder fill mass is from about 1.0 mg to about 40 mg, such as from about 3 mg to about 20 mg, or from about 10 mg to about 15 mg in a size 3 or size 2 capsule.

[0147] In certain embodiments, the powder fill mass has good precision (eg, RSD < 3%) and accuracy to the target fill mass.

[0148] method

[0149] In certain aspects, a method of treating a disease or disorder is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to any embodiment described herein; wherein the administration is pulmonary administration. In certain embodiments, the disease or disorder comprises cystic fibrosis, non-cystic fibrosis bronchiectasis (NCFBE), or chronic obstructive pulmonary disease (COPD). In certain embodiments, the disease or disorder is cystic fibrosis.

[0150] A method of restoring host defense characteristics of ASL (e.g., increase in bicarbonate secretion, pH, antimicrobial activity, and / or decrease in viscosity) is provided, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby improving lung function and quality of life.

[0151] Also provided is a method of increasing the pH of airway surface fluid, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby increasing the pH of the subject's airway surface fluid; wherein the administration is pulmonary administration.

[0152] Also provided is a method of increasing bicarbonate secretion into airway surface fluid, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby increasing bicarbonate secretion into the airway surface fluid of the subject; wherein the administration is pulmonary administration.

[0153] Also provided is a method of increasing forced expiratory volume in one second (FEV1), comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby increasing the subject's FEV1; wherein the administration is pulmonary administration.

[0154] In some embodiments, a subject's FEV1 is referred to as increased when the subject's FEV1 is measurably greater than the same subject's FEV1 measured prior to or temporally remote from treatment according to the methods of the present disclosure.

[0155] In certain embodiments, the subject's FEV1 is increased by about 3% to about 20%, about 5% to about 15%, or about 6% to about 10%.

[0156] In certain embodiments, the subject has CF.

[0157] In certain embodiments, the pharmaceutical composition is administered to the airway of a subject.

[0158] In certain embodiments, the pharmaceutical composition is administered to the bronchial airways of a subject.

[0159] In certain embodiments, the pharmaceutical composition is administered as an aerosol.

[0160] In certain embodiments, the pharmaceutical composition is administered as a dry powder aerosol.

[0161] In certain embodiments, the subject is a human.

[0162] In certain embodiments, the subject is an adult.

[0163] In certain embodiments, the subject is a human less than 12 years old.

[0164] In certain embodiments, the subject is a human who is at least 12 years old.

[0165] In certain embodiments, the subject is a human who is at least 4 years old.

[0166] In certain embodiments, the CF is refractory to ivacaftor; or a combination of elexacaftor, tezacaftor, and ivacaftor.

[0167] In certain embodiments, the subject with CF has a Class 1 or other mutation that cannot be effectively treated with a current modulator (including combination therapy). This includes nonsense mutations or other mutations in which no functional CFTR protein is produced.

[0168] In certain embodiments, the subject has two mutations in the CF transmembrane conductance regulator (CFTR) anion channel; and the two mutations are each independently selected from the mutations listed in Table 1.

[0169] Table 1. Exemplary CTFR mutations

[0170]

[0171]

[0172]

[0173] In certain embodiments, the two mutations are each independently selected from 2184delA, F508del, V520F, 1717-1G->A, E60X, G551D, R553X and D259G.

[0174] In certain embodiments, the two mutations are a pair of CFTR mutations selected from F508del / F508del, G551D / F508del, R553X / E60X, F508del / 1717-1G->A, F508del / 2184delA, and D259G / V520F.

[0175] In certain embodiments, the two mutations are a pair of CFTR mutations selected from F508del / F508del, R553X / E60X, F508del / 1717-1G->A, F508del / 2184delA, and D259G / V520F.

[0176] In certain embodiments, the nominal or metered dose of the pharmaceutical composition is 0.01 mg to 10 mg.

[0177] In certain embodiments, the nominal or metered dose of the pharmaceutical composition is about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.0 mg, or about 4.0 mg.

[0178] In certain embodiments, the composition is administered at least once a day, at least twice a day, or at least three times a day.

[0179] In certain embodiments, the composition is administered once daily.

[0180] In certain embodiments, the composition is administered at least once a week, at least twice a week, or at least three times a week.

[0181] In certain embodiments, administration comprises initially delivering a loading dose of the pharmaceutical composition; subsequently delivering a maintenance dose of the pharmaceutical composition; wherein the mass ratio of the loading dose to the maintenance dose is about 2:1 to about 5:1. In certain embodiments, the mass ratio of the loading dose to the maintenance dose is about 2.5:1 to about 3:1. In certain embodiments, the loading dose is about 1.0 mg to about 20 mg, about 3 mg to about 15 mg, or about 6 mg to about 10 mg. In certain embodiments, the maintenance dose is about 0.1 mg to about 5 mg, about 0.5 mg to about 5 mg, about 1 mg to about 4 mg, or about 2 mg. In certain embodiments, the loading dose is about 1.5 mg, and the maintenance dose is about 0.5 mg. In certain embodiments, the loading dose is about 6 mg, and the maintenance dose is about 2 mg. In certain embodiments, the loading dose is about 10 mg, and the maintenance dose is about 4 mg.

[0182] All masses described above for the loading dose and the maintenance dose refer to the mass of the nominal dose or metered dose (ie, not the emitted dose) of the pharmaceutical composition.

[0183] In certain embodiments, a method of treating a subject having CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a lipid-coated crystal composition comprising AmB and Chol as provided in the present disclosure,

[0184] The lipid-coated crystalline composition is administered to a subject by oral inhalation into the bronchial airways in the lungs.

[0185] In certain embodiments, a method of treating a subject having CF, NCFBE, or COPD comprises administering to a subject in need thereof an effective amount of a lipid-coated crystalline composition comprising AmB and Chol as provided in the present disclosure, wherein the composition provides an increase in ASL pH of about 0.2 to about 1.0 pH units.

[0186] In certain embodiments, a method of treating a subject having CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a lipid-coated crystal composition comprising AmB and Chol as provided in the present disclosure,

[0187] wherein the composition provides a clinically significant improvement in lung function (FEV1) relative to placebo.

[0188] In some embodiments, provided herein is a method of reducing the viscosity of airway surface liquid in an individual with CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby increasing the hydration and height of the airway surface liquid while reducing the viscosity of the airway surface liquid of the subject; wherein the administration is pulmonary administration.

[0189] In some embodiments, provided herein is a method of increasing the antibacterial activity of airway surface fluid in an individual with CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby increasing the antibacterial activity of the airway surface fluid of the subject; wherein the administration is pulmonary administration.

[0190] In some embodiments, provided herein is a method of improving mucociliary clearance in an individual with CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby improving mucociliary clearance in the subject; wherein the administration is pulmonary administration.

[0191] In some embodiments, provided herein is a method of enhancing lung function in an individual with CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby enhancing the subject's lung function; wherein the administration is pulmonary administration.

[0192] In some embodiments, provided herein is a method of improving the health-related quality of life (HRQoL) of an individual with CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any embodiment described herein, thereby improving the subject's HRQoL; wherein the administration is pulmonary administration.

[0193] In certain embodiments, provided herein is a method of treating a subject having CF, NCFBE or COPD, comprising administering to a subject in need thereof an effective amount of a lipid-coated crystal composition comprising AmB and Chol as provided in the present disclosure,

[0194] wherein the lipid-coated crystalline composition provides a clinically significant improvement in health-related quality of life relative to placebo.

[0195] In certain embodiments, provided herein is a method of improving lung function, comprising administering an effective amount of a pharmaceutical composition of the present disclosure to a subject in need thereof, thereby improving the subject's lung function; wherein the administration is pulmonary administration.

[0196] In certain embodiments, the subject has CF, NCFBE, or COPD.

[0197] In certain embodiments, the subject has CF.

[0198] In certain embodiments, the pharmaceutical composition is a dry powder.

[0199] In certain embodiments, the dry powder is administered with a portable dry powder inhaler.

[0200] In certain embodiments, the dry powder inhaler utilizes the subject's inspiratory flow to fluidize and disperse the powder (ie, the device is a passive dry powder inhaler).

[0201] In certain embodiments, the dry powder inhaler preferably has a moderate to high airflow resistance (ie, about 0.10 to 0.30 cm H2O 0.5 L -1 min).

[0202] In certain embodiments, the portable dry powder inhaler comprises a capsule-based unit dose DPI, a single-use disposable DPI, or a blister-based multi-dose dry powder inhaler.

[0203] More than 1900 different CFTR mutations have been found in CF patients, hundreds of which have been shown to be pathogenic through at least five different loss-of-function mechanisms. Important recent progress has been made in the development of genotype-specific small molecule drugs that bind to certain mutant forms of CFTR and thereby increase its activity. However, approximately 10% of CF patients have a CFTR genotype that is unresponsive to current small molecule treatments. These include major truncations that produce a complete lack of functional CFTR protein and very rare mutations whose mechanistic basis for their functional defects is unknown.

[0204] In some embodiments, any of the methods disclosed herein treat a CF mutation class selected from I, II, III, IV, V, VI, U, and combinations thereof. In some embodiments, the mutation class is selected from I / I, II / II, II / U, and U / III.

[0205] Without intending to be bound by theory, the compositions described herein do not modulate, correct or alter the activity of the CFTR channel. It is believed that the molecular prosthesis comprising lipid-coated AmB crystals creates a new ion channel whose activity is independent of the specific genetic mutation of CFTR and the subject.

[0206] In some embodiments, any of the methods disclosed herein is a genotype-independent treatment.

[0207] As used herein, the phrase "genotype-independent" or "genotype-agnostic" refers to any treatment that is independent of the exact nature of the genetic mutation that leads to reduced CFTR expression or reduced CFTR function in CF.

[0208] In certain embodiments, the methods described herein can be used to treat various CF genotypes that are generally unresponsive or minimally responsive to treatment with conventional CF therapies. For example, the V520F allele in patients with the D259G / V520F CTFR mutation pair is refractory to ivacaftor; or a combination of elexacaftor, tezacaftor, and ivacaftor.

[0209] Thus, in certain embodiments, the methods of treating CF described herein are independent of CF genotype.

[0210] In certain embodiments, any of the methods disclosed herein treat refractory or resistant CF. In certain embodiments, the CF is refractory or resistant to one or more CF treatments.

[0211] The pH of the airway surface liquid (ASL) of a subject can be measured using any technique known to those skilled in the art. For example, airway pH can be measured by placing a planar pH-sensitive probe on the tracheal surface. Pezzulo AA et al. (2012) Nature 487: 109-113.

[0212] The pH of a subject's ASL is referred to as elevated when the pH of the subject's ASL is measurably greater than the pH of an untreated subject's ASL. In one embodiment, the pH of a subject's ASL is referred to as elevated when the pH of the subject's ASL is measurably greater than the pH of the same subject's ASL measured prior to or temporally remote from treatment according to the methods of the present disclosure.

[0213] This method of increasing the pH of the airway surface liquid is described in Example 1. Notably, restoration of ASL pH was observed over a wide range of AmB:sterol concentrations. Therefore, the method of increasing ASL pH described herein has great significance for clinical applications.

[0214] In certain embodiments, the increase in pH may be from 0.1 pH units to 2.0 pH units. In certain embodiments, the increase in pH may be from 0.1 pH units to 1.0 pH units. In certain embodiments, the increase in pH may be from 0.3 pH units to 1.0 pH units. In certain embodiments, the increase in pH may be from 0.3 pH units to 1.0 pH units. In certain embodiments, the increase in pH may be from 0.5 pH units to 0.9 pH units. In certain embodiments, the increase in pH may be from 0.5 pH units to 0.8 pH units. In certain embodiments, the increase in pH may be from 0.6 pH units to 0.8 pH units. In certain embodiments, the increase in pH may be about 0.7 pH units. In certain embodiments, the increase in pH may be from 0.1 pH units to 0.5 pH units. In certain embodiments, the increase in pH may be from 0.1 pH units to 0.4 pH units. In certain embodiments, the increase in pH may be from 0.2 pH units to 0.4 pH units. In certain embodiments, the increase in pH may be from 0.1 pH units to 0.3 pH units. In certain embodiments, the increase in pH may be from 0.2 pH units to 0.3 pH units. In certain embodiments, the increase in pH may be from 0.1 pH units to 0.2 pH units.

[0215] In certain embodiments, the pH is increased by top-addition of any of the compositions disclosed herein.

[0216] In certain embodiments, the methods described herein can be used to increase the pH of the airway surface fluid in patients regardless of their genotype, including patients with any of the various CF genotypes that generally do not respond or respond minimally to treatment with conventional CF modulator / corrector therapies.

[0217] Thus, in certain embodiments, the methods described herein for increasing ASLpH are independent of the patient's CF genotype.

[0218] In certain embodiments, any of the methods disclosed herein increases ASLpH in a patient with refractory or resistant CF. In some embodiments, the CF is refractory or resistant to one or more CF treatments, such as ivacaftor; or a combination of elexacaftor, tezacaftor, and ivacaftor.

[0219] In certain aspects, provided herein is a method of restoring anion transport, increasing airway surface liquid hydration and height, thereby reducing the viscosity of airway surface liquid in CF patients, comprising administering to a CF patient a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0220] In some embodiments, restoration of anion secretion (e.g., bicarbonate secretion) results in increased airway surface liquid pH, increased airway hydration, increased airway surface liquid height, increased antimicrobial activity of airway surface liquid, or a combination thereof. These improvements in the properties of airway surface liquid can result in clinical improvements in mucociliary clearance, lung function measures (e.g., FEV1), patient health-related quality of life (HRQoL), or a combination thereof.

[0221] In certain embodiments, HRQoL is measured using the Cystic Fibrosis Questionnaire (CFQ). Henry, B. et al., Qual Life Res. 2003 February; 12(1): 63-76. In some embodiments, HRQoL is measured using the Cystic Fibrosis Questionnaire Revised (CFQ-R). Wenninger, K. et al., Qual Life Res. 2003 NIAN 2 YUE; 12(1): 77-85. In certain embodiments, administration of the present composition to a patient provides an improvement in HRQoL domain scores of at least 0.5, at least 0.8, at least 1, or at least 2 as measured by the CFQ or CFQ-R. In some embodiments, the HRQoL domain score of a subject is referred to as improved when the HRQoL domain score of the subject is measurably greater than the HRQoL domain score of the same subject measured prior to treatment according to the methods of the present disclosure or at a time remote from the treatment. In certain embodiments, a subject's HRQoL domain score is referred to as improved when the subject's HRQoL domain score is measurably greater than the HRQoL domain score of an untreated subject.

[0222] A subject with CF may have a reduced ASL height compared to a healthy subject. Muraglia, Katrina A. et al., Nature 2019 Mar; 567(7748): 405-408. In certain embodiments, a subject with CF has an ASL height of about 3 μm to about 4 μm. In some embodiments, the methods described herein increase the ASL height of a subject with CF to that of a healthy subject (e.g., to an ASL height of about 9 μm to about 10 μm).

[0223] In certain embodiments, the methods described herein increase the ASL height of a subject by about 1 μm to about 8 μm, about 1 μm to about 5 μm, about 1 μm to about 3 μm, about 2 μm to about 8 μm, about 2 μm to about 5 μm, about 3 μm to about 8 μm, about 3 μm to about 5 μm, about 5 μm to about 7 μm, or about 6 μm. In some embodiments, the ASL height of a subject is referred to as increased when the ASL height of the subject is measurably greater than the ASL height of the same subject measured prior to treatment according to the methods of the present disclosure or at a time remote from the treatment. In certain embodiments, the ASL height of a subject is referred to as increased when the ASL height of the subject is measurably greater than the ASL height of an untreated subject.

[0224] In certain aspects, provided herein is a method of reducing the viscosity of airway surface liquid, comprising administering an effective amount of a pharmaceutical composition of the present disclosure to a subject in need thereof, thereby reducing the viscosity of the airway surface liquid of the subject; wherein the administration is pulmonary administration.

[0225] In certain embodiments, the subject has CF, NCFBE, or COPD.

[0226] In certain embodiments, the subject has CF.

[0227] In certain embodiments, the pharmaceutical composition is administered to the airways of the subject via a dry powder aerosol.

[0228] In certain embodiments, the pharmaceutical composition is administered to the bronchial airways of a subject via a dry powder aerosol.

[0229] In certain embodiments, the airways of a subject refer to the so-called conducting or bronchial airways, ie, any or all of the following lung structures: trachea, bronchi, and bronchioles in the lower respiratory tract.

[0230] In certain embodiments, the methods described herein can be used to reduce the viscosity of airway surface fluid in patients with any of the various CF genotypes that are generally unresponsive or minimally responsive to treatment with conventional CF therapies.

[0231] Thus, in certain embodiments, the methods described herein for reducing ASL viscosity are independent of the patient's CF genotype.

[0232] In some embodiments, any of the methods disclosed herein reduce ASL viscosity in a refractory or resistant CF patient. In some embodiments, the CF is refractory or resistant to one or more CF treatments, such as ivacaftor; or a combination of elexacaftor, tezacaftor, and ivacaftor.

[0233] According to each of the preceding embodiments, in certain embodiments, the patient is a human.

[0234] In certain embodiments, the age range suitable for treatment with the compositions described herein depends on the ability to effectively utilize a passive dry powder inhaler (DPI). Subjects under the age of 6 may not have the cognitive ability to correctly perform the steps involved in the use of a passive DPI. In addition, they may lack the respiratory muscle strength required to produce the required inspiratory pressure. Nevertheless, some passive DPIs have been approved for subjects as young as 4 years old. Similarly, some elderly patients may also have cognitive or atrophic muscles that hinder the correct use of passive DPIs. Inspiratory pressure tends to increase with age, reaching a maximum at about 25 years old, until it stabilizes at about 40 years old, after which they steadily decline. Men tend to achieve higher inspiratory pressures than women. The increase in airway diseases may also adversely affect the ability of subjects to achieve the pressure drop (~1.0kPa) required for effective operation of passive DPIs. In-Check dial can be used to identify subjects with poor inspiratory muscle strength based on its ability to achieve 1.0kPa inspiratory pressure. According to each of the aforementioned embodiments, in certain embodiments, the age of the patient is between 4 and 80 years old.

[0235] In certain embodiments, the patient is at least 4 years old. In certain embodiments, the patient is at least 6 to about 18 years old. In some embodiments, the patient is under about 80 years old with acceptable inspiratory muscle strength. In certain embodiments, the patient is about 60 to about 70 years old. In some embodiments, the patient is about 70 to about 80 years old.

[0236] In certain embodiments, any of the methods disclosed herein permeabilize the apical membrane. In certain embodiments, any of the methods disclosed herein permeabilize the apical membrane to protons. In certain embodiments, any of the methods disclosed herein permeabilize the apical membrane to bicarbonate anions.

[0237] API

[0238] The composition described herein may be referred to as Amphotericin B Cystetic for Inhalation (ABCI), which may be used as an ion channel prosthesis in CF patients to restore anion transport and host defense, thereby improving lung function and quality of life.

[0239] In certain embodiments, the compositions described herein comprise highly crystalline micronized particles of amphotericin B coated with a porous layer of lipids comprising cholesterol.

[0240] AmB is a heptene macrolide containing seven conjugated double bonds in the trans position and 3-amino-3,6-dideoxymannose (trehalosamine) linked to the main ring via a glycosidic bond.

[0241] AmB is produced by a strain of Streptomyces nodosus as a fermentation product. Intravenous AmB is the only approved fungicidal compound currently used to treat systemic fungal infections. Although not approved for administration by inhalation, inhaled AmB has been extensively studied for the treatment of invasive pulmonary aspergillosis.

[0242] In fact, more than 500,000 doses of inhaled AmB have been administered with no serious or severe side effects reported. Most research publications have described inhaled AmB as well tolerated. Adverse events (AEs) reported were primarily related to respiratory irritation (e.g., cough, bronchospasm, dyspnea, wheezing). Other AEs included unpleasant taste (dysgeusia), nausea, and vomiting. Slow absorption of the drug from the lungs results in very low systemic drug levels, which are typically close to or below the limit of quantitation. Concentrations of serum creatinine (a biomarker of renal function) do not typically change with aerosol AmB administration. Therefore, AmB targeting the respiratory tract significantly improves the systemic tolerability of AmB.

[0243] At neutral pH, AmB is a zwitterionic form in which the carboxylate group (pKa = 5.7) is deprotonated, while the amine group on the trehalose amine ring (pKa = 10.0) is protonated. The highly crystalline zwitterionic form of AmB is almost insoluble in aqueous solution and is soluble in water, Gamble solution and dilute trehalose at 37°C. The solubilities in the samples were 0.5, 0.2 and 0.2 μg / mL, respectively.

[0244] The dose number (Do) is a simple metric used to predict whether a compound will be reasonably absorbed from the lung based on its solubility at the expected clinical dose.

[0245]

[0246] In this M o is the AmB dose in ASL, V ASL is the volume of ASL fluid, and S ASL is the solubility of AmB in ASL fluid. Do values ​​greater than 1.0 are consistent with dissolution-limited absorption from the lungs. ASL =0.2 μg / mL and V ASL =25mL, at M o Dissolution-limited absorption occurs at >5 μg. Therapeutic doses of AmB are expected to be greater than about 1.0 mg. In this case, the absorption of AmB will be dissolution-limited.

[0247] The slow appearance of AmB in plasma (e.g., t max=4 to 8 hr) and the overall systemic exposure is significantly reduced relative to the intravenous composition of AmB, demonstrating the slow dissolution of the compositions of the present disclosure. In some embodiments, the systemic exposure of the inhaled composition described herein is 1 / 100-1 / 1,000 times the systemic exposure measured after intravenous administration of a comparable AmB dose.

[0248] In certain embodiments, the absolute bioavailability of AmB in the methods described herein is about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2%, or about 0.1% to about 1%. In some embodiments, the clearance of AmB from plasma is biphasic, with an initial half-life of about 5-20 hours for AmB dissolved in ASL, followed by a very slow terminal clearance (t ) caused by redistribution of the drug from lung tissue. 1 / 2 A similar two-phase clearance process was observed for AmB after intravenous administration of microparticles, which were initially cleared by cells of the reticuloendothelial system and later redistributed back into the plasma.

[0249] The nominal dose, particle size, and physical form of AmB may have an impact on the toxicity of AmB after oral inhalation in rats (US2012 / 0128728 A1). Rats that received highly amorphous AmB (i.e., ABIP, Nektar Therapeutics) formulated in lipid-coated crystals containing 50% AmB and 50% DSPC / calcium chloride in a 2:1 molar ratio (US 7,326,691; US ​​8,404,217; US 2012 / 0128728) showed clinical symptoms of respiratory distress, tracheal and bronchial hypertrophy, tracheal inflammation, bronchial mucosal cell infiltration, and signs of tracheal and bronchial luminal exudates. The incidence of these effects was significantly reduced for rats that received highly crystalline AmB with a smaller particle size (≥76% crystallinity in ABIP).

[0250] ABIP compositions containing highly crystalline AmB and administered at appropriate doses and schedules were well tolerated in nonclinical toxicity studies. They were also well tolerated in single-dose and multiple-dose studies in healthy volunteers (Kugler et al.: Amphotericin B Inhalation Powder (ABIP) Achieves Significant Lung Amphotericin B Concentrations and Low Systemic Amphotericin B Concentrations.16 thCongress of the International Society of Human and Animal Mycology (ISHAM), Paris, France, Abstract O-0011, June 26, 2006); Lee JD et al.: Amphotericin B inhalation powder (ABIP) is well tolerated in healthy subjects with low systemic amphotericin B exposure. In: Program and Abstracts of the 2 nd Meeting of Advances Against Aspergillosis (AAA), Abstract P-118, pages 214-215, Athens, Greece, 2006).

[0251] An increase in amorphous content may also result in reduced grindability of the drug substance, which may lead to an increase in particle size. This observation is exemplified for the wet milling process in Example 3. These increases in crystal size have the potential to adversely affect critical quality attributes related to aerodynamic particle size distribution.

[0252] The crystallinity of AmB decreased from 96% to 75% after wet grinding in water. 50 From 1.06 to 2.16 μm, and X 90 The increase in AmB crystal size had no significant effect on the physicochemical properties and aerosol performance of the formulated ABCI-002 drug product. The MMAD increased from 2.9 to 3.1 μm, while the FPF <5μm Increased from 61% to 63%.

[0253] In some embodiments, the drug substance has a degree of crystallinity of at least 75%, at least 90%, or at least 95%.

[0254] In certain embodiments, the small primary particle size distribution of the micronized drug particles can provide efficient delivery of the drug product to the lungs. AmB crystals have an X-ray diffraction range of about 0.5 μm to about 4 μm, such as about 1 μm to about 3 μm, as determined by laser diffraction. 50 In some embodiments, the AmB crystals have an X of about 1.5 μm to about 10 μm, such as about 2.0 μm to about 8.0 μm. 90 .

[0255] excipient

[0256] In certain embodiments, the compositions described herein comprise highly crystalline AmB particles coated with a porous layer comprising two phospholipids (HSPC and DSPG), cholesterol, and calcium chloride. These materials may exhibit complex phase behavior that directly affects the physicochemical and aerosol properties of the composition. This in turn may have an impact on the efficacy, safety, and tolerability of the formulated drug product.

[0257] Phospholipids are a class of lipids that contain a glycerol or sphingosine backbone to which one or more fatty acids and a phosphate group with an alcohol attached thereto. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine or serine. Phospholipids are amphiphilic molecules in which the two fatty acid acyl chains are lipophilic and the modified phosphate group is hydrophilic. Phospholipids self-assemble in water to form membrane structures. They are ubiquitous in mammalian cell membranes.

[0258] Hydrated phospholipid bilayers exhibit thermotropic phase behavior. As temperature increases, the acyl chains undergo a phase transition from a "gel phase" (SO), in which the acyl chains exist in a highly ordered, solid-like, all-trans configuration, to a disordered "liquid crystal phase" (LD), in which an increase in the content of gauche conformers in the acyl chains results in a disordered, liquid-like packing. The gel-to-liquid crystal phase transition temperature is sometimes called the main transition temperature, or T m .

[0259] Phospholipid T m The T values ​​of various hydrated phospholipids are given in Table 2. m Comparison of values.

[0260] Table 2. Main transition temperatures of hydrated gel to liquid crystal of various phospholipids

[0261]

[0262] The increase in the length of the acyl chain of phosphatidylcholine from 12 to 18 results in the hydration of m from -2 to 55 °C. The introduction of unsaturation in the acyl chain leads to T m The change in the nature of the head group also leads to a decrease in T m The difference, where T m Phosphatidylethanolamine (PE) > phosphatidylserine (PS) > phosphatidylglycerol (PG) ~ phosphatidylcholine (PC). Hydration of HSPC and DSPG m The values ​​are 53.6 and 55°C respectively.

[0263] Cell membranes are inherently multicomponent systems containing various classes of lipids and proteins. The physical properties and biological functions of cell membranes are closely related to the arrangement and distribution of lipids within the phospholipid bilayer. In fact, lipids may phase separate into different domains within the bilayer depending on their composition. Chol is abundantly present in cell membranes and plays a key role in maintaining structural integrity and regulating membrane fluidity.

[0264] In certain embodiments, the compositions described herein include Chol. Chol can contribute to the safety and ion channel activity of the formulated drug product. The addition of Chol to saturated PL can have a significant effect on the PL phase behavior in cell membranes and spray-dried particles.

[0265] Figure 1 Phase diagram of hydrated DPPC-Chol mixture (Ipsen JH, G, Mouritsen OG et al., Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim Biophys Acta. 1987; 905:162-172).

[0266] like Figure 1 As depicted in Figure 1, the addition of Chol leads to the formation of a new phase called the ordered liquid phase (Io). Incorporation of less than ~6 mol% Chol into so results in T m The broadening of the phase transition and the increase in disorder in the acyl chain stacking. m The phospholipid acyl chains present in the disordered ld phase above have an increased degree of order with the addition of Chol. At Chol concentrations between about 6 and 20 mol%, the two phases coexist. m Above, the disordered liquid phase and the ordered liquid phase coexist (ld-lo). m Below this, the solid ordered phase coexists with the ordered liquid phase (so-lo). At high Chol concentrations (20-25 mol%), the synergistic T m phase transition, only the lo phase exists. For HSPC (longer acyl chains), a similar diagram is expected, where Figure 1 The temperature on the vertical axis in the figure shifts up by about 13°C.

[0267] Compared to the fully hydrated PL described above, the compositions according to the embodiments described herein comprise partially dehydrated PL.For the proposed use as ion channel prostheses, the lipids in the compositions described herein may be spray dried to form a respirable dry powder having a low water content.

[0268] In the absence of water, the spacing between phospholipid head groups decreases, thereby increasing van der Waals interactions between lipid acyl chains and increasing T m For example, after freeze-drying, the T m The value increases from about 41°C (fully hydrated) to 105°C (Ohtake S, Schebor C, Palacek SP, de Pablo JJ: Phase behavior of freeze-dried phospholipid-cholesterol mixtures stabilized with trehalose. Biochim Biophys Acta. 2005, 1713: 57-64).

[0269] For spray-dried DSPC powder, T m From 55°C (fully hydrated) to 71°C at 75% RH, 85°C at 11% RH, and 102°C at <3% RH (Pikal-Cleland KA, Zhang J, Lechuga-Ballesteros D, Tarara TE, Weers JG: The impact of Ca 2+ binding on the packing structure of dryphospholipids.Presented at CRSAnnualMeeting,Seoul,Korea,2002)

[0270] For currently commercially available spray-dried compositions containing PL (e.g., Podhaler TM , Aerosphere, Aerosphere and ), the goal is to maintain the PL in the so phase during particle formation, in the collector during spray drying, and when stored during the shelf life of the product. Therefore, these products utilize long-chain saturated phosphatidylcholines such as DPPC and DSPC as the main shell-forming excipients.

[0271] During spray drying of aqueous feedstocks, evaporative cooling of the atomized droplets maintains the droplet temperature just above room temperature and well below the hydration T of the PL. m (41 to 55°C). Even when the inlet temperature on the spray dryer exceeds T m After particle formation, the outlet temperature in the spray dryer and the collector jacket temperature are kept below T in the dehydration state. mTherefore, the acyl chains remain in the SO phase throughout the manufacturing process and during storage.

[0272] For liquid feedstocks containing DPPC or DSPC, spray drying of the so phase enables the formation of discrete spray-dried particles, in which the surface composition, surface morphology, size, density, and porosity of the particles can be effectively controlled. In addition, due to their presence in endogenous lung surfactant and the natural rapid clearance pathway from the lung, long-chain saturated PLs are considered to be biocompatible in the lung.

[0273] In contrast, T m Spray drying of aqueous feedstocks with unsaturated PL values ​​< 0 °C poses significant challenges. Water evaporation during spray drying may require significantly higher temperatures than T. m In this case, the PL acyl chains are present in the ld phase during particle formation, and the drying process may therefore result in large agglomerates of sintered particles. This may result in low production yields and aerodynamic particle size distributions that are not suitable for inhalation as a dry powder aerosol.

[0274] In certain embodiments, the compositions described herein comprise saturated phospholipids with longer acyl chains (e.g., 16:0 / 16:0 or 18:0 / 18:0). In some embodiments, the compositions described herein comprise DPPC, DSPC, HSPC, DSPG, or a combination thereof.

[0275] Calcium ions can bind to phosphate groups in phospholipids to displace water molecules and compress the packing between PL molecules. For spray-dried DSPC powder, the addition of Ca ions can improve the environmental robustness (T, RH) of the dehydrated powder, especially at RH values ​​(e.g., 75% RH) that typically result in a significant increase in capillary forces. In some embodiments, the PL / Ca ratio is less than 4.0 mol / mol, less than 3.0 mol / mol, or about 2.0 mol / mol (US 8,709,484; US 7,442,388; incorporated herein by reference). In some embodiments, it may not be desirable to reduce the PL / Ca ratio to less than the stoichiometric ratio of 2.0 mol / mol, because excess calcium chloride below this ratio may increase the hygroscopicity of the powder.

[0276] T in ld or lo phase m Above, spray-dried powders can become "sticky" viscous liquids. These cohesive powders tend to negatively affect powder flow and aerosol performance. This observation may be similar to the highly disordered amorphous solids above their glass transition temperature T gSpray-dried powders with phase separated SO and Io domains tend to have poor powder properties. Therefore, it may be desirable to keep the lipids in the SO phase.

[0277] Little is known about the effect of added Chol on the phase behavior of PL in dry powder formulations. Differential scanning calorimetry thermograms of compositions according to embodiments described herein indicate that the Io phase completely dissolves into the SO phase at Chol / PL ratios below about 0.05 w / w (≤ 9.4 mol % Chol) (Example 4). Thus, in the dehydrated state, the SO phase can dissolve a greater percentage of Chol than observed for hydrated DPPC-Chol mixtures ( Figure 1 ). The maintenance of the so phase leads to T m Value>90℃.

[0278] The DSPG content in ABCI powder varies from Reducing the HSPC / DSPG ratio from 2.3 mol / mol (-7 / 3 mol / mol) used in some lung surfactant formulations to 9.0 w / w or higher HSPC / DSPG ratios can also improve the physicochemical properties and aerosol properties of spray-dried AmB / Chol powders (Example 12).

[0279] In some embodiments, the increase in the HSPC / DSPG ratio reduces the hygroscopicity of the compositions described herein at high relative humidity (RH) (Example 5). DSPG is an anionic phospholipid that can be provided as a sodium salt. The addition of calcium ions can displace sodium ions, which can then combine with chloride ions from calcium chloride to form a hygroscopic sodium chloride domain in the spray-dried powder. At high RH, NaCl deliquesces (i.e., absorbs moisture from the air and dissolves therein). This may contribute to the viscosity of the compositions according to the embodiments described herein at high RH.

[0280] Other PLs can provide improvements in ASL pH and acceptable powder and aerosol properties. Their effectiveness can be determined using the characterization methods described in the Examples section. In certain embodiments, the compositions described herein comprise sphingomyelin, phosphatidylserine (PS), or a combination thereof. In some embodiments, the compositions described herein comprise phospholipids with longer saturated acyl chains (e.g., n≥16).

[0281] Drug product composition

[0282] In some embodiments, the compositions described herein comprise drug particles that are phase separated from the lipid excipient in their own crystalline domains. That is, AmB and Chol are not complexed in amorphous nanoparticles as in the case of US 2019 / 0083517, US 2020 / 0352970. AmB is also not The protein is encapsulated in liposomes as in the case of (US 5,965,156).

[0283] The presence of Chol in the compositions described herein may be critical from both an efficacy and safety perspective.

[0284] From the perspective of efficacy, the presence of Chol can maintain ion channel activity at both low and high AmB concentrations. Although the mechanism is not yet clearly understood, in the absence of Chol, ion channel activity tends to decrease at higher AmB concentrations (Example 10). This trend is observed for both pure AmB and ABIP without Chol.

[0285] In US 2019 / 0083517, Burke et al. disclose a composition of AmB and Chol for restoring airway surface physiology in CF lung epithelium, wherein the molar ratio of Chol / AmB is claimed to be in the range of 2.6 to 50 mol / mol. In US2020 / 0352970, Burke et al. disclose a method for treating CF with a small molecule bicarbonate channel. In this case, the Chol / AmB ratio is between 1.0 and 12.0 mol / mol. In the present disclosure, it has been surprisingly found that ion channel activity is maintained at a Chol / AmB ratio significantly lower than 1.0 mol / mol (Example 10). In some embodiments, the nominal ratio of the Chol / AmB ratio is between 0.4 and 1.2 mol / mol. In some embodiments, due to the enrichment of the bulk drug during the spray drying process, the actual range of the Chol / AmB ratio in the final spray-dried powder is about 0.33 to about 1.0 mol / mol. In certain embodiments, a significant improvement in ASL pH (ie, >0.1 pH unit) comparable to that achieved with the AmB:Chol complex is maintained down to a nominal Chol / AmB ratio of 0.05 mol / mol.

[0286] Maintaining ion channel activity at lower Chol / AmB ratios in spray-dried powders containing PL can minimize the phase separation of the Chol-rich lo phase due to its negative impact on powder properties.

[0287] The AmB:Chol complex disclosed in US 2019 / 0083517 and US 2020 / 0352970 achieves an improvement of ~0.1 to 0.2 pH units in ASL pH in CuFi-1 and CuFi-4 epithelial cells. The ASL pH remains slightly acidic (pH ~6.90-6.95). In CuFi-4 cells, the CFTR modulator ivacaftor achieves an ASL pH improvement comparable to that of the AmB:Chol complex. In contrast, the ASL pH improvement observed in CuFi-1 cells with the composition according to the embodiments described herein was about 0.4 to 0.5 pH units, i.e., about 2 to 5 times that observed with the AmB:Chol complex and ivacaftor. In some embodiments, the ASL pH is increased from slightly acidic when using the AmB:Chol complex to slightly alkaline (pH ~7.2) when using the composition described herein. Achieving a slightly alkaline pH can improve the activity of many cationic peptides (e.g., defensins) involved in host defense.

[0288] From a safety perspective, it is known that the presence of Chol in the compositions according to the embodiments described herein prevents sterols from being extracted from mammalian cell membranes into the extramembrane sterol sponge phase. This has been shown to be the primary mechanism of toxicity in Chol-free compositions of AmB. It has been proposed in US 2019 / 0083517, US 2020 / 0352970 to maintain this protective effect at a Chol / AmB ratio as low as 1.0 mol / mol. It has been surprisingly found that this protective effect for erythrocyte membranes is maintained at a Chol / AmB ratio as low as 0.4 mol / mol or less (Example 15).

[0289] Figure 2 is a ternary phase diagram detailing the composition of various AmB / lipid compositions. In certain embodiments, the compositions described herein are defined by a shaded triangle. Two sides of the triangle are defined by the range of Chol / AmB molar ratios (e.g., 1.2 mol / mol and 0.4 mol / mol). The third side of the triangle is defined by the Chol / PL ratio (0.05 w / w) that eliminates the phase separation of the lo phase. Phase diagrams and various compositions are discussed in more detail in Example 1.

[0290] In some embodiments, the nominal daily dose of AmB for restoring ASL pH in CF patients is from about 0.1 to about 10 mg, or from about 0.2 mg to about 6 mg.

[0291] In some embodiments, estimates of drug loading based on the nominal dose and tap density of the powder are given in Example 16. In certain embodiments, the drug loading is about 0.1% to about 50% w / w, such as about 0.5% to about 20% w / w.

[0292] Physicochemical properties

[0293] The physicochemical properties of the compositions described herein can depend on the raw material composition (eg, solids content, PFOB content) and the drying conditions in the spray dryer. The ranges described below are based on the results of the composition batches given in the Examples.

[0294] In some embodiments, the small porous particles have a relatively low density, although not as low as compositions spray dried from solution-based liquid feedstocks. In some embodiments, the poured bulk density is from about 0.04 to about 0.12 g / cm 3 , and a tap density of about 0.08 to about 0.20 g / cm 3 .

[0295] The Carr Index provides a measure of the flow properties of bulk powders (Table 3). Fine particles with geometric dimensions less than 5 μm, such as those according to embodiments described herein, are highly cohesive and have very poor powder flow properties.

[0296] Table 3. Relationship between Carr's Index and bulk powder flow properties

[0297]

[0298]

[0299] However, it is possible to use a drum loader (e.g. from Harro The machine) accurately and precisely fills fine cohesive powders to achieve a relative standard deviation of less than about 3% for fill masses as low as about 1.0 mg.

[0300] In some embodiments, the powders described herein have a Carr's index of about 20 to about 50, such as about 24 to about 30. Powders with lower Carr's index values ​​can have significantly improved powder flow properties, reduced variability in size and density measurements, and increased production yields. Compositions with a 10 range in spray-dried powders have a Carr's index of about 40, which tends to result in low yields and difficult to handle (e.g., fill and fluidize) powders.

[0301] In certain embodiments, the relatively high Carr's Index values ​​observed (indicative of poor cohesive flow, ie, Carr's Index values ​​of 20-32) do not negatively impact flow properties that are important to the development of a portable dry powder inhaler.

[0302] The primary particle size distribution can be determined by laser diffraction (see, e.g., Characterization Methods below). In certain embodiments, the powders described herein have an X distribution of about 0.40 to about 1.2 μm. 10 , X from about 1.5 to about 3.5 μm50 and X of about 4.0 to about 8.0 μm 90 .

[0303] The water content can be determined by coulometric Karl Fischer titration.In certain embodiments, the powders described herein have a water content of about 1.5% to about 6% w / w.

[0304] In certain embodiments, the physicochemical properties of the powders can be tuned throughout the described ranges by variations in the manufacturing process parameters.

[0305] Aerosol performance

[0306] In certain embodiments, the compositions described herein target the bronchial airways. In this regard, it is often beneficial to minimize extrathoracic drug deposition in the mouth and throat and alveoli while maximizing deposition in the large and small airways within the lungs.

[0307] In the context of in vitro aerosol performance indicators, the compositions according to the embodiments described herein have an ejection dose from a portable dry powder inhaler at a 4 kPa pressure drop and a 4 L inhalation volume of at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the compositions described herein are formulated to pass the delivery dose uniformity (DDU) regulatory requirements as described in the FDA's draft guidance on "Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Products-Quality Considerations (April 2018)", wherein the standard deviation of the mean ED value is 6% or less.

[0308] In certain embodiments, the compositions described herein have a mass median aerodynamic diameter (MMAD) of about 1.5 to about 4.0 μm, such as about 2.0 to about 3.5 μm, in a Next Generation Impactor (NGI) operating at 4 kPa pressure drop and 4 L intake volume.

[0309] The deposition pattern within the NGI may be important. According to the desired pattern of regional deposition detailed above, it is beneficial to minimize deposition in the USP throat and impactor 1 and 2 stages (referred to as the "coarse fraction"), and to minimize deposition in the "ultrafine fraction" on stage 6 to the filter, while maximizing deposition on stages 3, 4, and 5 (referred to as the "airway fraction"). In certain embodiments, deposition in the coarse fraction is less than 30%, less than 20%, or less than 10% w / w of the nominal dose. In some embodiments, deposition in the ultrafine fraction is less than 16%, less than 12%, or less than 8% of the nominal dose. In certain embodiments, deposition in the airway fraction is at least 40%, at least 50%, at least 60%, or at least 70% of the nominal dose.

[0310] In certain embodiments, the fine particle fraction (FPF) smaller than 5 μm expressed as a percentage of the emitted dose is <5μm ) is at least 40%, at least 50%, at least 70% or at least 90% w / w.

[0311] In certain embodiments, the flow rate dependence of total lung dose (TLD) in the Alberta Idealized Throat (AIT) model between pressure drops of 1.0 and 6.0 kPa is less than 40% or less than 15%.

[0312] In certain embodiments, the target fill mass is about 5 to about 40 mg, such as about 10 to about 20 mg. In some embodiments, the compositions described herein are administered to a subject using a portable capsule-type dry powder inhaler (DPI). In certain embodiments, the DPI has a volume of about 0.30 cm 3 (Capsule No. 3) to about 0.37cm 3 (Capsule No. 2) container.

[0313] In certain embodiments, the DPI is a variant of the RS01 DPI (Plastiape, Osnago, Italy). The variants of RS01 differ in the size of the capsule they contain (size 3 to size 0) and their resistance to airflow (R = 0.06 to 0.16 cm H2O 0.5 L -1 In some embodiments, the DPI is medium to high resistance (e.g., R is about 0.10 to about 0.30 cm H2O 0.5 L -1 min). In certain embodiments, the DPI is about 0.14 cm H2O 0.5 L -1A variation of the RS01 DPI with a resistance of 1.5 to 2.0 min. In certain embodiments, medium to high resistance DPIs limit extrathoracic deposition while also limiting the potential for post-inhalation coughing.

[0314] In some embodiments, the compositions described herein are prepared using Handihaler (Boehringer Ingelheim), RS00 (Plastiape), or (Novartis) (U.S. Patent 3,991,761; incorporated herein by reference), (US 2007 / 0295332; incorporated herein by reference) (Novartis). In certain embodiments, the compositions described herein are administered with Turbospin (PH&T) or a variant thereof, such as T-326 (Podhaler TM ) (Novartis) or AIR inhaler (Acorda Therapeutics).

[0315] In certain embodiments, the compositions described herein are administered with a single-use disposable inhaler. In some embodiments, the single-use disposable inhaler is TwinCaps (Hovione), (MicroEngineering Solutions)、ICOone TM (Iconovo) or (Pure IMS).

[0316] In some embodiments, dry powder compositions of the present disclosure are administered intranasally to increase ASL pH in the nose and increase nasal potential difference. Contemplated devices include Aptar's Unidose system.

[0317] manufacture

[0318] A mixture of cholesterol and phospholipids can be prepared by dissolving in an organic solvent. In certain embodiments, a lipid mixture comprising HSPC / DSPG / Chol in a 54 / 24 / 22 w / w / w ratio is prepared. Depending on the desired composition, the lipid mixture can be supplemented with additional PL and calcium chloride.

[0319] In some embodiments, the method of preparing the compositions described herein comprises dispersing the lipid mixture in water to form multilamellar liposomes. In certain embodiments, this step involves adding the lipids to a liposome at a temperature above the hydration temperature of the lipids. m In some embodiments, the temperature of the water is between 65°C and 90°C. In some embodiments, a high shear mixer such as T-50 achieves dispersion.

[0320] In some embodiments, the method further comprises adding a fluorinated (FC) blowing agent (e.g., perfluorooctyl bromide (PFOB, Perflubron), perfluorooctyl ethane (PFOE), or perfluorodecalin (PFD)) while mixing to form a coarse FC-in-water emulsion containing micron-sized emulsion droplets. In certain embodiments, the coarse emulsion is or piston-gap homogenizer (e.g. Avestin ) is homogenized under high pressure to form a submicron emulsion. In some embodiments, the pressure during homogenization is about 10,000 to about 20,000 psi, and the homogenization is performed for a set time (depending on the batch size) or a specified number of independent passes. In some embodiments, a single independent pass is performed at the end of a set cycle process to ensure that all droplets pass through the homogenizer at least once.

[0321] In some embodiments, the method further comprises adding the drug substance to the submicron emulsion under mixing. In certain embodiments, the addition is performed in a one-pot process. In some embodiments, the resulting complex dispersion of the emulsion droplets and the suspended drug particles is mixed with a high shear mixer or passed through a homogenizer.

[0322] In some embodiments, the emulsion preparation and wet milling of the drug are performed in separate tanks in a two-pot process. In certain embodiments, this enables in-process sizing of the micronized drug particles by laser diffraction or dynamic light scattering. In some embodiments, the two pots are then combined into one with simultaneous mixing.

[0323] In certain embodiments, the emulsion preparation and wet milling steps are performed with a concentrated emulsion / suspension to limit the amount of liquid processed through the homogenizer. In some embodiments, water is added in the final step to achieve the target solids content and PFOB volume fraction in the final liquid feedstock.

[0324] In some embodiments, the raw material is sprayed into a warm filtered air stream to evaporate the solvent and convey the dried product to a cyclone separator or bag filter house. In certain embodiments, the exhaust gas is then discharged together with the evaporated solvent. In some embodiments, the operating conditions of the spray dryer such as inlet and outlet temperature, feed rate, atomization pressure, flow rate of dry air and nozzle configuration can be adjusted to produce the desired particle size, moisture content and production yield of the resulting dry particles. In certain embodiments, the method further includes setting the Niro PSD-1 scale spray dryer to have an inlet air temperature between 80°C and 200°C, an air outlet temperature between about 40°C and 120°C, a liquid feed rate between 30g / min and 120g / min, a total air flow between 50scfm and 230scfm and an atomizing airflow between 30 and 90scfm. The solid content in the spray-dried raw material will generally be between 0.5% w / v and 10% w / v, and the foaming agent concentration will generally be between 3% and 30% v / v. In some embodiments, the desired configuration depends at least in part on the size and type of equipment used.

[0325] As the water in the atomized droplets evaporates, the diameter of the aqueous atomized droplets decreases, and the slowly diffusing emulsion droplets and AmB crystals concentrate at the air / water interface to form a hollow droplet core. When the higher boiling oil phase evaporates, it leaves pores in the spray-dried particles. The final particle morphology includes porous lipid particles with entrapped drug particles.

[0326] In certain embodiments, the outlet temperature and the collector jacket temperature (if used to control the RH in the collector) must be below the lowest lipid major transition temperature. In certain embodiments where the Chol / PL ratio is <0.05 w / w, the lowest lipid major transition temperature is greater than 80°C to provide an outlet and collector jacket temperature of 70°C or higher. A reduction in the Chol / PL ratio and an increase in the HSPC / DSPG ratio (Examples 9 and 12) may eliminate the Chol-rich phase, thereby providing a higher outlet temperature, an increase in production rate, and a reduction in residual solvent in the spray-dried powder.

[0327] If the atomized droplets dry too quickly, the benefits of the miniemulsion droplets may be lost. Therefore, controlling the drying rate can be beneficial.

[0328] definition

[0329] As used herein, the terms "treat" and "treating" refer to performing an intervention that results in (a) preventing a condition or disease from occurring in a subject who may be at risk for developing the condition or disease or who is susceptible to the condition or disease but has not yet been diagnosed as having the condition or disease; (b) inhibiting the condition or disease, e.g., slowing or arresting its development; or (c) relieving or ameliorating the condition or disease, e.g., causing regression of the condition or disease. In one embodiment, the term "treat" refers to performing an intervention that results in (a) inhibiting the condition or disease, e.g., slowing or arresting its development; or (b) relieving or ameliorating the condition or disease, e.g., causing regression of the condition or disease.

[0330] As used herein, "subject" or "patient" refers to a living mammal. In various embodiments, the patient is a non-human mammal, including but not limited to a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, cat, dog, pig, horse, cow, or non-human primate. In certain embodiments, the patient is a human.

[0331] As used herein, "effective amount" refers to any amount sufficient to achieve a desired biological effect.

[0332] As used herein, "therapeutically effective amount" refers to any amount sufficient to achieve a desired therapeutic effect, eg, treating CF.

[0333] As used herein, "active ingredient," "therapeutically active ingredient," "active agent," "drug," or "drug substance" refers to the active component of a drug, also known as an active pharmaceutical ingredient (API).

[0334] "Amorphous" as used herein refers to a state in which a material lacks long-range order at the molecular level and can exhibit the physical properties of either a solid or a liquid, depending on the temperature. Typically, such materials do not give a characteristic X-ray diffraction pattern and, although exhibiting the properties of a solid, are more formally described as liquids. Upon heating, a change from solid to liquid-like properties occurs at the "glass transition," which is generally defined as a second-order phase transition.

[0335] As used herein, "crystalline" refers to a solid phase in which the material has a regular, ordered internal structure at the molecular level and gives a characteristic X-ray diffraction pattern with defined peaks. Such a material will also exhibit the properties of a liquid when heated sufficiently, but the change from solid to liquid is characterized by a phase transition, usually a first order phase transition ("melting point"). In the context of the present disclosure, a crystalline active ingredient refers to an active ingredient with a crystallinity greater than 75%. In certain embodiments, the crystallinity is suitably greater than 90%. In other embodiments, the crystallinity is greater than 95%. In other embodiments, the crystallinity is less than 10% or less than 5%.

[0336] As used herein, "drug loading" refers to the percentage of active ingredient(s) in the total mass of the composition on a mass basis.

[0337] As used herein, "mass median diameter" or "MMD" or "X 50 " refers to the median diameter of a plurality of particles, typically in a polydisperse population of particles, i.e., consisting of a range of particle sizes. Unless the context indicates otherwise, X as reported herein 50 The values ​​were determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany).

[0338] As used herein, "tap density" or p tapped Similar to USP <616> The tapped density is measured as described in Bulk Density and Tapped Density of Powders in Method I. The tapped density represents a closer approximation of the particle density than the poured bulk density, and the measured value is approximately 20% less than the actual particle density.

[0339] As used herein, "mass median aerodynamic diameter" or "MMAD" refers to the median aerodynamic size of a plurality of particles, typically in a polydisperse population. "Aerodynamic diameter" is the diameter of a unit density sphere that typically has the same settling velocity in air as the powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle composition in terms of its settling behavior. Aerodynamic particle size distribution (APSD) and MMAD are described herein by using the NEXTGENERATION IMPACTOR TM (Copley Scientific) Cascade Impaction Assay. In general, if the particles are aerodynamically too large, fewer particles will reach a particular area of ​​the lungs. If the particles are too small, a greater percentage of the particles may be exhaled. In contrast, d a Represents the aerodynamic diameter of a single particle.

[0340] As used herein, "nominal dose" or "ND" refers to the mass of drug loaded into a container (eg, capsule or blister) in a non-reservoir based dry powder inhaler. ND is sometimes also referred to as a metered dose.

[0341] As used herein, "emitted dose" or "ED" refers to an indication of dry powder delivered from an inhaler device following an actuation or dispersion event from a powder unit. ED is defined as the ratio of the dose delivered by the inhaler device to the nominal or metered dose. ED is an experimentally determined parameter and can be determined using an in vitro device setup that simulates patient dosing. ED is sometimes also referred to as delivered dose (DD).

[0342] As used herein, "fine particle fraction" (FPF) refers to the percentage of active ingredient in the sprayed dose that has an aerodynamic size less than 5 μm. Aerodynamic particle size distribution (APSD) is determined herein by using the NEXT GENERATION IMPACTOR TM Cascade impact assay.

[0343] As used herein, "solid content" refers to the concentration of the active ingredient(s) and excipients dissolved or dispersed in the liquid solution or dispersion to be spray dried.

[0344] As used herein, "the airways of a subject" refers to any or all of the following lung structures: trachea, bronchi, and bronchioles.

[0345] The term "about" refers to variations in numerical values ​​that are commonly encountered by those skilled in the art of inhalable compositions, including variations of plus or minus 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the numerical values ​​recited herein.

[0346] Throughout the specification and the claims that follow, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to mean the inclusion of a specified integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0347] Unless otherwise stated or clear from the context, numerical ranges include the endpoints and any values ​​therebetween. Example

[0348] Various aspects and embodiments of the present disclosure are further clarified with reference to the following examples. These examples are exemplary in nature and should not be construed as limiting the subject matter of the present disclosure.

[0349] Characterization methods

[0350] Amphotericin B (AmB) content and purity. The AmB content and purity in the formulated bulk powder or aerodynamic particle size distribution (APSD) samples were determined by reverse phase high performance liquid chromatography (RP-HPLC) with detection at 383 nm. The samples were analyzed using an Agilent 1260 InfinityII HPLC system (Wilmington, DE, USA). Separation was achieved using an Agilent InfinityLab Poroshell 120 EC-C18, 3.0x150mm, (2.7μm) column using a gradient method (solvent A = 10mM acetate buffer, pH 4.2; solvent B = acetonitrile / methanol, 1.0 v / v). AmB was quantified using a single point calibration using USP certified reference standards.

[0351] 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). TM Separation was achieved on a C18 column (5 μm) using an isocratic method (acetonitrile / isopropanol, 1.0 v / v). Cholesterol was quantified using Cholesterol HP, Ph. Eur / USP-NF stock (Carbogen Amcis, Beuvry-la-Foret, France) using a single point calibration.

[0352] 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 micrometering unit, and a RODOS / M dry powder dispersion unit. About 2 mg to 5 mg of powder was filled into an ASPIROS tube and fed at 5 mm / s into a RODOS operated with a 4 bar dispersion pressure and a 65 mbar vacuum. The powder was introduced at an optical concentration of about 1% to 5%, and data were collected over a measurement duration of up to 15 seconds. The particle size distribution was calculated using the Fraünhofer model by the instrument software. The reported values ​​represent the average of three independent measurements for each collector.

[0353] Tap density. Using the known volume (0.593 cm 3 ) of a cylindrical cavity to measure the tap density (ρ tapped). Use a micro spatula to fill the sample holder with powder. Then tap the sample cell gently on the benchtop. 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 no longer consolidates with further tapping. The tapped density is defined as the mass of the powder bed divided by the volume of the cavity.

[0354] Bulk density. Bulk density (ρ bulk ) represents the mass of powder loaded into the sample container to the required volume without compaction.

[0355] Carr's Index. The Carr's Index C provides an indication of the compressibility of a powder. It is given by the following formula:

[0356]

[0357] As the Carr's Index increases, the flowability of the powder is considered to decrease. Values ​​<10% indicate an excellent free-flowing powder, values ​​between 11-15% are associated with a good free-flowing powder, values ​​between 16-20% are associated with moderate powder flow, values ​​between 21-25% are associated with passable powder flow, values ​​between 26-31% are associated with poor flow cohesion, values ​​between 32-37% are associated with very poor flow, and ≥38% are associated with almost no flow.

[0358] Water content. The water content was determined by Karl Fischer titration using a Nittoseiko Analytech Moisture Meter CA-310 with a fritless cathode and vaporizer model VA-300.

[0359] Dynamic Vapor Sorption. Moisture sorption isotherms at 25°C were measured using a dynamic vapor sorption (DVS) instrument manufactured by Surface Measurement Systems, UK. The instrument measures the absorption and loss of water vapor by a material by 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 allow the sample to reach a constant mass. The instrument was then programmed to increase the RH from 0 to 2% RH, to 5% RH, and then increase the RH to 90% RH in steps of 5% RH and decrease from 90% to 0% RH in steps of 5% RH. A balance standard of dm / dt=0.005% / min was selected for the system to achieve each RH step before automatically proceeding to the next RH step. Sample masses between 10 and 15 mg were used in this study.

[0360] Differential Scanning Calorimetry. DSC thermograms of given samples were measured using a TA Instruments Model Q2000 Differential Scanning Calorimeter equipped with a refrigerated cooling system (New Castle, Delaware). Dry nitrogen was used at 50 cm 3 The sample pool was purged at a flow rate of 110 cm / min. The refrigeration control system (RCS) used a flow rate of 110 cm 3 / min of nitrogen. A Tzero aluminum pan containing about 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 5°C / min.

[0361] Aerodynamic particle size distribution. Using RS01 dry powder inhaler (Mod.7 Ultra High Resistance 2 model), USP induction port (IP) and Next Generation Impactor TM or NGI TM Determines aerodynamic particle size distribution (APSD) in accordance with USP <601> and Ph. Eur. 2.9.18. The flow control device was adjusted to operate at a 4 kPa pressure drop and a 4 L total volume through the inhaler. The RS01 DPI variant used had a 0.143 cmH2O 0.5 L -1 min(0.045kPa 0.5 L -1 min). This corresponds to 44.2 L min at a 4 kPa pressure drop. -1 flow rate. Approximately 10 mg was manually filled into #3 size inhalation grade HPLC capsules (Vcaps, Qualicaps). The aerosol powder sprayed from the inhaler was aspirated through USPIP and graded in NGI. At each level of NGI, the emptied capsules and devices were extracted with a sample dissolution solution containing methanol. Further dilutions were performed to reduce the AmB concentration to within the linearity of the detection range. The AmB mass of each level was determined using the above HPLC method, and the fine particle dose (FPD) less than 5 μm was calculated. <5μm ) and mass median aerodynamic diameter (MMAD).

[0362] Cell lines and growth conditions. 75 cm cells treated with Thermo Scientific BioLite Cell Culture 2NuLi, CuFi-1 and CuFi-4 cells (Welsh Laboratory, University of Iowa) were grown on flasks from cryostock. These flasks were pre-coated with 4 mL 60 μg / mL human placental collagen type IV (Sigma-Aldrich) at 37°C for at least 1 hour, rinsed twice with PBS, then dried, and then inoculated. The cells were cultured with 12 mL bronchial epithelial cell growth medium (BEGM) BulletKit (Lonza CC-3170), which included basal medium and 8 SingleQuots of supplements (bovine pituitary extract (BPE) 2 mL; hydrocortisone 0.5 mL; hEGF 0.5 mL; epinephrine 0.5 mL; transferrin 0.5 mL; insulin 0.5 mL; retinoic acid 0.5 mL; triiodothyronine 0.5 mL). Gentomycin-AmB aliquots were 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) instead. The original CF transplant donor was genotyped by Integrated Genetics. The cell line was confirmed to have the correct genotype and no mycoplasma contamination by the ATCC library. The MycoAlert Mycoplasma Detection Kit (Lonza LT07-418) was used to detect any RNA transcripts common to a wide spectrum of mycoplasmas. The cell line was confirmed to be mycoplasma-free. The cells were grown in 5% CO2 at 37°C to> 90% confluence, the culture medium was changed every 2-3 days, and then trypsinized with 4mL 0.25% trypsin containing 1mM EDTA (Gibco 25200-056). Trypsin was inactivated with 10mL HEPES buffered saline solution (Lonza CC-5024) containing 1% fetal bovine serum. The cells were spun at 1,500rpm for 5 minutes at room temperature in an Eppendorf centrifuge 5430R, and resuspended in BEGM culture medium for passage. In order to culture on a membrane carrier to differentiate, the cells were resuspended in Ultroser G culture medium after centrifugation. This included 1:1 DMEM: Ham's F-12, supplemented with 4% v / v Ultroser G (Crescent Chemical) and 50μg / mL penicillin-streptomycin (Corning Cellgro), 50μg / mL gentamicin (Sigma-Aldrich G1397) and 2μg / mL fluconazole (Sigma-Aldrich).For all studies, the membrane support used was Corning Costar 0.4-μm 24-well Transwell clear polyester membrane inserts (0.33 cm. 2 ) (Corning 3470). The membranes were coated with collagen in the same manner as the flasks detailed above, except that 100 mL of collagen was used and only one rinse with PBS was used. The inserts were inoculated with 115,000 cells each. The membranes were allowed to mature at the air-liquid interface for a minimum of 14 days to achieve complete differentiation, with the Ultroser G culture medium being replaced once or more per week as needed. After maturation, the culture medium was replaced every 7 days. For covariate control, the membranes used in the experiment were as close as possible in age and maturity.

[0363] For primary cultured airway epithelial cells, lung tissue was obtained from individuals with CF undergoing lung transplantation or organ donation. The tissue was dissociated and the dissociated cells were seeded directly onto transwell filters and cultured at the air-liquid interface. The cultures were used at greater than three weeks after seeding, when the epithelial cells differentiated into typical cell types of the airway and the electrical properties of the cells reflected the tissue isolated from the body.

[0364] Fluorescence microscopy for measuring airway surface fluid pH. For each experiment, a fresh suspension of ABCI was 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 suspension was measured in triplicate by absorption spectroscopy. The pH of the airway surface fluid was determined using Beer's law from the absorbance at 406 nm (ε 406 =164,000M -1 cm -1 ) The concentration was calculated. Next, the stock suspension was diluted with PFH to achieve a suspension AmB concentration of -0.5 to 50 μM.

[0365] Small diameter NuLi and CuFi cells were used for the measurement of ASL pH. The ratiometric pH indicator SNARF coupled to dextran (Molecular Probes) was used to measure ASL pH. SNARF powder was suspended in PFH by sonication and distributed onto the apical surface of the cells. ASL pH was measured after 2 hours. SNARF was excited at 514 nm, and the ejection was recorded at 580 nm and 640 nm at 40x magnification using a Zeiss LSM 800 microscope equipped with a water immersion lens for cell line cultures. To generate a standard curve for pH determination, SNARF was dissolved in a colorless pH standard and the fluorescence ratio was converted to pH. The powder tested in this assay was suspended in an appropriate volume of PFH, which was sonicated for 1 minute to aid suspension. AmBisome was suspended by vortexing in PFH. Subsequently, 20 μL of the suspension was applied to cultured airway epithelial cells (A=0.33 cm 2 ). In all experiments, the ASL pH of compound-treated epithelial cells was measured and compared with the results from vehicle-treated epithelial cells. For apical compound administration, cultured airway epithelial cells were incubated at 37° C. for approximately 22 hours prior to measuring ASL pH.

[0366] Preparation of erythrocyte stock suspension. 1 mL of human whole blood (sodium heparin 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 in 1 mL of 0.9% (m / v) saline by gentle inversion (the erythrocytes will be lysed by pipetting 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 two more times. After the last wash, the supernatant was removed and the erythrocyte 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 make an erythrocyte stock suspension.

[0367] Minimum hemolytic concentration assay (MHC). Compounds to be tested were prepared in a dilution series in a DMSO (D6-99.9%; Cambridge Isotope Laboratories) solution, each concentration being 25.63x the final concentration. The compound dilution series was diluted 1:25 in resuspension buffer to a total of 100 μl in a 0.2 mL microcentrifuge tube and vortexed to mix the solution. The negative control (0% lysis) contained DMSO only in the resuspension buffer, while the positive control (100% lysis) contained DMSO only in water, as this resulted in complete lysis of erythrocytes due to osmotic pressure. A 2.52 μL volume of erythrocyte suspension was added to each tube (including the control), each tube was mixed by gentle inversion, and statically incubated at 37°C for 2 hours. After incubation, each sample was mixed again by gentle inversion and centrifuged at 3,214 x g for 6 minutes. After centrifugation, 60 μL of supernatant was removed, added to a 96-well plate, and the absorbance was read at 540 nm. Data were normalized to negative controls and processed as % total hemolysis relative to positive controls.

[0368] Example 1: Composition of lipid-coated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004)

[0369] The nominal anhydrous compositions of four compositions of lipid-encapsulated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004) are shown in Table 4. Three controls are also shown: (i.e., liposomal amphotericin B, L-AmB); lyophilized AmB / Chol complex prepared by Burke et al. (US 2019 / 0083517; US 2020 / 0352970); and amphotericin B inhalation powder (ABIP), a dry powder composition of AmB developed by Nektar Therapeutics for the treatment of invasive pulmonary aspergillosis (US 7,326,691; US ​​8,404,217; US 2012 / 0128728).

[0370] Table 4. Theoretical anhydrous inhalable AmB composition

[0371]

[0372] a In addition to lipids and AmB, AmBisome contains 67.8% sucrose, 2.0% disodium succinate hexahydrate, and 0.2% tocopherol.

[0373] b ABIP utilizes synthetic DSPCs instead of HSPCs

[0374] The AmB / lipid compositions detailed in Table 4 were plotted on Figure 2 The pseudo-ternary phase diagram in . The PL components at the vertices of the ternary diagram represent the sum of the masses of various PL components (e.g., HSPC, DSPC, DSPG).

[0375] Although commercial L-AMB contains the lipid components (i.e. HSPC, DSPG, Chol) identical with ABCI compositions, they exist in different ratios. In addition, compared with ABCI compositions, the organization of the medicine in L-AMB is different from that of lipid. The bulk drug in L-AMB is encapsulated in small unilamellar vesicles (liposomes). Said composition has the Chol / AmB ratio of 2.5mol / mol and the HSPC / DSPG ratio of 2.3 (i.e. 7 / 3w / w). Lipid particles are lyophilized to dry powder in the presence of large percentage of sucrose, and the sucrose serves as a cryoprotectant to maintain liposome integrity during lyophilization. The L-AMB of 60mg reconstruction is passed through four CFTR - / - 1cm in pig 2 Application of tracheal windows to the apical side of airway epithelial cells increased ASL pH by ∼0.2 pH units, from pH 6.8 to 7.0 (US2020 / 0352970).

[0376] The lyophilized AmB:Chol complexes studied by Burke et al. (US 2019 / 0083517; US 2020 / 0352970) had an even higher Chol / AmB ratio of 5.0 mol / mol, but contained no added PL. The AmB:Chol complexes were formed by rapid nanoprecipitation from a solution of the material in dimethyl sulfoxide / chloroform upon rapid injection into a nonsolvent (water). The resulting suspension was lyophilized to form a dry powder. The AmB:Chol complexes exhibited a significant increase in bicarbonate secretion and ASL pH (~0.1-0.2 pH units) in CuFi-1 cells.

[0377] ABIP is a spray-dried composition comprising wet-milled AmB crystals coated with DSPC / calcium chloride in a 2:1 molar ratio. This composition does not contain cholesterol (i.e., Chol / AmB=0 mol / mol). It also contains only saturated phosphatidylcholine (DSPC) without the addition of DSPG. Although ABIP showed an improvement in ASL pH in CuFi-1 cells at low doses (2 μM in FC-70), it did not at high doses (50 μM) (Examples 10 and 12). This result is consistent with previous studies using pure AmB and indicates that a certain amount of Chol is necessary to maintain ion channel activity over a wide range of AmB concentrations. ABIP compositions containing highly crystalline bulk drugs have demonstrated good tolerability in early clinical development.

[0378] Also presented are the compositions of three ABCI compositions (ABCI-001, ABCI-002, and ABCI-003) that have been used in nonclinical toxicology studies. These powders have a Chol / AmB ratio of 0.4 to 1.2 mol / mol. Without intending to be bound by theory, the high Chol / AmB ratios used in L-AMB and AmB:Chol complexes may not be suitable for dry powder compositions containing PL because the presence of large amounts of Chol may lead to disordering of the packing of PL acyl chains, resulting in an unacceptable increase in interparticle cohesion and a "sticky" powder. Both ABCI-003 and ABCI-004 contain Figure 2 is within the shaded triangle, while ABCI-001 and ABCI-002 are not.

[0379] As described, ABCI-001 and ABCI-002 were Figure 2 The lipids in these compositions are outside the shaded triangle because they are in the so+lo two-phase region. In contrast, ABCI-003 and ABCI-004 are in the so phase. The compositions of other inhaled AmB compositions investigated to date (i.e., ABIP, L-AMB (AmBisome), and AmB:Chol complex) are entirely in the so phase. Figure 2 outside the shadow triangle.

[0380] The DSC thermogram of ABCI-001 (30% w / w AmB, Chol / AmB=1.2 mol / mol) contains a significant proportion of a Chol-rich lo phase (Example 4). For ABCI-002, the drug content was reduced to 14% w / w while keeping the Chol / AmB molar ratio constant, which resulted in a decrease in the cholesterol / PL ratio from 0.29 to 0.09 w / w. Despite this, a small proportion of a Chol-rich lo phase was still retained. Dry powders with a phase-separated lo phase can exhibit strong inter-particle cohesion and increased hygroscopicity and deliquescence at high relative humidity (Example 5). These characteristics may have a negative impact on the powder yield during spray drying and may result in a relatively large mass median aerodynamic diameter (MMAD ~ 4 μm) (Example 9).

[0381] ABCI-003 maintains the 14% w / w drug loading in ABCI-002, but has a reduced nominal Chol / AmB ratio of 0.4 mol / mol. Overall, the Chol content is reduced from 14.6% in ABCI-001 to 2.4% in ABCI-003, while the Chol / PL ratio is reduced to 0.03 w / w. At this Chol / PL ratio, the lo phase dissolves in the so phase (Example 4). In ABCI-003, the HSPC / DSPG ratio is also increased from 2.3 w / w to 9.0 w / w, resulting in improved solubility of Chol in the PL phase, reduced NaCl formation, and reduced powder hygroscopicity (Example 5).

[0382] Unlike ABIP, all ABCI compositions containing added Chol showed significant improvements in ASL pH at both low and high AmB concentrations (Example 10). Furthermore, the presence of DSPG has been shown to enhance the magnitude of the observed ASL pH improvements (Example 12).

[0383] Many of the restrictions on Chol / AMB, Chol / PL, and HSPC / DSPG ratios may be specific to the development of dry powder compositions for inhalation and may not apply to liquid-based aerosols, such as inhaled L-AMB. They are also not directly applicable to compositions that do not contain PL.

[0384] Example 2: Preparation of ABCI-001 by spray drying

[0385] Preparation of raw materials. First, lipids and calcium chloride were dispersed in hot water using a high shear mixer (UltraTurrax T-50) to form multilamellar vesicles (MLVs). The aqueous phase must be above the main transition temperature (T m) to promote MLV formation (T>65°C). The MLV dispersion was cooled (T<30°C) and filtered and perfluorooctyl bromide (PFOB) was added using a Watson-Marlow peristaltic pump while mixing to form a coarse PFOB-in-water emulsion droplet stabilized by a monolayer of lipids. The emulsion droplets act as pore formers to form a porous coating of lipids on the crystalline drug particles. The coarse emulsion was then homogenized in a single independent pass using an M-100 microfluidizer under high pressure to form nanoemulsion droplets (diameter ~200-500nm). Next, the drug substance was added to the nanoemulsion under high shear mixing. The composite dispersion containing the suspended drug and nanoemulsion droplets was passed through the homogenizer for another two independent passes. The homogenization process wet-milled the AmB particles to a size suitable for pulmonary delivery. On a quantitative basis, most wet-milled AmB crystals have a diameter of less than 1000nm. 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 of feedstock) or 424.2 g (12 liters of feedstock).

[0386] Dry powders were 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 two-fluid atomizer (0.8 mm id), a DorrClone cyclone separator, and a lower end geometry including a straight tube and a Brewer valve. A 1 L Agele collector attached below the Brewer valve was jacketed and maintained at 50°C.

[0387] The liquid feed was pumped into the spray dryer with a Watson-Marlow peristaltic pump. The atomizer was 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 a dryer outlet temperature of 55 ± 3 °C. The total gas flow rate was approximately 140 N m 3 / h(~85scfm).

[0388] In a suspension-based feed, each atomized droplet (mass median diameter ~10 μm) contains dispersed drug crystals and approximately 1000 submicron emulsion droplets. At the initial moment of the drying process, the more volatile aqueous phase begins to evaporate. The rapidly receding atomized droplet interface drives the slowly diffusing drug and emulsion particles to be enriched at the interface. This results in the formation of void space in the center of the drying droplet. As the drying process continues, the less volatile oil phase in the emulsion droplets evaporates, so that hollow pores are formed in its place. Overall, the resulting hollow spray-dried composite particles contain drug crystals embedded in the interfacial layer of the porous lipid matrix.

[0389] ABCI-002, ABCI-003, and ABCI-004 were made using the same general process, although the composition of the liquid feed was different.

[0390] Example 3: Wet grinding of AmB and its effect on the properties of ABCI-002

[0391] The physical form of AmB (crystalline vs. amorphous) can have an impact on its wet milling behavior. For this study, two batches of AmB were obtained from North China Pharmaceutical Group Corp. (Hebei, China). The two batches differed in crystallinity, with batches '202 and '203 having crystallinity of 77% and 96%, respectively (determined by quantitative XRPD).

[0392] In fact, the reduced crystallinity of batch '202 had a negative impact on the wet milling process to significantly increase the X-ray diffraction of the wet milled drug as measured by laser diffraction on the drug suspension using a Malvern Mastersizer. 50 and X 90 (Table 5).

[0393] Table 5. Effect of AmB crystallinity on primary particle size distribution during wet grinding

[0394]

[0395] ABCI-002 batches were made on a Niro Mobile Mino using AmB batches '202 (ABCI-002 batch FP21060) and '203 (ABCI-002 batch FP21059) using the method described in Example 2. The total solids content and %PFOB in the liquid feed were 2% w / w and 10% v / v, respectively. The liquid feed rate was about 44.5 g / min. The atomizer was at 11.5 ± 1.0 Nm 3 / h (7.0 scfm) gas flow and 104 ± 5 ​​°C inlet temperature. This is equivalent to about 44.5 g / min. The total gas flow is about 140 N m 3 / h (~85scfm). Target batch size is 40g.

[0396] The physicochemical properties and aerosol performance of the two batches are detailed in Table 6. No significant differences in powder properties and aerosol performance were observed, indicating that crystallinity as low as 77% is still suitable for preparing ABCI-002 compositions.

[0397] Table 6. Comparison of physicochemical properties and aerosol performance of ABCI-002 batches prepared from AmB batches with different crystallinity

[0398]

[0399] Example 4: Effect of lipids on the phase behavior of lipid-coated AmB crystal compositions

[0400] Comparison of DSC thermograms of ABCI-001, ABCI-002, ABCI-003 and ABIP is presented in Figure 3 middle.

[0401] ABIP has a simple thermogram with a single sharp collaborative T m , with an extrapolated onset temperature of 96.2° C. The addition of Chol and DSPG to the ABCI composition resulted in a more complex thermogram with broadened transitions and the presence of multiple peaks.

[0402] At a Chol / PL ratio of 0.29 w / w (ABCI-001), two phase-separated domains were observed, a Chol-rich lo phase with an onset temperature of 62.5 °C and a broad PL-rich so phase with an onset temperature of 83.9 °C. As demonstrated for hydrated DPPC bilayers ( Figure 1 ), the addition of Chol to the so phase leads to increased disorder and eventual phase separation of the coexisting lo phase.

[0403] The Chol-rich phase transition is very broad and contains overlapping features. The presence of Chol crystals in the lo phase is confirmed by X-ray powder diffraction (XRPD) patterns, which show a diffraction peak of the ABCI variant at about 5.2° 2θ. The phase separation of cholesterol crystals is in PLCI-001 ( Figure 4 ) are more easily distinguishable (ie, fewer diffraction peaks in the same area and no interference from the amphotericin B peak). The PLCI composition is a placebo composition that does not contain AmB but contains the other components of the ABCI composition.

[0404] As the Chol content in the powder composition increases, a linear increase in the enthalpy of the Chol-rich phase transition is observed ( Figure 5 ). A linear regression was performed on the data points where Chol / (Chol+PL)>10% w / w. The data were extrapolated to the x-axis at a Chol / (Chol+PL) weight ratio of about 4.9% w / w. This corresponds to a Chol / PL ratio of 0.05 w / w or ~9.4 mol% Chol. Therefore, compositions with less than about 9.4 mol% Chol can be expected to have undetectable low temperature peaks.

[0405] In the dehydrated state, the onset of the so-lo two-phase region tends to occur at much higher Chol concentrations (i.e., relative to Figure 1Thus, the ordered SO phase in the ABCI composition is maintained at much higher Chol contents (~9.4 mol% vs. 6 mol% for a hydrated DPPC bilayer). Similarly, although the SO phase is eliminated in hydrated DPPC / Chol mixtures above about 20 mol% Chol ( Figure 1 ), but in dehydrated ABCI-001 the so-lo two-phase region extends beyond 36.5 mol% Chol. Figure 1 Phase diagram of hydrated DPPC-Chol mixture in Figure 1. The phase diagram of ABCI-001 shifts to a higher T m values ​​and move to the right toward higher cholesterol levels.

[0406] High-T in ABCI-001 m The peak is also very broad and contains multiple overlapping features. High T m The enthalpy of the peak tends to increase with increasing PL content and tends to decrease with increasing Chol content, indicating that below T m This peak is related to the so phase.

[0407] The binary mixture of HSPC / DSPG in 20% AmB composition (without Chol) exhibited similar overlapping features, indicating the coexistence of immiscible so phases. The phase separation of PC and PG domains in the presence of calcium ions is consistent with observations in other studies. m The onset temperature and enthalpy of the peak are 83.9 °C and 19.74 J / g, respectively.

[0408] 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 of AmB and Chol resulted in an increase in PL content and a decrease in the Chol / PL ratio from 0.28 w / w to 0.092 w / w. Despite the small amount of low T m The peak is still visible in ABCI-002, but the Chol-rich T m From 62.5℃ to 64.6℃, while ΔH decreased from 7.73J / g to 1.93J / g ( Figure 3 ). High T m The peaks remain broad with overlapping features. Relative to ABCI-001, the onset temperature increases from 83.9°C to 88.5°C, indicating that the acyl chains become more ordered as the Chol content decreases.

[0409] In ABCI-003, the drug loading was maintained at 14% w / w, but the Chol / AmB ratio was reduced from 1.2 to 0.4 mol / mol. This enabled the Chol / PL ratio to be reduced from 0.092 to 0.030 w / w relative to ABCI-002. m The increase in peak area ( Figure 3 ) shows that the Chol-rich peak has been eliminated. High T m The peak is also much sharper, and the onset temperature is increased by 7.2°C to 91.1°C relative to ABCI-001.

[0410] For the long-term stability of amorphous solids, the glass transition temperature T g Exceeding the storage temperature T s At least 50°C may be beneficial. By analogy, T m Represents order-disorder transition. T of ABCI-003 m 91.1℃ means T m Accelerated T over 40°C s 50℃.

[0411] As previously mentioned, a reduction in the Chol / PL ratio had a significant impact on the powder properties, increasing the production yield from 74.5% to 82.4%, reducing the Carr index from 41.4 to 27.0, and significantly reducing the inter-particle cohesion as evidenced by a nearly two-fold reduction in MMAD.

[0412] As the Chol content in the powder composition increases, a linear decrease in the enthalpy of the Io phase transition is observed ( Figure 5 ). Regression onto these points shows that the x-intercept (i.e., where the enthalpy is zero) occurs at a Chol / PL ratio of 0.05 w / w. This ratio is used to define Figure 2 The shadow area.

[0413] Example 5: Effect of lipid composition on hygroscopicity of AmB compositions at high relative humidity

[0414] Figure 6 The dynamic vapor sorption characteristics of various compositions are shown. Relative to ABIP, a significant increase in the hygroscopicity of the compositions containing Chol and DSPG is observed. Without intending to be bound by theory, it is believed that the increase in hygroscopicity is due to the presence of NaCl in the composition, which is caused by the interaction of DSPG and calcium chloride. The divalent calcium ions can strongly bind to the anion DSPG Na to displace the sodium ions, which can then interact with the chloride ions from CaCl to form NaCl. Under high RH, the compositions containing DSPG / CaCl deliquesce, and the magnitude of hygroscopicity and deliquesce is proportional to the amount of NaCl formed.

[0415] ABCI-003, in which the PL component contained a HSPC / DSPG ratio of 9.0 w / w, exhibited significantly reduced hygroscopicity compared to ABCI-001 and ABCI-002, in which the HSPC / DSPG ratio was 2.3 (ie, 7 / 3) w / w.

[0416] Example 6: Physicochemical properties of ABCI-001

[0417] Spray-dried powder of ABCI-001 was produced 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, Carr's index, and water content) are detailed in Table 7.

[0418] As a characteristic of the powder spray-dried from the drug suspension, the ABCI-001 powder is enriched with AmB to approximately 10% of the nominal drug content. This enrichment results in a reduction in the Chol / AmB molar ratio to 1.0 mol / mol. Despite the enrichment, AmB and Chol assays were consistent across the five batches, with relative standard deviations (RSDs) of 5% and 1%, respectively. The purity of the introduced AmB drug substance was 96.7%. The purity of AmB was maintained throughout the manufacturing process, with an average purity of 97.2±0.5% for five ABCI-001 batches. In addition, no new degradation peaks were observed in the RP-HPLC chromatogram.

[0419] Table 7. Physicochemical properties of ABCI-001 bulk powder batches

[0420]

[0421]

[0422] The primary particle size distribution obtained by laser diffraction is typical of spray-dried particles from emulsion-based feedstock, where the average X of five batches is 50 The value is 1.9±0.1μm, and the average X 90 is 4.7±0.5μm. Within the batch, X 50 The RSDs varied between 4.7% and 9.6%, with most of the variability coming from the first collector before equilibrium is established in the spray dryer.

[0423] The observed low bulk density (0.071 ± 0.008 g / cm 3 ) and tap density (0.121±0.015g / cm 3) is also unique to powders made from emulsion-based liquid raw materials (Table 7). The high compressibility of the fine, low-density particles is demonstrated by the average Carr's Index value of 41.5 ± 2.0%. This value indicates that the ABCI-001 powder has almost no flowability. The average residual water content in the powder is 3.2 ± 0.2%.

[0424] Example 7: Physicochemical properties of ABCI-002

[0425] The physicochemical properties of ABCI-002 are shown in Table 7. The results are somewhat similar to those presented for ABCI-001. This is not surprising since 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.

[0426] Table 8. Physicochemical properties of ABCI-002 bulk powder batches

[0427]

[0428]

[0429] Example 8: Physicochemical properties of ABCI-003

[0430] The physicochemical properties of ABCI-003 are detailed in Table 9. Relative to ABCI-001, the drug loading has been reduced from 30% w / w to 14% w / w, and the nominal Chol / AmB ratio has been reduced from 1.2 mol / mol to 0.4 mol / mol.

[0431] Table 9. Physicochemical properties of ABCI-003 bulk powder batches

[0432]

[0433] Example 9: Comparison of three ABCI compositions

[0434] Comparison of the physicochemical properties and aerosol performance of the three ABCI compositions is shown in Table 10.

[0435] Table 10. Comparison of physicochemical properties and aerosol performance of ABCI-001, ABCI-002 and ABCI-003

[0436]

[0437]

[0438] As PL / Chol increases from 3.5 to 10.9 to 33.1 w / w (ABCI-001 to ABCI-003), there are several clear trends. First, the yield increases from 74.5% to 82.4% (+10.6%). Second, the Carr Index decreases from 41.4 to 27.0, indicating a significant improvement in powder flowability.

[0439] There were also significant changes in aerosol performance. This may reflect the significant reduction in interparticle cohesion and improved 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 ED increased from 56% to 92%, while the MMAD decreased from 3.9 μm to 2.1 μm. The large reduction in MMAD may also reduce deposition in the rodent nose during nasal-only aerosol delivery.

[0440] Example 10: Improvement of ASL pH after apical application of various compositions of AmB to cultured airway epithelial cells

[0441] A cell-based assay was used to study the efficacy of various lipid-coated AmB crystal compositions. The ASL pH observed for normal lung epithelial cells (NuLi) was approximately 7.6. In contrast, the ASL pH observed in CuFi-1 cells (ΔF508 / ΔF508 CF mutation) was approximately 6.85. Reductions in ASL pH have a significant impact on host defense in CF lungs. ASL pH values ​​greater than 7.0 result in improved effectiveness of defensins (peptides that are critical in host defense with antimicrobial activity in the lungs). Therefore, measurement of ASL pH is a useful proxy for evaluating the effectiveness of different inhaled AmB compositions.

[0442] Table 11 compares the improvement in ASL pH observed for immortalized and primary cultured airway epithelial cells with various AmB compositions. The ASL pH observed for the normal immortalized airway epithelial cell line (NuLi) developed at the University of Iowa was approximately pH 7.5. Two immortalized CF airway epithelial cell lines were studied: CuFi-1 with homozygous ΔF508 / ΔF508 mutations and CuFi-4 cells with heterozygous G551D / ΔF508 mutations. In various studies, the ASL pH measured in these cells varied between approximately pH 6.7 and pH 7.0. Primary cell cultures of airway epithelial cells derived from individuals with CF (including those with nonsense mutations) were also studied. The ASL pH measured in primary airway epithelial cells derived from control subjects without CF has a pH of approximately 6.9 (Shah et al. Airway acidification initiates host defense abnormalities in cystic fibrosis mice. Science 2016, 351: 503-507)).

[0443] Findings from these studies include: AmB compositions without added Chol (e.g., pure AmB, ABIP) were effective at 2 μM concentrations in perfluorohexane vehicles, but ineffective at 50 μM AmB concentrations ( FIG. 7 b ); AmB / Chol complexes exhibited an improvement of ˜0.1 pH units relative to vehicle controls at 2 μM and 50 μM AmB concentrations ( Table 11 ); ASL pH improvements of 0.1 to 0.2 pH units were observed following apical administration of 2 μM AmB to primary cultured airway epithelial cells from subjects with CF, regardless of CFTR genotype ( Table 11 ); L-AMB containing added PL (HSPC / DSPG=7 / 3 w / w) exhibited an ASL pH improvement of ˜0.4 pH units, which was significantly different at 2 μM and 50 μM The results showed that the ASL pH of CuFi-4 cells was maintained at 2 μM and 50 μM AmB concentrations (Table 11); Ivacaftor increased ASL pH by approximately 0.2 pH units in CuFi-4 cells, but had minimal increase in ASL pH in CuFi-1 cells with the homozygous ΔF508 mutation as expected (Table 11); Trikafta brought about an increase in ASL pH similar to ABCI-003 in CuFi-1 cells (Table 11); ABCI compositions comprising a Chol / AmB ratio ≥ 0.4 mol / mol showed the greatest improvement in ASL pH in CuFi-1 cells of approximately 0.4 to 0.5 pH units at both 2 μM and 50 μM AmB concentrations, but ASL pH was not significantly increased at Chol / AmB ratios less than 0.4 mol / mol. The magnitude of improvement in pH was reduced (Fig. 7a; Fig. 7c); improvements of ≥0.2 pH units were observed for Chol / AmB ratios as low as 0.05 mol / mol (Fig. 7b; Fig. 7c); the presence of DSPG in the composition enhanced the improvement in ASL pH in comparison to HSPCs alone (Fig. 7b; Fig. 7c); no significant differences in ASL pH were observed after administration of ABCI-001, ABCI-002, and ABCI-003 (Fig. 7a); the average improvement in ASL pH in primary cultured airway epithelial cells from individuals with CF was ~0.7 pH units, from pH 6.3 to pH 7.0; and a pH of 6.9 or higher was achieved in six of seven primary cultured airway epithelial samples from individuals with CF (i.e., complete restoration of ASL pH relative to healthy volunteers), including two cell cultures derived from individuals with nonsense mutations ( Figure 8 Based on these results, the optimal Chol / AmB ratio of the ABCI composition may be between 0.4 and 1.2 mol / mol ( Figure 2 ).

[0444] Table 11. Improvement of ASL pH in CuFi-1, CuFi-4 and primary epithelial cells from CF patients after administration of AmB compositions

[0445]

[0446]

[0447] 1 US 2019 / 0083517; US 2020 / 0352970; 2 the present disclosure; 3 Shah, 2016

[0448] Example 11: Dose response observed with ABCI-001

[0449] In the case of ABCI-003, the ASL pH improvement was dose-dependent. Fig. 9 The concentrations on the abscissa represent the concentrations of AmB suspended in the perfluorohexane vehicle for application to CuFi-1 cells, not the actual ASL concentrations. The EC based on the perfluorohexane concentration 50 and EC 90 About 0.5 μM and 2.0 μM respectively.

[0450] Example 12: ABCI compositions containing various Chol / AmB ratios and DSPG contents

[0451] Burke et al. demonstrated that an AmB:Chol complex with a Chol / AmB ratio of 5.0 mol / mol maintained improvement in ASL pH in CuFi-1 cells over a suspension concentration range of 2 μM to 50 μM in perfluorohexane (US 2019 / 0083517; US 2020 / 0352970; both incorporated herein by reference).

[0452] Similar results were observed with the lipid-encapsulated AmB compositions of the present disclosure, provided they contained added Chol. At 50 μM AmB concentration, ABIP (which contained no added Chol) showed no improvement in ASL pH.

[0453] In contrast, ABCI compositions with a theoretical Chol / AmB ratio of -1.2 (eg, ABCI-001, ABCI-002) exhibited a significant improvement in ASL pH at 50 μM (eg, 0.35 to 0.45 pH units).

[0454] Previous studies have shown that the benefits achieved by adding Chol may be lost at Chol / AmB ratios below 1.0 mol / mol (US 2019 / 0083517; US 2020 / 0352970). It has been surprisingly found that for ABCI compositions with a theoretical Chol / AmB ratio of 0.4 mol / mol (nominal measured ratio in powder = 0.32 mol / mol), the improvement in ASL pH can be maintained at a 50 μM AmB concentration.

[0455] Thus, while Chol may be necessary to maintain effective improvement in ASL pH over a wide range of delivered doses, Chol / AmB ratios as low as 0.4 mol / mol are still effective. The efficacy observed for lower Chol / AmB ratios was established in a small Design of Experiments (DoE) study ( FIG. 7A ). The study also explored the effect of reducing the percentage of DSPG in the composition. The theoretical anhydrous composition of the spray-dried powder manufactured on a Buchi B-191 spray dryer is detailed in Table 12. The physicochemical properties of the ABCI variants are presented in Table 13. The aerosol performance of selected ABCI variants is presented in Table 14.

[0456] Table 12. Nominal anhydrous compositions of ABCI variants containing lower Chol / AmB ratios and reduced DSPG content

[0457]

[0458] a Batch R21062 contains amorphous AmB particles

[0459] b PL / CaCl2 ratio is 2.0 mol / mol

[0460] Table 13. Physicochemical properties of 14% w / w ABCI variants containing various Chol / AmB ratios and DSPG percentages

[0461]

[0462] Table 14. Aerosol performance indicators for ABCI variants

[0463]

[0464] The inter-particle cohesion tends to decrease as the Chol and DSPG content in the powder decreases. This is reflected in the significant reduction in the coarse particle fraction as shown in the USP throat in the impactor and the deposition on stage 1 and stage 2. The coarse particle fraction decreases from 35.9% in ABCI-001 to 21.1-21.8% in ABCI-002, and to only 6.2% in batch R21058 (ABCI-003). This is further reflected in the shift in the stage deposition pattern. The APSD of ABCI-001 is centered on stages 2 and 3, with an MMAD of 3.9μm. ABCI-002 shows major deposition on stages 3 and 4, with the MMAD shifting down to 2.6 to 2.7μm. Finally, for batch R21058, the Chol / AmB ratio is reduced to 0.4mol / mol and the HSPC content in the PL is increased to 90% w / w, with the major deposition now peaking on stages 4 and 5 of the impactor, with an MMAD of 2.1μm.

[0465] When the data for batch R21058 are expressed as a percentage of the ejected dose, FPF <5μm Considering that 98% of the ejected dose of batch R21058 was deposited on the impactor stage (i.e., only 2% was deposited on the inlet or USP throat), the impaction parameter for the entire unimodal particle size distribution can be calculated (i.e., ). 195μm 2 The value of L / min can be used to predict particle deposition in the upper respiratory tract (URT) and the total lung dose (TLD). The impact parameter results show that about 90% of the ejected dose is expected to be deposited in the lungs, and only about 10% is deposited in the URT. As evidenced by the large percentage of dose deposited in the airway fractions on stages 3 to 5 and the relatively low percentage of particles deposited in the ultrafine fractions on stages 6 to the filter, a large percentage of TLD can be deposited in the large and small airways in the lungs.

[0466] The significant reduction in MMAD can not only reduce URT deposition in humans, but also reduce nasopharyngeal deposition in rats in non-clinical studies. The improvement in ASL pH achieved with various ABCI variants is plotted in Figure 7. Data were collected after 22 hours of incubation at a concentration of 50 μM AmB in a fluorocarbon vehicle (N=12 replicates for each composition). Data were also collected at 2 μM for batch R21058 (N=6 replicates). The ASL pH of ABCI variants with CuFi-1 cells was between about 7.25 and 7.35 (ΔpH~0.4 to 0.5). Therefore, all batches showed a significant increase in ASL pH (p<0.0001 relative to CuFi-1 controls). The magnitude of the improvement in ΔpH observed with the lipid-coated crystal composition was significantly greater than the improvement of ~0.15-0.2 pH units observed with the AmB:Chol complex adopted by Burke et al. (US2019 / 0083517; US 2020 / 0352970).

[0467] Example 13: Evaluation of the relative viscosity of ASL fluid in primary cultured epithelial cells from humans with CF following administration of ABCI-003

[0468] Defective ion transport due to loss or dysfunction of the CFTR protein – Potential defects result in ASL depletion (ie, increased ASL thickness), increased ASL viscosity, and impaired mucociliary clearance.

[0469] The ability of ABCI to reduce the viscosity of ASL fluid in primary cultures of CF airway epithelial cells was evaluated relative to FC-72 delivery vehicle alone. Apical addition of ABCI to primary cultures of CF airway epithelial cells resulted in an average ASL viscosity (τ ASL / τ saline ) values ​​decreased from 3.3-fold to approximately 1.9 to 2.2-fold that of saline; similar activities were observed regardless of CFTR genotype ( Fig.10 ).

[0470] The relative viscosity of ASL in non-CF airway epithelium is 2.5 times that of saline. Therefore, these data demonstrate that ABCI can reduce the relative viscosity of ASL in CF airway epithelium to a level similar to that observed in airway epithelial cells from individuals without CF. Since increased viscosity of ASL is associated with impaired mucociliary clearance and increased risk of infection / inflammation in subjects with CF, these data suggest the potential of compositions according to embodiments described herein to alleviate one of the most serious CF sequelae in the airways.

[0471] Example 14: Antimicrobial killing activity of ASL fluid in primary cultured epithelial cells from humans with CF following administration of ABCI

[0472] Many aspects of the innate host defense system of the airway are pH sensitive, including the rheological properties of secreted mucins, mucociliary clearance, the activity of proteases, and the activity of antimicrobial peptides (e.g., defensins). In primary airway epithelium derived from subjects without CF, ASL reduced the antimicrobial activity of Staphylococcus aureus on gold-coated grids by approximately 50%.

[0473] The effect of apical administration of ABCI-003 on the antimicrobial activity of ASL relative to FC-72 delivery vehicle alone was evaluated in primary cultured airway epithelial cells derived from six subjects with CF with different CFTR mutations. Exposure of CF-derived airway epithelial cells to ABCI-003 increased the percent killing of S. aureus from approximately 22% in the vehicle control to approximately 40% in the high-dose ABCI-003 group (i.e., the antimicrobial activity of ASL nearly doubled to levels similar to those measured in non-CF cells) ( Fig.11 ).

[0474] The CFTR mutation status of the subjects with CF as the source of cells is shown in Fig.11 Notably, responses in cells from two individuals with two nonsense mutations, for whom modulator drugs were ineffective, were at least as high as those with the more common ΔF508 mutation, who were candidates for modulator therapy. Furthermore, improved bacterial killing by ASL at the 50 μM ABCI-003 dose was similar to that of non-CF controls.

[0475] Example 15: Effect of lipid composition and AmB crystallinity on hemolysis

[0476] Toxicity of AMB. Intravenous administration of amphotericin B (Fungizone) causes a number of acute systemic reactions, including fever, shivering, chills, hypotension, hypertension, anorexia, nausea, vomiting, headache, and shortness of breath, leading to its nickname "amphoterrible". Its major chronic side effect is nephrotoxicity. Lipid-based compositions (e.g., L-AMB) reduce the acute systemic effects, but nephrotoxicity remains the dose-limiting toxicity.

[0477] Recent studies from the Burke lab have shown that the toxic effects of AmB in mammalian cells are not the result of ion channel formation as has been assumed, but rather are due to the extraction of cholesterol from the cell membrane into an extramembrane sterol sponge phase (Anderson et al., Nature Chem Biol. 2014; 10:400-406; Gray et al., PNAS. 2012; 109:2234-2239). Toxicity caused by Chol extraction is thought to occur only when the ratio of sterol:AmB is less than ˜1.0 mol / mol.

[0478] The physical form of the API (i.e., amorphous vs. crystalline) and the size of the particles may affect the "apparent" solubility of AmB in water. This in turn may affect the safety profile of the drug after parenteral administration (Ghielmetti et al., J Pharm Sci. 1976; 65: 905-907; Bennett et al., Antimicrob Agents Chemother. 1963; 161: 745-752) and after oral inhalation (US 2012 / 0128728). In the context of the present disclosure, hemolysis assays are used to evaluate the effects of the physical form and compositional attributes of the API on the extraction of Chol from erythrocyte membranes.

[0479] Table 15 compares hemolysis following administration of various compositions comprising AmB. A micellar composition of AMB dissolved in sodium deoxycholate micelles without Chol serves as a positive control, while L-AMB serves as a negative control. ABCI-001, ABCI-002 and ABCI-003 (all comprising highly crystalline AmB and Chol) show hemolysis similar to that of the negative control. Surprisingly, even if the Chol / AmB ratio is significantly less than 1.0, this is also true. For example, ABCI-003, whose theoretical Chol / AmB ratio is only 0.4 mol / mol (actual = 0.33 mol / mol), has hemolysis comparable to that of L-AMB.

[0480] Table 15. Hemolysis of compositions comprising AmB

[0481]

[0482]

[0483] The case of ABIP compositions is instructive. ABIP formulated with fully amorphous AmB was highly hemolytic, whereas ABIP formulated with crystalline drug was not highly hemolytic, suggesting that the physical form or “apparent solubility” of the amorphous phase can enhance efficacy.

[0484] Example 16: Drug Loading and Target Density in Dry Powder Compositions

[0485] Nominal dose (ND), tap density (ρ tapped ) and the container volume of the dry powder inhaler (V r ) can be used to calculate drug loading according to the following equation:

[0486]

[0487] The equation assumes that 50% of the container volume is filled. Optimum performance of the capsule may occur at fill volumes between 25% and 75% of the capsule volume, although fill volumes as low as about 10% and as high as 90% may be used. Too low a fill volume may result in reduced emitted dose (ED) values ​​due to fixation losses in the capsule and device during aerosol administration.

[0488] For a 2 mg nominal dose (ND) (size 3 capsule) of a powder with a tap density of 0.1 g / mL, the %AmB was -13.4% w / w at half-full capsules and 6.7% to 20.1% w / w at 25% to 75% fill volume. Thus, ABCI-003 powder with a tap density of 0.11 g / mL can be well designed to achieve the target 2 mg dose.

[0489] For 0.67 mg Nd at a tap density of 0.1 g / mL, the %AmB is 4.5%, which is 2.25-6.75% w / w when filling 25% to 75% of the volume of a size 3 capsule.

[0490] For a 0.1 mg nominal dose, the drug load required to achieve 50% fill volume is approximately 0.7% (tapped density = 0.1 g / mL).

[0491] Figure 12 provides an estimate of the drug loading requirement based on the nominal dose and tap density of the powder.

[0492] Generally, for a nominal dose of less than about 10 mg, the %AmB varies from 0.1% to 50%, such as 0.5% to 20%. For a nominal dose of about 10 mg, it may be advantageous to adjust the tap density to about 0.2 g / cm 3 , while for lower doses less than 3 mg, it is advantageous to use about 0.1 g / cm 3 The tap density of the spray-dried powder can be controlled by changing the spray drying conditions, the solid content and the concentration of the pore-forming processing aid PFOB in the composition.

[0493] Example 17: Removal of Compositions from Airway Surface Liquids

[0494] Administration of the composition according to the present disclosure by oral inhalation deposits AmB in the ASL on the apical side of the airway epithelial cells, where ion channel formation in the epithelial cell membrane occurs. Considering that only drugs dissolved in the ASL may contribute to ion channel formation, it is desirable to select a dose and regimen that maintains an ASL trough concentration well above the equilibrium solubility of AmB in the ASL (i.e., >0.2 μg / mL).

[0495] Drug particles deposited in the airway lumen after inhaled (IH) administration are distributed from the ASL to other compartments via multiple clearance pathways. These include: (1) absorption of dissolved AmB from the ASL into the systemic circulation; (2) clearance of particles on the mucociliary escalator or by coughing; (3) phagocytosis of particles by circulating and tissue-resident alveolar macrophages, and (4) binding or association of AmB with lung tissue.

[0496] Due to the low solubility of AmB, systemic absorption is dissolution limited, and most of the drug not cleared across the mucociliary escalator will be distributed into the lung tissue where it accumulates over time due to its slow clearance. Similar to intravenous administration, IH administration of AmB results in biphasic elimination kinetics from ASL and plasma due to the slow redistribution of the drug from lung tissue.

[0497] The clearance of ABCI-001 from ASL was evaluated in bronchoalveolar lavage fluid (BALF) samples obtained from Sprague-Dawley rats after IT (study FY21-211A) and IH (study FY21-211B). Both FY21-211A and FY21-211B are non-GLP single-dose studies. Study FY21-211A evaluates IT administration of ABCI-001 and ABIP at a 5 mg / kg delivery dose. Study FY21-211B evaluates three delivery doses of ABCI-001 (6.8, 13.6, and 23 mg / kg) by nasal inhalation only. BALF collection was evaluated at six time points (0.5, 1, 2, 4, 8, and 16 hours after administration) in both studies. The bronchoalveolar lavage procedure significantly dilutes the AmB concentration in the ASL. Urea can be used as an endogenous marker of ASL dilution because the rapid diffusion of urea through the body results in the same concentration in plasma and ASL. This enables the calculation of the ASL volume in BALF samples by simple dilution principles. The AmB concentration in ASL was quantified using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method, while the urea concentration in ASL and plasma was quantified using a colorimetric blood urea nitrogen assay.

[0498] The ASLAmB concentration vs. time curves for the three delivered doses of ABCI-001 (6.8, 13.6, and 23.0 mg / kg) administered in FY21-211B were combined on a single graph, and the results were obtained after intratracheal administration of 5 mg / kg doses of ABCI-001 and AB1P in FY21-211A. For this reason, ASL concentrations were normalized to the 12 mg / kg dose level. Since the dose-normalized ASL concentration vs. time curves for each of the five treatments overlapped significantly and appeared to share similar rates of decline, the aggregated data were fitted with a single exponential decline equation to estimate this initial rate of ASL clearance in rats. The results show that the exposure and initial disposition of AmB in ASL are essentially independent of the dose of ABCI-001 administered and are comparable to ABIP. In addition, the half-life of the initial decline of AmB in ASL after administration of ABCI-001 and ABIP to rats in this study was ~16 hours. This clearance rate of AmB from ASL can support once-daily dosing of the compositions according to the embodiments described herein.

[0499] Example 18: Lung tissue distribution and clearance of AmB in rats and dogs after IH administration of the composition

[0500] After IH administration, a significant proportion of AmB present in ASL is distributed to lung tissue. In a 28-day GLP toxicology study, lung tissue concentrations were measured using ABCI-001 in Sprague-Dawley rats (study FY21-235) and using ABCI-003 in rats (study FY22-071) and using ABCI-003 in beagle dogs (study FY21-234). Lung tissue concentrations were also measured in rats after administering a single dose of ABCI-001 (study FY21-211B) and multiple doses of ABCI-003 (study FY22-070) in non-GLP studies. Average lung tissue concentrations at autopsy are detailed in Table 16.

[0501] Table 16. Lung tissue concentrations of AmB in rats and dogs after IH administration of the composition

[0502]

[0503]

[0504] SD = standard deviation; QW = once a week

[0505] 1 All lung tissue concentrations were quantified 24 h after the last dose, except for measurements on days 42 and 57.

[0506] 2Administered once weekly (QW) as a loading / maintenance dose in Study FY22-071

[0507] After repeated delivery of doses of ≥1.8 mg / kg / days in 14 or 28 days, the average lung concentration of AmB in rats and dogs usually exceeds 1.0 mg / g. The lung concentration in these two species increases with the increase of dosage, but in a disproportionate manner. At the end of repeated administration, the average lung tissue concentration of AmB from ABCI-001 and ABCI-003 in these two species was about 80 to 100 times the reported concentration of AmB in the lung tissue after repeated IV administration of liposomal AmB. The lung tissue concentration observed with ABCI-001 and ABCI-003 and IV administration are suitable for the concentration (1-10 mg / g) measured by L-AMB in liver and spleen. The lung tissue concentrations observed with repeated dosing of ABCI-001 and ABCI-003 (>1.0 mg / g) were significantly greater than those measured after administration of a single dose of ABCI-001 and ABCI-003 (<0.1 mg / g), even at single doses as high as 23.0 mg / kg (Study FY21-211B). The substantial increase in lung tissue levels with repeated dosing is due to accumulation of the nearly insoluble drug substance in the lung. The accumulation ratio AR represents the relationship between the dosing interval and the drug elimination rate at steady state, i.e.: AR = 1 / 1-e -kτ , where k is the terminal elimination rate constant and τ is the dosing interval.

[0508] The t1 / 2 of ABCI-003 measured in lung tissue in FY21-234 in dogs was about 10 days. In the medium and high dose groups, the lung tissue t1 / 2 measured during the ABCI-003 washout in rats (study FY22-071) was 20-25 days. In the 14-day GLP study in dogs, significant accumulation in lung tissue was also observed for ABIP. After stopping dosing, the t1 / 2 of AmB redistribution from lung tissue was 19 days. In the corresponding 14-day GLP study in rats, for the delivered doses of 1.1 and 12.4 mg / kg, the t1 / 2 of redistribution from lung tissue was 22 and 34 days, respectively. These results are consistent with the 1 to 4 weeks t1 / 2 measured for the clearance of AmB from the liver after IV administration. Due to the flip PK (wherein the rate at which the drug enters the ASL or plasma compartment is slower than its absorption or elimination rate), the slow redistribution of the drug from the lung tissue leads to the biphasic clearance of the drug from the ASL and plasma.

[0509] If the terminal t1 / 2 of AmB is assumed to be ~20 days, then k ~ 0.035 days -1For once-daily administration, the AR was equal to 29.4, indicating that a daily dose of AmB would accumulate up to 30-fold in lung tissue at steady state. This degree of accumulation is reflected in the high lung tissue concentrations measured for ABCI-IP, ABCI-001, and ABCI-003 in rats and dogs.

[0510] The lower AmB exposure in lung tissue observed in the FY22-071 study was attributed to the lower delivered dose and once-weekly dosing regimen. For once-weekly dosing of ABCI-003, the AR was equal to 4.6. A loading dose 5 times higher than the maintenance dose can be used to maintain constant lung tissue concentrations during dosing. Considering that our therapeutic target is the ASL, rapidly reaching equilibrium concentrations of AmB in lung tissue is not as important as in the ASL.

[0511] Maintaining a trough concentration in the ASL > 0.2 μg / mL may be important for the efficacy of the compositions described herein. As discussed, the measured t1 / 2 of AmB in the ASL is ~16 hours. This results in an AR of approximately 2 at steady state. Therefore, a loading dose of 2.5 to 3 times the maintenance dose can achieve consistent ASL peak and trough AmB concentrations during administration of the compositions described herein.

[0512] Example 19: Plasma toxicokinetics of AmB in rats and dogs after IH administration

[0513] Plasma AmB elimination in male and female Sprague-Dawley rats was evaluated after inhalation of single or repeated doses of ABCI-001 in a 28-day GLP toxicology study (study FY21-235). The study included three treatment groups, with the average delivered doses of 4.7 mg / kg (low dose), 9.3 mg / kg (medium dose) and 17.3 mg / kg (high dose). Plasma samples were collected before and after administration at 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 24.0 hours, and plasma AmB concentrations were quantified using a validated LC-MS / MS method with a 1 ng / mL lower limit of quantification. Toxicokinetic parameters were determined using Phoenix WinNonlin version 8.3 software.

[0514] Although females consistently showed higher exposures than males, there was limited evidence of differences between the sexes. Therefore, combined means for males and females are presented in Table 17. Due to the low solubility of AmB, systemic absorption of AmB is slow after single or repeated inhalation, with median times to maximum concentrations (t) generally observed between 4 and 8 hours. max) values, regardless of dose, animal sex, and day of administration. Minimal accumulation of AmB in plasma was observed over the 28-day dosing period, with the dose-normalized area under the time-concentration curve (AUC) being proportional to the last measurable concentration (AUC last ) and the maximum concentration (C max ) ranged from 0.699 to 1.51. On Day 1, low, medium, and high dose males passed reporting criteria such that their plasma ABCI-001 elimination t1 / 2 could be estimated to be values ​​of 5.28 to 8.55 hours. On Day 28, the elimination t1 / 2 for low and medium dose females was between 7.16 and 9.33 hours. These values ​​are consistent with the 8 hour t1 / 2 value reported for AmBisome at a 4 mg / kg IV dose in rats with a 24 hour sampling period.

[0515] Table 17. Summary of mean TK parameters in rat plasma on day 1 after ABCI-001 dosing or on day 28 after 28 days of continuous daily dosing for low, medium, and high doses (Study FY21-235)

[0516]

[0517] AUC 0-24hr = area under the time-concentration curve from 0 to 24 hours; Cmax = maximum concentration; t 1 / 2 = Half-life

[0518] At comparable doses, the mean plasma C max The values ​​in the case of IV liposome AmB (AmBisome) were about two orders of magnitude higher than those in IHABCI-001 (Table 18), while the values ​​in lung tissue C max The value is 1 / 83 times. Assuming a lung deposition fraction of 0.1 in rats, the absolute bioavailability of ABCI after systemic absorption for 24 hours on day 1 is about 3%. This is additional evidence that the drug is highly distributed into tissues.

[0519] IH administration provides significant targeting of the drug to the lung, with lung tissue concentrations nearly two orders of magnitude higher than those achieved following IV administration, and plasma concentrations two orders of magnitude lower.

[0520] Table 18. Comparison of plasma and lung tissue exposure of IHABCI-001 and IV AmBisome at comparable doses in Sprague-Dawley rats

[0521]

[0522] Plasma TK in male and female beagles was also evaluated after inhalation of single or repeated doses of ABCI-003 in a 28-day GLP toxicology study (study FY21-234). The study included three dose groups with average delivered doses of: 0.51 mg / kg (low dose), 1.8 mg / kg (medium dose), and 4.9 mg / kg (high dose). Sample collection time, AmB quantification, and non-compartmental data analysis were performed in the same manner as described above for rats.

[0523] There was limited evidence of overt sex differences, so combined means for males and females are presented in Table 19. As in rats, plasma exposure increased in a less than dose proportional manner on days 1 and 28, with AmB concentrations remaining elevated at or near the Cmax level until the 24-hour time point, especially for the mid-dose and high-dose groups. Concentrations were still quantifiable in all animals at necropsy on day 28 and after 14 days of recovery from inhalation on day 42.

[0524] Table 19. Summary of ABCI mean TK parameters (± SD) in dog plasma on Day 1 or Day 28 after a single dose (Study FY21-234)

[0525]

[0526] There was consistent evidence of plasma AmB accumulation in dogs over the 28-day dosing period compared with rats, with the dose-normalized area under the time-concentration curve (AUC 0-24hr ) and C max The mean accumulation ratios of the values ​​ranged from 1.54 to 3.58. This difference in drug disposition may reflect differences in lung anatomy and physiology between the two species. The magnitude of measured plasma concentrations and accumulation observed with ABCI-003 is consistent with previous studies with ABIP.

[0527] Plasma ABCI-003 elimination t1 / 2 could only be estimated for two high-dose female animals in the recovery study and by reporting standards, the values ​​were 103 and 105 hours. As discussed for the IV composition, the t1 / 2 measured using the non-compartmental method is severely affected by the sampling time due to the slow clearance of AmB from the plasma caused by redistribution from tissues. The longer t1 / 2 observed in dogs relative to rats (~100 hours vs. 5-10 hours) is simply a reflection of the longer sampling period (i.e., to day 42 for recovery animals).

[0528] At comparable doses, mean plasma C in dogs was significantly higher than that in IH ABCI-003. max IVAmB deoxycholate The case was about two orders of magnitude higher and in IV liposomal AmB The lung tissue concentrations were 19 to 100 times higher for ABCI-003. Assuming a deposition fraction of 0.25 in dogs, the absolute bioavailability at 24 hours was 0.6%. The low bioavailability observed in rats and dogs is consistent with the low bioavailability observed in and This is consistent with the low systemic toxicity observed with IH administration of and expected for IH of the compositions according to the embodiments described herein.

[0529] Table 20. Comparison of plasma and lung tissue exposure of IH ABCI with IV Fungizone and AmBisome at comparable doses in beagle dogs

[0530]

[0531] Example 20: Toxicology studies in rats and dogs

[0532] Toxicity observed in study FY21-211B (single-dose study in rats). In study FY21-211B, single doses of ABCI-001 of 6.8, 13.6, and 23.0 mg / kg were administered to 54 Sprague-Dawley rats by nasal inhalation only in a non-GLP study. The ABCI-001 composition comprises 30% w / w AmB and a Chol / AmB ratio of 1.2 mol / mol. ABCI-001 was well tolerated, with no abnormal clinical or macroscopic observations at necropsy. AmB exposure in plasma, ASL, and lung tissue tended to increase with increasing dose, although in a less than dose-proportional manner. The measured lung tissue concentrations were 0.010, 0.014, and 0.016 mg / g, respectively. Although a dose-dependent increase in alveolar macrophages was observed, no adverse changes (e.g., an increase in inflammatory cells) were noted in histopathology. The increase in macrophages was attributed to normal particle clearance of the inhaled dry powder. Although a non-GLP study, the 23.0 mg / kg dose supports a single human dose of -46 mg of ABCI with a 10-fold safety margin.

[0533] Toxicity observed in study FY21-235 (28-dGLP study in rats). After a successful single-dose study, a 28-day GLP repeated-dose toxicology study with 14-day recovery was conducted in rats with ABCI-001 (study FY21-235). The slow clearance of the nearly insoluble AmB drug substance resulted in significant accumulation of AmB in lung tissue after daily doses of 4.7, 9.3, and 17.3 mg / kg / day. The lung tissue concentrations of AmB in the low-dose, medium-dose, and high-dose groups were 1.2, 2.0, and 2.9 mg / g, respectively.

[0534] Peak plasma drug concentrations remained less than 55 ng / mL, 2 to 3 orders of magnitude lower than observed after intravenous administration of comparable concentrations of AmB and well below the systemic toxicity threshold of 1000 ng / mL.

[0535] In the low, medium and high dose groups, early death occurred in 4 / 38, 3 / 38 and 13 / 47 animals, respectively. It is speculated that this is due to the large airway obstruction caused by excessive secretion of mucous substances. Some animals in the ABCI-001 treatment group intermittently had respiratory related symptoms (wheezing, palpable rales, shortness of breath) during the administration phase. In the high dose group, the first symptoms appeared on the 4th day and appeared more frequently with continued exposure. However, the correlation between clinical signs and mortality is poor.

[0536] No important differences in body weight, food consumption, hematology, clinical chemistry, and coagulation were observed.

[0537] Histopathological evaluation revealed lesions in the larynx, trachea and lungs of males and females in all dose groups. Found to include minimal to moderate increase of macrophages with yellow / gold pigmentation, minimal to moderate mixed cell inflammation in the larynx, trachea and lungs, minimal to moderate accumulation of mucous material, and minimal to moderate increase of respiratory epithelial cell hyperplasia. Due to significant morbidity and mortality observed in research FY 21-235, it is not possible to determine the dose (noobservable adverse effect level) (NOAEL) that no adverse effects are observed. The lowest dose (lowest adverse effect level) (LOAEL) of adverse effects is the lowest dose studied (4.7mg / kg).

[0538] Study FY22-071 (28-dGLP study in rats). A second 28-day GLP study (study FY22-071) with 14 days of recovery was conducted in Sprague-Dawley rats. This study utilized ABCI-003 and achieved much lower AmB loading in lung tissue. The ABCI-003 composition contained 14% w / w AmB and a Chol / AmB ratio of 0.4 mol / mol. Compared to ABCI-001, ABCI-003 may have reduced inter-particle cohesion due to lipids being maintained in a highly ordered so phase. The three dose groups used a loading dose followed by a weekly maintenance dose. The low, medium, and high doses were: 1.08 / 0.17 mg / kg, 2.54 / 0.41 mg / kg, and 5.74 / 1.06 mg / kg, respectively. For the low, medium, and high dose groups, lung tissue concentrations were 0.019, 0.050, and 0.088 mg / g, respectively.

[0539] Compared with study FY21-235, the in-life portion of study FY22-071 showed no evidence of morbidity or mortality. Gross pathology results were also not significant. Although a slight increase in macrophages was observed, these findings were not accompanied by adverse findings (mucous material, epithelial hyperplasia, or inflammation) noted in study FY21-235. No significant differences in body weight, food consumption, hematology, clinical chemistry, and coagulation were observed. The NOAEL was the highest administered dose, i.e., 5.74 / 1.06 mg / kg.

[0540] Study FY21-234 (28-d GLP study in dogs). Study FY21-234 administered ABCI-003 to beagles at daily doses of 0.5, 1.8, and 4.9 mg / kg. The high dose in dogs was comparable to the low dose in study FY21-235 in rats (where NOAEL was not established). In addition, similar to rats, exposure in lung tissue was high (0.25, 1.38, and 3.54 mg / g). However, compared to the rat study, no morbidity or mortality was observed in dogs at any dose. Clinical signs, body weight, food consumption, clinical pathology (hematology, coagulation, urinalysis), ophthalmology, and ECG parameters were not affected by treatment with ABCI-003. The clinical pathology results on the 29th day at low doses showed no effect. Unfortunately, due to a malfunction of the clinical chemistry analyzer, the data of the medium and high dose groups and the recovery groups (air, placebo control, and high dose) on the 29th day were not evaluable. Concerns that clinical pathology findings might have been missed are unlikely given the known clinical profile of the effects of systemic AmB administration on clinical chemistry at larger magnitude plasma exposures and the fact that no clinical chemistry findings were observed in any other inhalation toxicology studies of this and AmB-related compositions (e.g., ABIP).

[0541] Furthermore, although an increase in the number of pigmented macrophages was observed, there was no evidence of increased inflammation, mucus hypersecretion, or epithelial hyperplasia. Histopathological findings were considered nonadverse and consistent with normal particle clearance from the lung. The NOAEL was the high dose group, i.e., 4.9 mg / kg / day.

[0542] Summary. Without intending to be bound by theory, it is believed that the slow clearance of AmB from lung tissue results in significant accumulation of the drug with daily dosing. Large amounts of undissolved particulate material of any kind within the lungs can lead to adaptive and ultimately adverse lung changes that are simply related to the physical load of foreign material present in the respiratory tract. These changes may be unrelated to any chemical-based or pharmacologically mediated toxicity of the drug.

[0543] As the amount of insoluble material increases, the number of macrophages increases, and excessive macrophage stimulation causes the recruitment of other inflammatory cells and ultimately can produce secondary tissue damage. This response is often referred to as "lung overload." It is important to distinguish it from a decrease in macrophage lung clearance caused by volume overload of less toxic particles (direct macrophage toxicity from compounds such as crystalline quartz that can kill macrophages or impair their function).

[0544] When the concentration of poorly soluble particles (e.g., titanium dioxide, carbon black) in lung tissue exceeds ~1 mg / g, adverse observations related to lung overload occur in rats. Adaptive responses (i.e., an increase in macrophages without an inflammatory response) are generally observed at concentrations below ~0.1 mg / g.

[0545] In study FY21-235, lung concentrations of AmB in the low-, medium-, and high-dose groups were 1.2, 2.0, and 2.9 mg / g, respectively, exceeding the threshold concentration for lung overload in rats. Consistent with the phenomenon of lung overload, these lung loads caused significant morbidity and mortality in all dose groups. This is consistent with other studies demonstrating that rat macrophages are particularly sensitive to overload.

[0546] In study FY22-071, the dose and regimen were adjusted to maintain AmB lung tissue concentrations below the 0.1 mg / g threshold, below which only adaptive macrophage responses were expected. Indeed, only a minor increase in macrophages was observed, with no evidence of toxicity related to lung overload.

[0547] It is also noteworthy that a single dose of ABCI-001 up to 23 mg / kg (study FY21-211B) also resulted in low lung load (0.016 mg / g) and adaptive macrophage responses in rats. This suggests that the adverse effects may not be an acute response to high AmB concentrations on the lung epithelium after administration, but may be caused by the accumulation of the drug in the lung tissue over time.

[0548] The adaptive and adverse reactions observed in rats with the composition at concentrations greater than 1.0 mg / g and less than 0.1 mg / g are consistent with those reported for insoluble compounds such as titanium dioxide and carbon black. These observations support the assertion that the toxicity observed with ABCI-001 in Study FY21-235 was not a chemically or pharmacologically mediated drug effect, but rather was due to a macrophage response associated with lung overload of virtually insoluble particles.

[0549] In general, under similar dosing conditions, rats are much more sensitive to lung overload effects than other species. This is clearly the case with ABCI-003, where a NOAEL was established at a deposition dose of 1.23 mg / kg in dogs, while lung overload was still observed at a deposition dose of about 1 / 3 times (0.47 mg / kg) in rats. Forbes and colleagues summarized the species differences associated with macrophage overload, reporting that rats are more sensitive to this phenomenon than mice or hamsters (Forbes et al. Adv Drug Deliv Rev. 2014, 71, 15-33). Despite the higher fraction deposition and slower clearance rate of poorly soluble particles (e.g., titanium dioxide, coal dust, silicon dioxide), lung overload is not common in humans. The reduced sensitivity seems to be related to anatomical differences (i.e., rats do not have respiratory bronchioles) and physiological differences (i.e., interstitial macrophages take up more particles and transport them to lymphatic vessels). Therefore, toxicity observed in rats may not be predictive of toxicity in humans, and dogs are considered the most appropriate species for evaluating human risk.

[0550] Although rats are a sensitive species, dogs are believed to be a more relevant species for assessing human risk. The NOAEL in dogs (4.9 mg / kg) provides a 12.6-fold safety margin relative to the maximum recommended nominal dose (i.e., 10 mg) in a Phase 1 single ascending dose clinical study in healthy volunteers. In a Phase 1 multiple ascending dose study in healthy volunteers, the margin to the maximum recommended daily dose increased to 31.5 times.

[0551] An alternative way to estimate the safety margin is described in detail below based on AmB exposure in lung tissue. After 28 days of administration of a 10 mg loading dose followed by a 4 mg daily maintenance dose in humans, the expected lung tissue concentration in human subjects is 0.08 mg / g. This is 1 / 12.5 times the lung tissue concentration in rats known to cause lung overload. In addition, it is less than the 0.1 mg / g lung tissue concentration, below which only adaptive responses are observed in rats.

[0552] Example 21: Comparison of Solid State Properties of Development Batches of PLCI-002, ABCI-003, and ABCI-004 with GMP Batches for Early Clinical Development

[0553] Comparable XRPD diffraction patterns were observed for development batches used for GLP toxicology studies and GMP batches used in early clinical development ( Fig.13 ) and the main transition temperature as determined by differential scanning calorimetry ( Fig.14 The results showed that wet-milled amphotericin B crystals retained their crystallinity when embedded in a shell comprising a mixture of phospholipids and cholesterol, which in the case of the dehydrated powder had a main transition temperature (ie, gel to liquid crystal phase transition temperature) exceeding 90°C.

[0554] Example 22: Aerosol Performance of GMP Batches of ABCI-003

[0555] Drum filling method (Harro ) The spray-dried ABCI-003 powder was filled into size 3 HPMC capsules with good precision (RSD < 3%) and accuracy (2 mg dose).

[0556] Capsule retention, device retention and the delivered dose of AmB from the RS01 dry powder inhaler were measured at a pressure drop of 4 kPa and an inhalation volume of 4 L. The average delivered dose was 83.1 ± 2.4%, with capsule and device retention being 6.3 ± 0.27% and 7.0 ± 2.0% (N = 5), respectively. The drug concentration was quantified using the RP-HPLC method.

[0557] The aerodynamic particle size distribution (APSD) of ABCI-003 was measured on a Next Generation Impactor operated under the same conditions as detailed above ( Fig.15 Batch MSV-ENG-129 represented fill development run using a GMP spray drying batch intended for Phase 1. Results from this batch were compared to development batches after 1 month stability under refrigerated conditions.

[0558] Key features of the APSD include: less than 5% deposition on stage 1 in the USP throat and impactor. The unimodal size distribution peaks on stage 4 with significant deposition on stages 3 and 5. The MMAD for the development batch was 2.5 μm, while two runs using clinical material produced an MMAD of 2.8 μm. The fine particle dose less than 5 μm was 69.6% of the nominal dose for the development batch and 66.8% for the clinical batch. The fine particle dose on stage 3 to the filter (a surrogate for the total lung dose) was 71.4% of the nominal dose for the development batch and 70.6% for the clinical batch. The development batch has greater deposition in the device and capsule. Overall, it can be expected that the clinical batches will achieve a total lung dose of 60-70% of the nominal dose and more than 75-85% of the delivered dose.

[0559] Incorporated by reference

[0560] All patents and published patent applications mentioned in the above specification are incorporated herein by reference in their entirety.

[0561] Equivalent

[0562] The present invention has been fully described in considerable detail by way of illustration and example for ease of understanding, but it will be apparent to one of ordinary skill in the art that the present invention may be practiced by modifying or changing the present invention within a broad and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and such modifications or changes are intended to be included within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising: (i) amphotericin B (AmB) or a pharmaceutically acceptable salt or hydrate thereof; (ii) Cholesterol (Chol); (iii) phospholipids comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2).

2. The pharmaceutical composition of claim 1, wherein the amount of AmB is about 0.5% to about 25% w / w.

3. The pharmaceutical composition of claim 2, wherein the amount of AmB is about 3% to about 16% w / w.

4. The pharmaceutical composition according to claim 3, wherein the amount of AmB is about 14% w / w.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the amount of Chol is about 0.1% to about 8% w / w.

6. The pharmaceutical composition of claim 5, wherein the amount of Chol is about 0.3% to about 6% w / w.

7. The pharmaceutical composition of claim 6, wherein the amount of Chol is about 0.5% to about 3% w / w.

8. The pharmaceutical composition of any one of claims 1-7, wherein the amount of CaCl2 is about 1% to about 10% w / w.

9. The pharmaceutical composition of claim 8, wherein the amount of CaCl2 is about 4% to about 7% w / w.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the amount of phospholipids is about 60% to about 95% w / w.

11. The pharmaceutical composition of claim 10, wherein the amount of phospholipids is about 70% to about 90% w / w.

12. The pharmaceutical composition of any one of claims 1-11, wherein the weight ratio of Chol to phospholipid is from about 0.001:1 to about 0.1:

1.

13. The pharmaceutical composition of any one of claims 1-11, wherein the weight ratio of Chol to phospholipid is from about 0.005:1 to about 0.05:

1.

14. The pharmaceutical composition according to any one of claims 1-13, wherein the weight ratio of soybean phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 2:1 to about 19:

1.

15. The pharmaceutical composition of claim 14, wherein the weight ratio of soybean phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is about 7:1 to about 12:

1.

16. The pharmaceutical composition of any one of claims 1-15, wherein the molar ratio of Chol to AmB is from about 0.05:1 to about 1.2:

1.

17. The pharmaceutical composition of claim 16, wherein the molar ratio of Chol to AmB is from about 0.4:1 to about 1.2:

1.

18. The pharmaceutical composition of claim 16, wherein the molar ratio of Chol to AmB is from about 0.05:1 to about 0.4:

1.

19. The pharmaceutical composition of claim 16, wherein the molar ratio of Chol to AmB is about 0.4:

1.

20. The pharmaceutical composition of any one of claims 1-19, wherein the molar ratio of phospholipid to CaCl2 is from about 4:1 to about 2:

1.

21. The pharmaceutical composition of claim 20, wherein the molar ratio of phospholipid to CaCl2 is about 2:

1.

22. The pharmaceutical composition of any one of claims 1-21, wherein the crystallinity of AmB is greater than about 75%.

23. The pharmaceutical composition of claim 22, wherein the crystallinity of AmB is greater than about 85%.

24. The pharmaceutical composition of claim 23, wherein the crystallinity of AmB is greater than about 95%.

25. The pharmaceutical composition of claim 1, comprising, consisting essentially of, or consisting of: (i) about 14.0% w / w amphotericin B (AmB); (ii) about 2.3% w / w cholesterol (Chol); (iii-a) about 70.3% w / w hydrogenated soy phosphatidylcholine (HSPC); (iii-b) about 7.8% w / w distearoylphosphatidylglycerol (DSPG); and (iv) about 5.52% w / w calcium chloride (CaCl2).

26. The pharmaceutical composition of claim 1, comprising, consisting essentially of, or consisting of: (i) about 14.0% w / w amphotericin B (AmB); (ii) about 6.81% w / w cholesterol (Chol); (iii-a) about 51.2% w / w hydrogenated soy phosphatidylcholine (HSPC); (iii-b) about 22.8% w / w distearoylphosphatidylglycerol (DSPG); and (iv) about 5.2% w / w calcium chloride (CaCl2).

27. The pharmaceutical composition of claim 1, comprising, consisting essentially of, or consisting of: (i) about 3.4% w / w amphotericin B (AmB); (ii) about 0.57% w / w cholesterol (Chol); (iii-a) about 80.72% w / w hydrogenated soy phosphatidylcholine (HSPC); (iii-b) about 8.97% w / w distearoylphosphatidylglycerol (DSPG); and (iv) about 6.34% w / w calcium chloride (CaCl2).

28. The pharmaceutical composition according to claim 1, comprising: (i) a Chol / AmB ratio of about 0.4 to 1.2 mol / mol; (ii) a Chol / PL ratio of less than about 0.05 w / w; (iii) a HSPC / DSPG ratio of about 2.3 to about 9.0 w / w; and (iv) PL / Ca of about 2:1 mol / mol 2+ Compare.

29. The pharmaceutical composition of any one of claims 1-28, wherein AmB and Chol are not complexed; and AmB is not encapsulated in liposomes.

30. The pharmaceutical composition according to any one of claims 1-29, wherein AmB is coated with a porous shell of phospholipids and Chol.

31. The pharmaceutical composition of any one of claims 1-30, wherein the pharmaceutical composition is formulated as a dry powder.

32. The pharmaceutical composition according to claim 31, wherein the mass median diameter X of the powder particles is 50 From about 1.0 to about 4.0 μm.

33. The pharmaceutical composition according to claim 32, wherein the mass median diameter X of the powder particles is 50 From about 1.5 to about 3.5 μm.

34. The pharmaceutical composition of claim 33, wherein the powder particles have a tap density of about 0.03 to about 0.4 g / mL.

35. The pharmaceutical composition of claim 34, wherein the powder particles have a tap density of about 0.06 to about 0.2 g / mL.

36. The pharmaceutical composition of any one of claims 31-35, wherein the powder particles have a Carr's Index of about 20 to about 32.

37. A pharmaceutical composition according to any one of claims 31-36, wherein the main transition temperature (T m ) is at least 80°C.

38. The pharmaceutical composition of any one of claims 31-37, wherein the powder has a water content of about 1.5 to about 6% w / w.

39. The pharmaceutical composition of any one of claims 1-38, wherein the composition is formulated for pulmonary or airway administration.

40. The pharmaceutical composition of claim 39, wherein the composition is formulated for aerosol administration.

41. The pharmaceutical composition of any one of claims 31-40, wherein the mass median aerodynamic diameter of the powder particles is from about 1.5 μm to about 4.0 μm.

42. The pharmaceutical composition of claim 41, wherein the powder particles have a mass median aerodynamic diameter of about 2.0 μm to about 3.5 μm.

43. A method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to any one of claims 1-42; wherein the administration is pulmonary administration.

44. The method of claim 43, wherein the disease or disorder is cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease.

45. The method of claim 44, wherein the disease or disorder is cystic fibrosis.

46. ​​A method of increasing the pH of airway surface fluid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any one of claims 1-42, thereby increasing the pH of the airway surface fluid of the subject; wherein the administration is pulmonary administration.

47. A method of increasing bicarbonate secretion into airway surface fluid, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of any one of claims 1-42, thereby increasing bicarbonate secretion into the airway surface fluid of the subject; wherein the administration is pulmonary administration.

48. A method of increasing forced expiratory volume in one second (FEV1), comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any one of claims 1-42, thereby increasing the subject's FEV1; wherein the administration is pulmonary administration.

49. The method of claim 48, wherein the subject's FEV1 is increased by about 3% to about 20%.

50. The method of any one of claims 43-49, wherein the subject has cystic fibrosis, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease.

51. The method of claim 50, wherein the subject has cystic fibrosis.

52. The method of any one of claims 43-51, wherein the composition is administered to the airway of a subject.

53. The method of any one of claims 43-52, wherein the composition is administered as an aerosol.

54. The method of any one of claims 43-53, wherein the subject is a human less than 12 years old.

55. The method of any one of claims 43-53, wherein the subject is a human who is at least 12 years old.

56. The method of any one of claims 43-55, wherein the cystic fibrosis is refractory to ivacaftor.

57. The method of any one of claims 43-56, wherein the cystic fibrosis is refractory to a combination of elexacaftor, tezacaftor, and ivacaftor.

58. The method of any one of claims 43-57, wherein the nominal or metered dose of the pharmaceutical composition is 0.01 mg to 10 mg.

59. The method of claim 58, wherein the nominal or metered dose of the pharmaceutical composition is 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, or 4.0 mg.

60. The method of any one of claims 43-59, wherein the composition is administered once daily.

61. The method of any one of claims 43-60, wherein the administering comprises initially delivering a loading dose of the pharmaceutical composition; followed by delivering a maintenance dose of the pharmaceutical composition; wherein the mass ratio of the loading dose to the maintenance dose is about 2:1 to about 5:

1.

62. The method of claim 61, wherein the mass ratio of the loading dose to the maintenance dose is about 2.5:1 to about 3:

1.

63. The method of any one of claims 43-62, wherein the absolute bioavailability of AmB is about 0.1% to about 5%.

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