Ion channel prosthetic composition comprising lipid-coated crystals of amphotericin B - Patent Application 20070229933

Abstract

A pharmaceutical composition is disclosed that includes (i) amphotericin B, or a pharma- ceutically acceptable salt or hydrate thereof; (ii) cholesterol; (iii) a phospholipid including hydrogenated soy phosphatidylcholine and distearoylphosphatidylglycerol; and (iv) calcium chloride (CaCl2). Also disclosed are methods of using the pharmaceutical composition to treat a disease or disorder, such as cystic fibrosis, to increase the pH of airway epithelial fluid, to increase bicarbonate secretion into airway epithelial fluid, and to increase forced forced expiratory volume in one second in a subject.

Description

Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 321,965, filed March 21, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0002] Cystic fibrosis (CF) is a progressive genetic disease that causes persistent lung infections and limits breathing ability over time. CF symptoms are caused by a defective protein known as the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Researchers are investigating possible therapies to restore proper function of the CFTR protein or to modify its production process so that normal protein is made. Current CFTR modulators and correctors improve lung function in approximately 90% of CF patients, but approximately 10% of patients have class I mutations or other mutations for which there are no effective treatments.

[0003] Burke and coworkers 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 monovalent ion channels. AmB-Chol complexes were prepared from solutions in organic solvents by flash nanoprecipitation in water (Patent Document 1; Patent Document 2). The resulting nanoparticles were then freeze-dried. Burke et al. claimed that ion channels formed from Chol and AmB (Chol / AmB=1.0-50.0 mol / mol) allowed the restoration of bicarbonate secretion, the increase of airway surface liquid (ASL) pH, and the improvement of host defense in CF cell lines (e.g., CuFi-1 cell line, CuFi-4, obtained from the University of Iowa), independent of genetic mutations. They also demonstrated that pre-decomposition of AmB with cholesterol increased the concentration range over which AmB was effective in restoring the pH of ASL: pure AmB was only effective at low AmB concentrations, whereas the AmB:Chol complex maintained activity over a wide range of AmB concentrations.

[0004] The above benefits of AmB were further established in differentiated primary cultures of human airway epithelium from CF patients with various CFTR mutations, including those resulting in no CFTR at all. Increased ASL pH was also observed in a CFTR-null pig model.

[0005] Finally, intranasal administration of Fungizone®, a commercially available parenteral composition of AmB, was able to alter the nasal transepithelial potential difference in CF patients, including those who cannot be treated with current therapies. The observed improvement in nasal transepithelial potential difference was comparable to that observed with modulators such as Ivacaftor.

[0006] Establishment of AmB / Chol ion channels in CF patients requires delivery of these substances to the apical side of airway epithelial cells, i.e., by oral inhalation. Similar challenges are observed in subjects with non-cystic fibrosis bronchiectasis bronchiectasis (NCFBE) and chronic obstructive pulmonary disease (COPD). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Patent Application Publication No. 2019 / 0083517 [Patent Document 2] US Patent Application Publication No. 2020 / 0352970 Summary of the Invention [Means for solving the problem]

[0008] In certain aspects, provided herein is a pharmaceutical composition comprising: (i) amphotericin B (AmB) or a pharma- ceutically acceptable salt or hydrate thereof; (ii) cholesterol (Chol); (iii) a phospholipid (PL) comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and (iv) calcium chloride (CaCl2).

[0009] In a further aspect, provided herein is a method of treating a disease or disorder comprising administering to a subject in need of treatment a therapeutically effective amount of the pharmaceutical composition, wherein the administration is pulmonary administration.

[0010] In a further aspect, provided herein is a method of increasing the pH of airway surface liquid comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition, thereby increasing the pH of airway surface liquid in the subject, wherein the administration is pulmonary administration.

[0011] In a further aspect, provided herein is a method of increasing bicarbonate secretion into airway surface liquids, comprising increasing bicarbonate secretion into airway surface liquids in a subject in need thereof by administering to said subject an effective amount of said pharmaceutical composition, said administration being pulmonary.

[0012] In a further aspect, provided herein is a method for increasing a subject's forced expiratory volume in one second (FEV1), comprising increasing the FEV1 in the subject by administering to the subject an effective amount of the pharmaceutical composition, wherein the administration is pulmonary administration. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows a phase diagram of a fully hydrated dipalmitoylphosphatidylcholine-cholesterol mixture. [Diagram 2] 1 shows a ternary phase diagram depicting various AmB / lipid compositions. The shaded triangles represent optimized lipid-coated crystal compositions of amphotericin B. [Diagram 3] 1 shows differential scanning calorimetry thermograms of amphotericin B inhalation powder (ABIP), ABCI-001, ABCI-002, and ABCI-003. [Figure 4] 1 shows X-Ray Powder Diffraction (XRPD) plots showing evidence of cholesterol crystallites in PLCI-001 and PLCI-002. [Diagram 5]1 shows a plot illustrating that the enthalpy of the Chol-rich phase is proportional to the Chol content. [Figure 6] 1 shows moisture sorption isotherms (25° C.) of various amphotericin B (AmB) and placebo compositions. [Figure 7A] FIG. 1 shows plots showing the increase in airway surface liquid (ASL) pH in CuFi-1 cells for various compositions containing HSPCs with Chol / AmB ratios ranging from 0.4 to 1.2 mol / mol and 70 w / w% to 90 w / w% of PL. [Figure 7B] 1 shows a plot illustrating the increase in pH of ASL in CuFi-1 cells for various compositions with Chol / AmB ratios ranging from 0 to 0.4 mol / mol, including compositions without Chol or DSPG. [Figure 7C] 13 shows plots depicting the effect of varying the ratio of Chol / AmB on the pH improvement of ASL in CuFi-1 cells. [Figure 8] 1 shows a plot showing the increase in pH of ASL following administration of ABCI-003 to primary cultured airway epithelial cells from individuals with CF, including those with nonsense mutations. [Figure 9] 1 shows a plot showing the increase in ASL pH in CuFi-1 for ABCI-003 as a function of increasing AmB concentration suspended in a perfluorohexane (FC-72) vehicle. [Figure 10] 1 shows plots demonstrating the reduction in viscosity of ASL following administration of ABCI-003 to primary cultured airway epithelial cells from individuals with CF, including those with nonsense mutations. [Figure 11] 1 shows plots demonstrating increased antibacterial activity of ASL following administration of ABCI-003 to primary cultured airway epithelial cells from individuals with CF, including those harboring nonsense mutations. [Figure 12]Figure 12a shows a plot illustrating the effect of nominal density and tapped density of spray dried powder compositions on the calculated drug loading for the bulk powder at a nominal dose range of 0 to 10 mg, and Figure 12b shows a plot illustrating the effect of nominal density and tapped density of spray dried powder compositions on the calculated drug loading for the bulk powder at a nominal dose range of 0 to 3 mg. [Figure 13] 1 shows an overlay of powder X-ray diffraction patterns of GMP and GLP batches of ABCI-003, ABCI-004, and PLCI-002. [Figure 14-1] 1 shows an overlay of DSC thermograms of GMP and GLP batches of ABCI-003, ABCI-004, and PLCI-002. [Figure 14-2] Continued from Figure 14-1. [Figure 15] The aerodynamic particle size distribution (APSD) of ABCI-003 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Surprisingly, it was discovered that it is not necessary to complex AmB with Chol to achieve the benefits summarized above: in fact, these two components can be phase separated into distinct domains within the core-shell particle.

[0015] Also surprisingly, it was discovered that improved host defense, including improved bicarbonate secretion, ASL pH, ASL viscosity, and / or ASL antibacterial activity, can be maintained at Chol / AmB ratios much lower than 1.0 mol / mol.

[0016] composition In some aspects, provided are: (i) amphotericin B (AmB), or a pharma- ceutically acceptable salt or hydrate thereof; (ii) Cholesterol (Chol); (iii) phospholipids; and (iv) Calcium chloride (CaCl2) A pharmaceutical composition comprising:

[0017] In a further aspect, there is provided: (i) amphotericin B (AmB), or a pharma- ceutically acceptable salt or hydrate thereof; (ii) Cholesterol (Chol); (iii) a phospholipid comprising hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), or dipalmitoylphosphatidylcholine (DPPC); and distearoylphosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2) A pharmaceutical composition comprising:

[0018] In a further aspect, there is provided: (i) amphotericin B (AmB), or a pharma- ceutically acceptable salt or hydrate thereof; (ii) Cholesterol (Chol); (iii) a phospholipid comprising hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl2) A pharmaceutical composition comprising:

[0019] In certain embodiments, the pharmaceutical composition comprises about 0.5% w / w to about 25% w / w AmB.

[0020] In certain embodiments, the pharmaceutical composition comprises about 1% w / w to about 22% w / w AmB.

[0021] In certain embodiments, the pharmaceutical composition comprises about 2 w / w% to about 16 w / w% AmB.

[0022] In certain embodiments, the pharmaceutical composition comprises about 14 w / w% AmB.

[0023] In a specific embodiment, the pharmaceutical composition comprises about 0.1 w / w% to about 8 w / w% Chol.

[0024] In a specific embodiment, the pharmaceutical composition comprises about 0.5 w / w% to about 3 w / w% Chol.

[0025] In a specific embodiment, the pharmaceutical composition comprises about 0.3 w / w% to about 6 w / w% Chol.

[0026] In a specific embodiment, the pharmaceutical composition comprises about 0.5 w / w% to about 3 w / w% Chol.

[0027] In certain embodiments, the pharmaceutical composition comprises about 1% w / w to about 10% w / w CaCl2.

[0028] In certain embodiments, the pharmaceutical composition comprises about 4% w / w to about 7% w / w CaCl2.

[0029] In certain embodiments, the pharmaceutical composition comprises about 60% to about 95% w / w of phospholipids.

[0030] In certain embodiments, the pharmaceutical composition comprises about 70 w / w% to about 90 w / w% phospholipid.

[0031] In certain embodiments, the pharmaceutical composition comprises about 75% to about 90% w / w of phospholipids.

[0032] In a particular embodiment, the weight ratio of Chol to phospholipid is from about 0.001:1 to about 0.1:1.

[0033] In a particular embodiment, the weight ratio of Chol to phospholipid is about 0.005:1 to about 0.05:1.

[0034] In certain embodiments, the weight ratio of soybean phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is from about 2:1 to about 19:1.

[0035] In certain embodiments, the weight ratio of soybean phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is from about 7:1 to about 12:1.

[0036] In certain embodiments, the weight ratio of soybean phosphatidylcholine (HSPC) to distearoylphosphatidylglycerol (DSPG) is from about 1:1 to about 3:1.

[0037] In certain embodiments, the molar ratio of Chol to AmB is from about 0.05:1 to about 1.2:1.

[0038] In certain embodiments, the molar ratio of Chol to AmB is from about 0.4:1 to about 1.2:1.

[0039] In certain embodiments, the molar ratio of Chol to AmB is from about 0.05:1 to about 0.4:1.

[0040] In certain embodiments, the molar ratio of Chol to AmB is about 0.4:1.

[0041] In certain embodiments, the molar ratio of phospholipid to CaCl 2 is from about 4:1 to about 2:1.

[0042] In certain embodiments, the molar ratio of phospholipid to CaCl2 is about 2:1.

[0043] In certain embodiments, the crystallinity of AmB is greater than about 75%.

[0044] In certain embodiments, the crystallinity of AmB is greater than about 85%.

[0045] In certain embodiments, the crystallinity of AmB is greater than about 95%.

[0046] In certain embodiments, the pharmaceutical composition comprises: (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) Calcium chloride (CaCl2) at about 5.52 w / w% Comprises, consists essentially of, or consists of.

[0047] In certain embodiments, the pharmaceutical composition comprises: (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) Calcium chloride (CaCl2) at approximately 5.2 w / w% Comprises, consists essentially of, or consists of.

[0048] In certain embodiments, the pharmaceutical composition comprises: (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) Calcium chloride (CaCl2) at about 6.34 w / w% comprises, consists essentially of, or consists of.

[0049] In certain embodiments, the pharmaceutical composition comprises: (i) Chol / AmB ratio of about 0.4–1.2 mol / mol; (ii) a Chol / PL ratio of less than about 0.05 w / w; (iii) an HSPC / DSPG ratio of about 2.3 to about 9.0 w / w; and (iv) PL / Ca ratio of about 2:1 mol / mol 2+ ratio has.

[0050] In certain embodiments, AmB and Chol are not complexed; AmB is not encapsulated in a liposome.

[0051] In certain embodiments, AmB is coated with a porous shell of phospholipids and Chol.

[0052] In certain embodiments, the pharmaceutical composition is formulated as a dry powder.

[0053] In certain embodiments, the tap density of the powder particles is from about 0.03 to about 0.4 g / mL.

[0054] In certain embodiments, the tap density of the powder particles is from about 0.06 to about 0.2 g / mL.

[0055] In certain embodiments, the powder particles have a Carr flowability index of about 20 to about 32.

[0056] 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 about 50°C to about 100°C, about 70°C to about 100°C, about 80°C to about 100°C, or about 85°C to about 95°C.

[0057] In certain embodiments, the water content of the powder is from about 1.5 to about 6 w / w%.

[0058] In certain embodiments, the compositions are formulated for pulmonary or airway administration.

[0059] In certain embodiments, the compositions are formulated for aerosol administration.

[0060] In certain embodiments, the compositions are formulated for aerosol administration as a dry powder.

[0061] In certain embodiments, a dry powder composition of artificial particles is provided comprising a plurality of AmB drug particles coated with a porous shell of phospholipid (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%.

[0062] In certain embodiments, the mass median diameter X of the powder particles 50 is about 0.5 μm to about 5.0 μm, about 1.0 μm to about 4.0 μm, or about 1.5 μm to about 3.5 μm.

[0063] In certain embodiments, the X of the powder particles 90 is about 1.5 μm to about 10.0 μm, for example, about 2.0 μm to about 8.0 μm.

[0064] In certain embodiments, the powder particles have a mass median aerodynamic diameter (MMAD) of about 1.5 μm to about 4.0 μm.

[0065] In certain embodiments, the powder particles have a mass median aerodynamic diameter (MMAD) of about 2.0 μm to about 3.5 μm.

[0066] In certain embodiments, a carrier-free dry powder composition of AmB, Chol, and PL is provided, in which AmB is not encapsulated in lipids, and AmB and lipids are each phase separated into unique domains.

[0067] In certain embodiments, a dry powder composition of artificial particles is provided comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein the Chol / AmB ratio is from about 0.05 to about 1.2 mol / mol, e.g., from about 0.2 to about 0.6 mol / mol.

[0068] In certain embodiments, a dry powder composition of artificial particles is 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.

[0069] In certain embodiments, a dry powder composition of artificial particles is provided that includes a plurality of AmB drug particles coated with a porous shell comprising PL and Chol, in some embodiments, the PL comprises hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylglycerol (DSPG), or a combination thereof.

[0070] In certain embodiments, a dry powder composition of artificial particles is provided comprising a plurality of AmB drug particles coated with a porous shell of PL and Chol, wherein the PL comprises a mixture of (1) HSPC or DSPC and (2) DSPG in a w / w ratio of about 2.3 w / w (i.e., 7 / 3 w / w) and about 19.0 w / w (i.e., 95 / 5 w / w), for example, from about 8 w / w to about 18 w / w.

[0071] In certain embodiments, a dry powder composition of artificial 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 shall not fall below about 2.0 mol / mol.

[0072] In certain embodiments, a dry powder composition of artificial 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 said composition is less than 60 w / w%, e.g., less than 30 w / w% or less than 20 w / w%. In some embodiments, the drug loading is about 0.5 w / w% to about 25 w / w%, and the nominal Chol / AmB ratio is about 0.05 to about 1.2 mol / mol.

[0073] In a particular embodiment, a dry powder composition of artificial particles is provided (ABCI-003), comprising spray-dried core-shell particles of microcrystalline AmB particles coated with a porous shell of PL and Chol (about 14.0 w / w%), 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.

[0074] In a particular embodiment, a dry powder composition of artificial particles is provided (ABCI-004), comprising spray-dried core-shell particles of microcrystalline AmB particles coated with a porous shell of PL and Chol (about 3.4 w / w%), 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.

[0075] In certain embodiments, a dry powder composition of artificial particles is provided comprising spray-dried core-shell particles (about 14.0 w / w%) of microcrystalline AmB particles coated with a porous shell of PL and Chol (FIG. 2), 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 PL is about 2.3 to about 9.0 w / w.

[0076] In certain embodiments, the maximum Chol / AmB ratio is about 1.2 mol / mol, but this high ratio may be acceptable only at relatively low drug loadings (e.g., 10.0 w / w% or less) where the lipids are maintained in the highly ordered So phase. Lowering Chol / AmB to 0.4 mol / mol allows for higher drug loadings (e.g., 22 w / w% or less) in the So phase.

[0077] In some embodiments, the compositions described herein occupy the shaded triangle in Figure 2. Two sides of the triangle represent the lines of nominal Chol / AmB ratios of 0.4 mol / mol and 1.2 mol / mol, and the third side represents a 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).

[0078] In certain embodiments, the compositions described herein have a HSPC / DSPG ratio of about 2.3 to about 9.0 w / w, PL / Ca 2+ The ratio is about 2.0 mol / mol.

[0079] In certain embodiments, the compositions described herein are selected to be single-phase (i.e., have a T of greater than 50° C., which is greater than the accelerated storage temperature of 40° C.). mIn some embodiments, compositions described herein are selected for their ability to maintain lipids in a gel phase (So) having a pH of 0.1 to 10 (Example 4)). In some embodiments, compositions described herein are selected for their ability to maximize the increase in pH of ASL maintained over a wide range of AmB concentrations (Example 11). In certain embodiments, compositions described herein are selected for their reduced hygroscopicity (Example 5) compared to compositions with HSPC / DSPG<9.0. In some embodiments, compositions described herein are selected for their ability to improve manufacturing yield (Example 9). In certain embodiments, compositions described herein are selected for their ability to improve powder flowability (Example 9). In some embodiments, compositions described herein are selected for their improved aerosol performance (Example 9) compared to powders containing a Lo phase. In certain embodiments, compositions described herein are selected for their superior ability to restore host defense in cell-based assays in both CuFi-1 cells and primary cultures from CF patients, including those with nonsense mutations (Example 10). In some embodiments, the compositions described herein are selected to avoid red blood cell hemolysis even at low Chol / AmB molar ratios (Example 15).

[0080] In certain embodiments, a dry powder composition of artificial 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.

[0081] In certain embodiments, a dry powder composition of artificial 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, for example, about 1.5 to about 4.0 μm.

[0082] In certain embodiments, a dry powder composition of artificial particles is provided, comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein X of said particles 90 is about 3 μm to about 10 μm, for example, about 3.5 μm to about 7 μm.

[0083] In certain embodiments, a dry powder composition of artificial 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, e.g., about 0.06 to about 0.20 g / mL.

[0084] In certain embodiments, a dry powder composition of artificial particles is provided comprising a plurality of AmB drug particles coated with a porous shell of PL, Chol, and calcium chloride (CaCl2), wherein the moisture content of the powder is from about 1.0% to about 10.0%, preferably from about 2.0% to about 5.0%.

[0085] In certain embodiments, a dry powder composition of artificial 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 aerodynamic diameter (MMAD) when administered from a handheld dry powder inhaler is between about 1.0 μm and about 6.0 μm, e.g., between about 2.0 μm and about 4.0 μm.

[0086] In certain embodiments, a dry powder composition of artificial 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 using a handheld dry powder inhaler, the fine particle fraction less than 5 μm expressed as a percentage of the nominal dose is at least 30 w / w%, at least 50 w / w%, or at least 60 w / w%.

[0087] In certain embodiments, a dry powder composition of artificial 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 a liquid feedstock comprising minute AmB crystals suspended in an oil-in-water emulsion stabilized by a monolayer of a mixture of lipids as described herein.

[0088] In certain embodiments, a dry powder composition of artificial 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 lipid main transition temperature (T m ) is at least 80°C, for example at least 90°C.

[0089] In certain embodiments, a dry powder composition of artificial 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 has a minimum T m In some embodiments, the outlet temperature is at least 50° C., at least 60° C., or at least 70° C.

[0090] In certain embodiments, a dry powder composition of artificial 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 has a minimum T m In some embodiments, the outlet temperature is at least 60° C., or at least 70° C.

[0091] In certain embodiments, a dry powder composition of artificial 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 liquid feedstock in a PSD-1 scale spray dryer with a total gas flow rate of about 70 to about 100 scfm.

[0092] In a particular embodiment, the above dry powder composition of artificial particles, comprising AmB drug particles coated with a porous shell of PL, Chol and calcium chloride (CaCl 2 ), is packed by a drum filler.

[0093] In certain embodiments, the powder fill mass is from about 1.0 mg to about 40 mg, for example, 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.

[0094] In certain embodiments, the powder fill mass has good precision (eg, RSD<3%) and accuracy relative to the target fill mass.

[0095] method In certain aspects, provided is a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein, said administering being pulmonary. In certain embodiments, said disease or disorder comprises cystic fibrosis, non-cystic fibrosis bronchiectasis (NCFBE), or chronic obstructive pulmonary disease (COPD). In certain embodiments, said disease or disorder is cystic fibrosis.

[0096] Provided are methods for restoring the host defense properties of ASL (e.g., increasing bicarbonate secretion, pH, antibacterial activity, and / or decreasing viscosity), comprising improving lung function and quality of life by administering to a subject in need of restoring the host defense properties of ASL a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein.

[0097] Also provided is a method of increasing the pH of airway surface liquid, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any one of the embodiments described herein, thereby increasing the pH of airway surface liquid in said subject, said administration being pulmonary administration.

[0098] Also provided is a method of increasing bicarbonate secretion into airway surface liquids, comprising increasing bicarbonate secretion into airway surface liquids in a subject in need thereof by administering to said subject an effective amount of a pharmaceutical composition according to any one of the embodiments described herein, said administration being pulmonary administration.

[0099] Also provided is a method for increasing forced forced expiratory volume in one second (FEV1), comprising increasing FEV1 in a subject in need thereof by administering to the subject an effective amount of a pharmaceutical composition according to any one of the embodiments described herein, wherein the administration is pulmonary administration.

[0100] In some embodiments, a subject's FEV1 is said to be "increased" if it is measurably greater than the FEV1 of the same subject measured prior to treatment with the methods of the present disclosure or at a time distant from treatment with the methods of the present disclosure.

[0101] 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%.

[0102] In certain embodiments, the subject is afflicted with CF.

[0103] In certain embodiments, the pharmaceutical composition is administered to the respiratory tract of the subject.

[0104] In certain embodiments, the pharmaceutical composition is administered to the bronchial airways of the subject.

[0105] In certain embodiments, the pharmaceutical composition is administered as an aerosol.

[0106] In certain embodiments, the pharmaceutical composition is administered as a dry powder aerosol.

[0107] In certain embodiments, the subject is a human.

[0108] In certain embodiments, the subject is an adult.

[0109] In certain embodiments, the subject is a human under the age of 12.

[0110] In certain embodiments, the subject is a human who is at least 12 years of age.

[0111] In certain embodiments, the subject is a human who is at least 4 years of age.

[0112] In certain embodiments, the CF is refractory to treatment with Ivacaftor, or a combination of Elexacaftor, Tezacaftor, and Ivacaftor.

[0113] In certain embodiments, subjects with CF have Class 1 or other mutations that are not effectively treated with current modulators, including combination therapies, including nonsense mutations or other mutations that result in the failure of a functional CFTR protein to be produced.

[0114] In certain embodiments, the subject has two mutations in the CF Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel, wherein the two mutations are each independently selected from the mutations listed in Table 1.

[0115] [Table 1] TIFF2025510096000002.tif235168TIFF2025510096000003.tif120168

[0116] In certain embodiments, the two mutations are independently selected from 2184delA, F508del, V520F, 1717-1G->A, E60X, G551D, R553X, and D259G.

[0117] 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.

[0118] 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.

[0119] In certain embodiments, the nominal or metered dose of the pharmaceutical composition is between 0.01 mg and 10 mg.

[0120] 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.

[0121] In certain embodiments, the composition is administered at least once a day, at least twice a day, or at least three times a day.

[0122] In certain embodiments, the composition is administered once a day.

[0123] In certain embodiments, the composition is administered at least once a week, at least twice a week, or at least three times a week.

[0124] In certain embodiments, the administration comprises initially delivering a loading dose of the pharmaceutical composition and subsequently delivering a maintenance dose of the pharmaceutical composition, the mass ratio of the loading dose to the maintenance dose being 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.

[0125] All masses stated above for the loading dose and maintenance dose refer to the mass of the nominal or metered dose (ie, not the emitted dose) of the pharmaceutical composition.

[0126] In certain embodiments, provided is a method of treating a subject suffering from CF, NCFBE, or COPD, comprising administering to a subject in need of treatment an effective amount of a lipid coated crystal composition comprising AmB and Chol, as provided herein, wherein the lipid coated crystal composition is administered to the subject by oral inhalation into the bronchial airways of the lungs.

[0127] In certain embodiments, provided is a method of treating a subject suffering from CF, NCFBE, or COPD, comprising administering to a subject in need of treatment an effective amount of a lipid coated crystal composition comprising AmB and Chol, as provided herein, wherein the composition provides an increase in the pH of ASL of about 0.2 to about 1.0 pH units.

[0128] In certain embodiments, provided is a method of treating a subject suffering from CF, NCFBE, or COPD, comprising administering to a subject in need of treatment an effective amount of a lipid coated crystal composition comprising AmB and Chol, as provided herein, wherein the composition provides a clinically significant improvement in lung function (FEV1) compared to placebo.

[0129] In some embodiments, provided herein is a method of reducing the viscosity of airway surface liquid in an individual suffering from CF, NCFBE, or COPD, comprising administering to a subject in need of reduced airway surface liquid viscosity a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein, thereby reducing the viscosity of said airway surface liquid in said subject while increasing the hydration and height of said airway surface liquid, wherein said administration is pulmonary administration.

[0130] In some embodiments, provided herein is a method of increasing antibacterial activity of airway surface liquid in an individual suffering from CF, NCFBE, or COPD, comprising increasing antibacterial activity of airway surface liquid in a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein, wherein said administration is pulmonary administration.

[0131] In some embodiments, provided herein is a method of improving mucociliary clearance in an individual suffering from CF, NCFBE, or COPD, comprising improving mucociliary clearance in a subject in need of increased mucociliary clearance by administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein, wherein the administration is pulmonary administration.

[0132] In some embodiments, provided herein is a method of improving pulmonary function in an individual suffering from CF, NCFBE, or COPD, comprising improving pulmonary function in a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein, wherein said administering is pulmonary administration.

[0133] In some embodiments, provided herein is a method of improving health related quality of life (HRQoL) of an individual suffering from CF, NCFBE, or COPD, comprising improving the HRQoL of a subject in need thereof by administering to said subject a therapeutically effective amount of a pharmaceutical composition according to any one of the embodiments described herein, wherein said administration is pulmonary administration.

[0134] In certain embodiments, provided is a method of treating a subject suffering from CF, NCFBE, or COPD, comprising administering to a subject in need of treatment an effective amount of a lipid coated crystal composition comprising AmB and Chol, as provided herein, wherein the lipid coated crystal composition provides a clinically significant improvement in health-related quality of life compared to placebo.

[0135] In certain embodiments, provided herein is a method of improving pulmonary function comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present disclosure, thereby improving pulmonary function in said subject, wherein said administration is pulmonary administration.

[0136] In certain embodiments, the subject has CF, NCFBE, or COPD.

[0137] In certain embodiments, the subject is afflicted with CF.

[0138] In certain embodiments, the pharmaceutical composition is a dry powder.

[0139] In certain embodiments, the dry powder is administered using a handheld dry powder inhaler.

[0140] In certain embodiments, the dry powder inhaler utilizes the subject's inspiratory airflow to fluidize and disperse the powder (ie, the device is a passive dry powder inhaler).

[0141] In certain embodiments, dry powder inhalers having a moderate or greater resistance to airflow are preferred (i.e., about 0.10-0.30 cmH2O 0.5 L -1 minutes).

[0142] 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.

[0143] Over 1900 different CFTR mutations have been found in CF patients, several hundred of which have been identified as causing disease through at least five different loss-of-function mechanisms. In recent years, important progress has been made in the development of genotype-specific small molecule drugs that bind to specific mutant forms of CFTR and enhance its activity. However, approximately 10% of CF patients have CFTR genotypes that do not respond to current small molecule treatments. These genotypes include major truncations that result in a complete lack of functional CFTR protein, as well as extremely rare mutations whose underlying mechanism of dysfunction is unclear.

[0144] In some embodiments, any of the methods disclosed herein treat a CF mutation class selected from the group consisting of I, II, III, IV, V, VI, U, and combinations thereof. In some embodiments, the mutation class is selected from the group consisting of I / I, II / II, II / U, and U / III.

[0145] Without intending to be bound by theory, the compositions described herein do not modulate, modify, or alter the activity of the CFTR channel. It is believed that molecular prostheses, including lipid-coated crystals of AmB, create new ion channels that have activity independent of CFTR and the subject's particular genetic mutation.

[0146] In some embodiments, any of the methods disclosed herein are genotype-independent treatment methods.

[0147] As used herein, the phrases "genotype-independent" or "genotype-agnostic" refer to any treatment that is independent of the precise nature of the genetic mutation that underlies the reduced expression or function of CFTR in CF.

[0148] In certain embodiments, the methods described herein are useful for treating various CF genotypes that are typically not or only minimally responsive to treatment with conventional CF therapeutics, for example, the V520F allele in patients with the D259G / V520F pair of CTFR mutations is refractory to treatment with Ivacaftor, or a combination of Elexacaftor, Tezacaftor, and Ivacaftor.

[0149] Thus, in certain embodiments, the methods of treating CF described herein are independent of the CF genotype.

[0150] In certain embodiments, any of the methods disclosed herein treat refractory or resistant CF, hi certain embodiments, the CF is refractory or resistant to one or more CF treatments.

[0151] 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.

[0152] The pH of a subject's ASL is said to be "elevated" when it is measurably greater than the pH of the ASL of an untreated subject. In one embodiment, the pH of a subject's ASL is said to be "elevated" when it is measurably greater than the pH of the ASL of the same subject measured prior to treatment with the disclosed methods or at a time distant from treatment with the disclosed methods.

[0153] Such a method of increasing the pH of airway surface liquid is described in Example 1. Notably, restoration of ASL pH is observed across a wide range of AmB:sterol concentrations. Thus, the method of increasing ASL pH described herein has significant implications for clinical applications.

[0154] In certain embodiments, the increase in pH can be between 0.1 pH units and 2.0 pH units. In certain embodiments, the increase in pH can be between 0.1 pH units and 1.0 pH units. In certain embodiments, the increase in pH can be between 0.3 pH units and 1.0 pH units. In certain embodiments, the increase in pH can be between 0.3 pH units and 1.0 pH units. In certain embodiments, the increase in pH can be between 0.5 pH units and 0.9 pH units. In certain embodiments, the increase in pH can be between 0.5 pH units and 0.8 pH units. In certain embodiments, the increase in pH can be between 0.6 pH units and 0.8 pH units. In certain embodiments, the increase in pH can be about 0.7 pH units. In certain embodiments, the increase in pH can be between 0.1 pH units and 0.5 pH units. In certain embodiments, the increase in pH can be between 0.1 pH units and 0.4 pH units. In certain embodiments, the increase in pH can be between 0.2 pH units and 0.4 pH units. In certain embodiments, the increase in pH can be between 0.1 pH units and 0.3 pH units. In certain embodiments, the increase in pH can be between 0.2 pH units and 0.3 pH units. In certain embodiments, the increase in pH can be between 0.1 pH units and 0.2 pH units.

[0155] In certain embodiments, the increase in pH is by adding any one of the compositions disclosed herein to the apical side.

[0156] In certain embodiments, the methods described herein are useful for increasing the pH of airway surface liquid in patients regardless of the patient's genotype, including patients with any one of a variety of CF genotypes that typically do not respond or only minimally respond to treatment with conventional CF modulator / corrector therapeutics.

[0157] Thus, in certain embodiments, the methods of increasing the pH of ASL described herein are independent of CF genotype.

[0158] In certain embodiments, any of the methods disclosed herein increase the pH of the ASL in patients 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.

[0159] In certain aspects, provided herein is a method of reducing the viscosity of airway surface liquid in a patient suffering from CF by restoring anion transport and increasing the hydration and height of the airway surface liquid, comprising administering to a patient suffering from CF a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0160] In some embodiments, restoration of anion secretion (e.g., bicarbonate secretion) leads to an increase in airway surface liquid pH, increased airway hydration, increased airway surface liquid height, increased airway surface liquid antibacterial activity, or a combination thereof. Improvements in airway surface liquid properties such as these can result in clinical improvements in mucociliary clearance, measures of pulmonary function (e.g., FEV1), the patient's health-related quality of life (HRQoL), or a combination thereof.

[0161] In certain embodiments, HRQoL is measured using the Cystic Fibrosis Questionnaire (CFQ). Henry, B. et al., Qual Life Res. 2003 Feb; 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 Feb; 12(1): 77-85. In certain embodiments, administering a composition of the invention to a patient provides an increase in HRQoL domain score of at least 0.5, at least 0.8, at least 1, or at least 2 as measured by the CFQ or CFQ-R. In some embodiments, a subject's HRQoL domain score is said to be "elevated" if it is measurably greater than the HRQoL domain score of the same subject measured prior to treatment with the methods of the present disclosure or at a time distant from treatment with the methods of the present disclosure.

[0162] A subject with CF may have a decreased ASL height compared to a healthy subject. Muraglia, Katrina A. et al., Nature 2019 Mar; 567(7748):405-408. In certain embodiments, the ASL height of a subject with CF is about 3 μm to about 4 μm. In some embodiments, the methods described herein increase the ASL height of a subject with CF to the ASL height of a healthy subject (e.g., to an ASL height of about 9 μm to about 10 μm).

[0163] In certain embodiments, the methods described herein increase the height of a subject's ASL 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, a subject's ASL height is said to be "increased" if it is measurably greater than the ASL height of the same subject measured prior to treatment with the disclosed methods or at a time point distant from treatment with the disclosed methods. In certain embodiments, a subject's ASL height is said to be "increased" if it is measurably greater than the ASL height of an untreated subject.

[0164] In certain embodiments, provided herein is a method of reducing the viscosity of airway surface liquids comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present disclosure, thereby reducing the viscosity of airway surface liquids in said subject, said administration being pulmonary administration.

[0165] In certain embodiments, the subject has CF, NCFBE, or COPD.

[0166] In certain embodiments, the subject is afflicted with CF.

[0167] In certain embodiments, the pharmaceutical composition is administered to the respiratory tract of the subject by dry powder aerosol.

[0168] In certain embodiments, the pharmaceutical composition is administered to the bronchial airways of the subject by dry powder aerosol.

[0169] In certain embodiments, the airway of a subject refers to the so-called conducting airway or bronchial airway, i.e., any or all of the following lung structures: trachea, bronchi, and bronchioles of the lower respiratory tract.

[0170] In certain embodiments, the methods described herein are useful for reducing the viscosity of airway surface liquid in patients with any one of a variety of CF genotypes that typically do not respond or only minimally respond to treatment with conventional CF therapeutics.

[0171] Thus, in certain embodiments, the methods of reducing the viscosity of ASL described herein are independent of CF genotype.

[0172] In some embodiments, any of the methods disclosed herein reduce the viscosity of ASL in patients 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.

[0173] According to each of the above embodiments, in certain embodiments the patient is a human.

[0174] In certain embodiments, the age range of subjects for treatment with the compositions described herein is determined based on the ability to effectively utilize a passive dry powder inhaler (DPI). Subjects under 6 years of age may not have the cognitive ability to properly perform the steps involved in using a passive DPI. Furthermore, subjects under 6 years of age may lack the respiratory muscle strength required to generate the necessary inspiratory pressure. Nevertheless, some passive DPIs have been approved for subjects up to 4 years of age. Similarly, some elderly patients may also have cognitive abilities or atrophied muscles that prevent proper use of a passive DPI. Inspiratory pressure tends to increase with age, reaching a maximum at about 25 years of age, remains constant until about 40 years of age, and then steadily declines thereafter. Men tend to achieve higher inspiratory pressures than women. Increased airway disease may also adversely affect a subject's ability to achieve the pressure drop (about 1.0 kPa) required to effectively operate a passive DPI. Subjects with poor inspiratory muscle strength can be identified based on whether they can achieve an inspiratory pressure of 1.0 kPa using the In-Check dial. According to each of the above embodiments, in certain embodiments, the patient is between 4 and 80 years old.

[0175] 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 less than about 80 years old with sufficient muscle strength for acceptable inspiration. 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.

[0176] 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 ions.

[0177] Drug substance The compositions described herein, sometimes referred to as Amphotericin B Cystetic for Inhalation (ABCI), can be used as ion channel prosthetics to restore anion transport and host defense to improve lung function and quality of life.

[0178] In certain embodiments, the compositions described herein comprise highly crystalline micronized particles of amphotericin B coated with a porous layer of lipid that includes cholesterol.

[0179] AmB is a heptaene macrolide containing seven conjugated double bonds in the trans position and 3-amino-3,6-dideoxymannose (mycosamine) attached to the main ring by a glycosidic bond.

[0180] AmB is produced as a fermentation product of a strain of Streptomyces nodosus. Intravenous AmB is the only fungicidal compound approved and is currently used to treat systemic fungal infections. Although not approved for inhalation administration, inhaled AmB has been extensively studied for the treatment of invasive pulmonary aspergillosis.

[0181] Indeed, over 500,000 doses of inhaled AmB have been administered, with no serious or severe adverse events reported. In the majority of published studies, inhaled AmB has been described as well tolerated. Reported adverse events (AEs) 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 through the lungs results in extremely low systemic drug levels, often close to or below the limit of quantification. Serum creatinine concentrations, a biomarker of renal function, are generally unchanged by aerosol AmB administration. Thus, targeting AmB to the respiratory tract dramatically improves its systemic tolerability.

[0182] At neutral pH, AmB is zwitterionic, with the carboxyl group (pKa=5.7) deprotonated and the amine group of the mycosamine ring (pKa=10.0) protonated. The highly crystalline zwitterionic form of AmB is virtually insoluble in aqueous solutions, with solubilities of 0.5, 0.2, and 0.2 μg / mL in water, Gamble's solution, and dilute Alveofact®, respectively, at 37°C.

[0183] Dose number (Do) is a simple metric used to predict whether a compound will be adequately absorbed from the lungs based on its solubility at the intended clinical dose.

number

[0184] Here, M o is the AmB dose in ASL, and V ASL is the volume of the ASL liquid, and S ASL is the solubility of AmB in ASL fluid. Values ​​of Do greater than 1.0 are consistent with dissolution-limited absorption from the lungs. S ASL = 0.2 μg / mL and V ASL = 25 mL, absorption is solubility limited by M o >5 μg. Therapeutic doses of AmB are expected to exceed about 1.0 mg. In this scenario, absorption of AmB becomes solubility-limited.

[0185] The slow dissolution of the compositions of the present disclosure allows for a slow appearance of AmB in plasma (e.g., t max =4-8 hours) and dramatically reduced systemic exposure compared to intravenous compositions of AmB. The systemic exposure of the inhaled compositions described herein is 100-1000 times lower than that measured after intravenous administration of an equivalent AmB dose.

[0186] 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 AmB dissolved in ASL on the order of 5 to 20 hours, followed by a very slow terminal clearance (t ) caused by redistribution of the drug from the lung tissue. 1 / 2 A similar biphasic clearance process is observed for AmB following intravenous administration of microparticles, which are redistributed into the plasma after initial clearance by cells of the reticuloendothelial system.

[0187] The nominal dose, particle size, and physical form of AmB can affect the toxicity of AmB after oral inhalation in rats (US Patent Application Publication No. 2012 / 0128728). Rats administered highly amorphous AmB formulated as lipid-coated crystals containing 50% AmB and 50% DSPC / calcium chloride in a 2:1 molar ratio (i.e., ABIP, Nektar Therapeutics) (US Patent Application Publication No. 7,326,691; US ​​Patent Application Publication No. 8,404,217; US Patent Application Publication No. 2012 / 0128728) showed clinical signs of dyspnea, tracheal and bronchial hypertrophy, tracheal inflammation, infiltration of bronchial mucosal cells, and evidence of tracheal and bronchial luminal exudate. The incidence of these effects was significantly reduced in rats administered small particle size and highly crystalline AmB (crystallinity in ABIP ≥ 76%).

[0188] ABIP compositions containing highly crystalline AmB administered at appropriate doses and regimens have been well tolerated in non-clinical toxicity studies. The compositions have also been well tolerated in single- and multiple-dose studies in healthy participants (Kugler et al.: Amphotericin B inhalation powder (ABIP) achieves significant pulmonary and low systemic amphotericin B concentrations. 16 th Congress of the International Society of Human and Animal Mycology (ISHAM), Paris, France, Abstract O-0011, 26-Jun-2006; Lee JD, et al.: Amphotericin B inhalation powder (ABIP) is well tolerated with low systemic amphotericin B exposure in healthy subjects. In: Program and Abstracts of the 2 nd Meeting of Advances Against Aspergillosis (AAA), Abstract P-118, pp. 214-215, Athens, Greece, 2006).

[0189] Increasing amorphous content can also result in decreased grindability of the drug substance, which can lead to increased particle size. This observation is illustrated for the wet milling process in Example 3. Such increases in crystal size can adversely affect important quality attributes related to aerodynamic particle size distribution.

[0190] When the crystallinity of AmB decreased from 96% to 75%, after wet grinding in water, X 50 increased from 1.06 μm to 2.16 μm, and X 90The AmB crystal size increased from 2.14 μm to 5.99 μm (Example 3). The increase in AmB crystal size did not significantly affect the physicochemical properties and aerosol performance of the formulated ABCI-002 drug product. The MMAD increased from 2.9 μm to 3.1 μm, but the FPF <5μm rose from 61% to 63%.

[0191] In some embodiments, the crystallinity of the drug substance is at least 75%, at least 90%, or at least 95%.

[0192] In certain embodiments, the micronized drug particles have a small primary particle size distribution, which can provide efficient delivery of the drug product to the lungs. 50 In some embodiments, the X of the AmB crystal is about 0.5 μm to about 4 μm, for example, about 1 μm to about 3 μm. 90 is about 1.5 μm to about 10 μm, for example, about 2.0 μm to about 8.0 μm.

[0193] Excipients In certain embodiments, the compositions described herein comprise highly crystalline AmB particles coated with a porous layer containing two phospholipids (HSPC and DSPG), cholesterol, and calcium chloride. These materials may exhibit complex phase behavior that directly affects the physicochemical properties and aerosol performance of the compositions. This, in turn, may affect the efficacy, safety, and tolerability of the formulated drug product.

[0194] Phospholipids are a type of lipid that contains a glycerol or sphingosine backbone to which one or more fatty acids and a phosphate group to which an alcohol is attached can be attached. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine. Phospholipids are amphipathic molecules, with the two fatty acyl chains being lipophilic and the modified phosphate group being hydrophilic. Phospholipids self-assemble in water to form membrane structures. Phospholipids are ubiquitous in the mammalian cell membrane.

[0195] Hydrated phospholipid bilayers exhibit thermotropic phase behavior. With increasing temperature, 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 crystalline phase" (Ld), in which an increase in the gauche conformers of the acyl chains leads to a disordered, liquid-like packing. The temperature of the gel-to-liquid crystalline phase transition is known as the main transition temperature, T m It is also sometimes called.

[0196] Phospholipid T m is highly dependent on the length and degree of saturation of the acyl chain, and to a lesser extent, on the nature of the head group. Table 2 shows the T m Provides a comparison of values.

[0197] [Table 2]

[0198] For phosphatidylcholine, increasing the acyl chain length from 12 to 18 increases the hydrated T m The T increases from -2°C to 55°C. By introducing unsaturation into the acyl chain, m The value drops dramatically below 0°C. The change in the nature of the head group also m This leads to differences in m The order of HSPC and DSPG is phosphatidylethanolamine (PE) > phosphatidylserine (PS) > phosphatidylglycerol (PG) to phosphatidylcholine (PC). m The values ​​are 53.6°C and 55°C, respectively.

[0199] Cell membranes are essentially multicomponent systems that contain a variety of lipid and protein species. The physical properties and biological functions of cell membranes are closely linked to the arrangement and distribution of phospholipids within the phospholipid bilayer. In fact, lipids may phase separate into different domains within the bilayer depending on their composition. Chol is abundant in cell membranes and plays an important role in maintaining structural integrity and regulating membrane fluidity.

[0200] In certain embodiments, the compositions described herein comprise Chol. Chol can contribute to the safety and ion channel activity of formulated drug products. Adding Chol to saturated PL can significantly affect the phase behavior of PL in cell membranes and spray-dried particles.

[0201] FIG. 1 shows the phase diagram of hydrated DPPC-Chol mixtures (Ipsen JH, Karlstroem G, Mouritsen OG, et al. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim Biophys Acta. 1987; 905: 162-172).

[0202] As shown in Figure 1, the addition of Chol leads to the formation of a new phase called the ordered liquid phase (Lo). By incorporating less than about 6 mol% Chol into So, the T m The phase transition broadens and the packing of the acyl chains becomes more disordered. In contrast, m The phospholipid acyl chains in the disordered Ld phase above T become more ordered by adding Chol. At Chol concentrations of about 6-20 mol%, the two phases coexist. m Above this temperature, the disordered and ordered liquid phases coexist (Ld-Lo). m Below 1000 K, the ordered solid phase coexists with the ordered liquid phase (So-Lo). At high Chol concentrations (20–25 mol%), the cooperative T m The phase transition is lost and only the Lo phase is present. For HSPC (with longer acyl chains), a similar diagram is expected, with the temperature on the vertical axis of Fig. 1 shifted upwards by about 13 °C.

[0203] In contrast to the fully hydrated PL described above, the compositions according to embodiments described herein comprise partially dehydrated PL. For use as ion channel prostheses as proposed herein, the lipids in the compositions described herein can be spray dried to form an inhalable dry powder with low moisture content.

[0204] In the absence of water, the spacing between phospholipid head groups is reduced, increasing van der Waals forces between lipid acyl chains and increasing T m For example, the T of DPPC increases. m The value increases from approximately 41°C (fully hydrated) to 105°C upon freeze-drying (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).

[0205] For spray-dried DSPC powder, m increases from 55°C (fully hydrated) to 71°C at 75% RH, 85°C at 11% RH, and 102°C at <3RH (Pikal-Cleland KA, Zhang J, Lechuga-Ballesteros D, Tarara TE, Weers JG: The impact of Ca 2+ binding on the packing structure of dry phospholipids. Presented at CRS Annual Meeting, Seoul, Korea, 2002).

[0206] For currently commercially available spray-dried compositions containing PL (e.g., TOBI® Podhaler™, Bevespi® Aerosphere, Breztri® Aerosphere, and Inbrija®), the goal has been to maintain the PL in the So phase during particle formation, in the collector during spray drying, and during storage over the shelf life of the product. As a result, these products utilize long-chain saturated phosphatidylcholines, such as DPPC and DSPC, as the primary shell-forming excipients.

[0207] During spray drying of the aqueous feedstock, the evaporative cooling of the atomized droplets results in a droplet temperature slightly above room temperature and above the hydration T m The inlet temperature of the spray dryer is kept much lower (41 to 55°C). m After particle formation, the spray dryer outlet temperature and collector jacket temperature are adjusted to the T m In this way, the acyl chains are maintained in the So phase throughout the entire manufacturing process and during storage.

[0208] For liquid feedstocks containing DPPC or DSPC, spray drying of the So phase allows for the formation of individualized spray-dried particles in which the surface composition, surface morphology, particle size, density, and porosity of the particles can all be effectively controlled. Furthermore, long-chain saturated PLs are believed to be biocompatible in the lungs because they are present in the endogenous pulmonary surfactant and within the rapid clearance pathway from the lung.

[0209] In contrast, T m There is a major problem with spray drying liquid feedstocks with unsaturated PL values ​​below 0°C. Water evaporates during spray drying, so T m In this scenario, since the acyl chains of PL exist in the Ld phase during particle formation, the drying process may result in large aggregates of fused particles, which may lead to low production yields and aerodynamic particle size distributions unsuitable for inhalation as dry powder aerosols.

[0210] In certain embodiments, the compositions described herein comprise saturated phospholipids with relatively long 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.

[0211] Calcium ions can bind with phosphate groups in phospholipids, replacing water molecules and allowing tighter packing between PL molecules. For spray-dried DSPC powders, the addition of Ca ions can improve the environmental robustness (T, RH) of the dehydrated powder, especially at RH values ​​that typically result in a large increase in capillary forces (e.g., 75% RH). In some embodiments, the ratio of PL / Ca is less than 4.0 mol / mol, less than 3.0 mol / mol, or about 2.0 mol / mol (U.S. Patent No. 8,709,484; U.S. Patent No. 7,442,388; incorporated herein by reference). In some embodiments, it may be undesirable to reduce the PL / Ca ratio below the stoichiometric ratio of 2.0 mol / mol, because excess calcium chloride at ratios below this can increase the hygroscopicity of the powder.

[0212] Ld or Lo phase T m Above the glass transition temperature T, spray-dried powders can become "sticky" viscous liquids. Such cohesive powders tend to adversely affect powder flow and aerosol performance. This observation is consistent with the glass transition temperature T g This may be similar to what occurs in highly disordered amorphous solids above 100 nm. Spray-dried powders with phase separation of So and Lo domains tend to have poor powder properties. It may therefore be desirable to maintain the lipids in the So phase.

[0213] The effect of added Chol on the phase behavior of PL in dry powder formulations is not well understood. Differential scanning calorimetry thermograms of compositions according to embodiments described herein suggest that the Lo phase is fully solubilized into the So phase at Chol / PL ratios below about 0.05 w / w (≦9.4 mol% Chol) (Example 4). Thus, in the dehydrated state, the So phase is capable of solubilizing a higher percentage of Chol than observed in hydrated DPPC-Chol mixtures ( FIG. 1 ). By maintaining the So phase, it is possible to achieve a T higher than 90° C. m The value is obtained.

[0214] Reducing the DSPG content in the ABCI powder from an HSPC / DSPG ratio of 2.3 mol / mol (approximately 7 / 3 mol / mol), as utilized in AmBisome® and some pulmonary surfactant preparations, to an HSPC / DSPG ratio of 9.0 w / w or higher can also improve the physicochemical and aerosol properties of the spray-dried AmB / Chol powder (Example 12).

[0215] In some embodiments, increasing 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. Calcium ions can be added to replace the sodium ions, which can combine with the chloride ions from calcium chloride to form hygroscopic sodium chloride domains in the spray-dried particles. At high RH, NaCl deliquesces (i.e., absorbs moisture from the air and dissolves in it). This can contribute to the stickiness of the compositions according to the embodiments described herein at high RH.

[0216] Other PLs can provide improved pH and acceptable powder and aerosol properties to ASL. Their usefulness 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).

[0217] Pharmaceutical Product Composition In some embodiments, the compositions described herein comprise drug particles that are phase separated from lipid excipients in distinct crystalline domains, i.e., AmB and Chol are not complexed in amorphous nanoparticles, as in U.S. Patent No. 5,965,156 and U.S. Patent No. 5,965,156, and AmB is not encapsulated in liposomes, as in AmBisome® (U.S. Patent No. 5,965,156).

[0218] The presence of Chol in the compositions described herein can be important from both an efficacy and safety standpoint.

[0219] From the viewpoint of efficacy, the presence of Chol can maintain ion channel activity at both low and high AmB concentrations. Although the mechanism is not clear, in the absence of Chol, ion channel activity tends to decrease at high AmB concentrations (Example 10). This tendency was observed for both pure AmB and ABIP without Chol.

[0220] In US Patent No. 5,399,136, Burke et al. disclose compositions of AmB and Chol for the restoration of physiological function of the airway surface of CF lung epithelium, with the molar ratio of Chol / AmB being claimed to be in the range of 2.6-50 mol / mol. In US Patent No. 5,399,136, Burke et al. disclose methods for treating CF with small molecule bicarbonate channels, with the Chol / AmB ratio being 1.0-12.0 mol / mol. Surprisingly, in this disclosure, it has been discovered that ion channel activity is maintained at Chol / AmB ratios significantly lower than 1.0 mol / mol (Example 10). In some embodiments, the nominal ratio of Chol / AmB is 0.4-1.2 mol / mol. In some embodiments, due to enrichment of the drug substance in the spray drying process, the Chol / AmB ratio of the final spray dried powder is about 0.33 to about 1.0 mol / mol. In certain embodiments, a significant improvement in the pH of ASL (ie, >0.1 pH units), comparable to that achieved with the AmB:Chol complex, is maintained down to a nominal Chol / AmB ratio of 0.05 mol / mol.

[0221] The maintenance of ion channels at lower Chol / AmB ratios in spray-dried powders containing PL minimizes phase separation of the Chol-rich Lo phase with adverse effects on powder properties.

[0222] The AmB:Chol complexes disclosed in US Pat. No. 5,399,433 and US Pat. No. 5,499,566 achieve an improvement in the pH of the ASL of CuFi-1 and CuFi-4 epithelia of about 0.1-0.2 pH units. The pH of the ASL remains slightly acidic (pH about 6.90-6.95). In CuFi-4 cells, the CFTR modulator Ivacaftor achieves an improvement in the pH of the ASL comparable to that of the AmB:Chol complex. In contrast, the improvement in the pH of the ASL of CuFi-1 cells observed with compositions according to embodiments described herein is about 0.4-0.5 pH units, i.e., about 2-5 times greater than that observed with the AmB:Chol complex and Ivacaftor. In some embodiments, the pH of the ASL is increased from slightly acidic with the AmB:Chol complex to slightly basic (pH about 7.2) with the compositions described herein. Achieving a slightly basic pH can improve the activity of many cationic peptides involved in host defense, such as defensins.

[0223] From a safety perspective, the presence of Chol in compositions according to embodiments described herein has been found to prevent the extraction of sterols from mammalian cell membranes into the extramembrane sterol sponge phase. This has been demonstrated to be the main mechanism of toxicity of Chol-free compositions of AmB. It was suggested that this protective effect was maintained up to Chol / AmB ratios of 1.0 mol / mol (Patent Document 1, Patent Document 2). Surprisingly, for red blood cell membranes, this protective effect was found to be maintained down to Chol / AmB ratios of 0.4 mol / mol or less (Example 15).

[0224] FIG. 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 a range of Chol / AmB molar ratios (e.g., 1.2 mol / mol and 0.4 mol / mol). The third leg of the triangle is defined by a Chol / PL ratio (0.05 w / w) that precludes phase separation of the Lo phase. The phase diagram and various compositions are described in more detail in Example 1.

[0225] In some embodiments, the nominal daily dose of AmB for restoration of ASL pH in patients with CF is from about 0.1 to about 10 mg, or from about 0.2 mg to about 6 mg.

[0226] In some embodiments, estimates of drug loading based on the nominal dose and tap density of the powder are provided in Example 16. In certain embodiments, the drug loading is from about 0.1 w / w% to about 50 w / w%, for example, from about 0.5 w / w% to about 20 w / w%.

[0227] Physicochemical properties The physicochemical properties of the compositions described herein may depend on the feed composition (e.g., solids content, PFOB content) and drying conditions in the spray dryer. The ranges described below are based on results for batches of the compositions shown in the examples.

[0228] In some embodiments, the small porous particles have a relatively low density, but not as low as compositions spray dried from solution-based liquid feedstocks. In some embodiments, the injected bulk density is about 0.04 to about 0.12 g / cm. 3 The tap density is about 0.08 to about 0.20 g / cm 3 It is.

[0229] The Carr flowability index provides a measure of the flow properties of bulk powders (Table 3). Particulates with a geometric size of less than 5 μm, such as particles according to embodiments described herein, are highly cohesive and have very poor powder flow properties.

[0230] [Table 3]

[0231] Nevertheless, by accurately and precisely filling cohesive fine powders using drum fillers (e.g., machines manufactured by Harro Hoefliger), a relative standard deviation of less than about 3% can be achieved even with a fill mass of only about 1.0 mg.

[0232] In some embodiments, the powders described herein have a Carr flowability index of about 20 to about 50, such as about 24 to about 30. Powders with low Carr flowability index values ​​can achieve significantly improved powder flow characteristics, reduced variability in particle size and density measurements, and increased production yields. Compositions with Lo domains in spray-dried powders have a Carr flowability index of about 40, which tends to lead to low yields and difficulties in powder handling (e.g., packing and fluidization).

[0233] In certain embodiments, the observed relatively high Carr flowability index values ​​(indicative of poor cohesive flow, i.e., Carr flowability index values ​​of 20-32) do not adversely affect flow properties important for the development of a portable dry powder inhaler.

[0234] The primary particle size distribution can be determined by laser diffraction (see, for example, "Characterization Methods" below). In certain embodiments, the powders described herein have a primary particle size distribution of X 10 is about 0.40 to about 1.2 μm, X 50 is about 1.5 to about 3.5 μm, X 90 is about 4.0 to about 8.0 μm.

[0235] The moisture content can be determined by Karl Fischer coulometric titration. In certain embodiments, the moisture content of the powders described herein is from about 1.5 w / w% to about 6 w / w%.

[0236] In certain embodiments, the physicochemical properties of the powder can be adjusted throughout the ranges mentioned above by varying the manufacturing process parameters.

[0237] Aerosol Performance In certain embodiments, the compositions described herein target the bronchial airways. In this regard, it would be beneficial to maximize deposition in the large and small airways within the lungs while minimizing extrathoracic drug deposition in the mouth and throat and alveoli.

[0238] In the context of in vitro aerosol performance metrics, compositions according to embodiments described herein have an emitted dose of at least 70%, at least 80%, at least 90%, or at least 95% from a handheld dry powder inhaler at a pressure drop of 4 kPa and an inhalation volume of 4 L. In some embodiments, compositions described herein are formulated to pass the delivered dose uniformity (DDU) regulatory requirements set forth in the FDA Draft Guidance: "Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Products- Quality Considerations (April, 2018)" with a standard deviation of the mean ED value of 6% or less.

[0239] In certain embodiments, in a Next Generation Impactor (NGI) operating at a pressure drop of 4 kPa and an inhalation volume of 4 L, the compositions described herein have a mass median aerodynamic diameter (MMAD) of about 1.5 to about 4.0 μm, e.g., about 2.0 to about 3.5 μm.

[0240] The pattern of deposition within the NGI can be important. Following the desired local deposition pattern detailed above, it is beneficial to maximize deposition in stages 3, 4, and 5 (called the "airways fraction") while minimizing deposition in the USP throat and impactor stages 1 and 2 (called the "coarse fraction"), and in the "extrafine fraction" of stages 6 through the filter. In certain embodiments, deposition in the coarse fraction is less than 30 w / w%, less than 20 w / w%, or less than 10 w / w% of the nominal dose. In some embodiments, deposition in the extrafine fraction is less than 16%, less than 12%, or less than 8% of the nominal dose. In certain embodiments, deposition in the airways fraction is at least 40%, at least 50%, at least 60%, or at least 70% of the nominal dose.

[0241] In certain embodiments, the fine particle fraction less than 5 μm (FPF), expressed as a percentage of the emitted dose, is <5μm ) is at least 40 w / w%, at least 50 w / w%, at least 70 w / w%, or at least 90 w / w%.

[0242] In certain embodiments, the flow rate dependence of the total lung dose (TLD) in an Alberta Idealized Throat (AIT) model at a pressure drop of 1.0 to 6.0 kPa is less than 40%, or less than 15%.

[0243] In certain embodiments, the target fill mass is about 5 to about 40 mg, e.g., about 10 to about 20 mg. In some embodiments, the compositions described herein are administered to a subject using a portable capsule-based dry powder inhaler (DPI). In certain embodiments, the DPI has a volume of about 0.30 cm. 3 (Size 3 capsule) ~ approx. 0.37cm 3 (Size 2 capsule) container.

[0244] In a particular embodiment, the DPI is a variant of the RS01 DPI (Plastiape, Osnago, Italy). Variations of the RS01 vary in size of capsule they house (size 3 to size 0) and resistance to airflow (R = 0.06 to 0.16 cmH2O 0.5 L -1 In some embodiments, the DPI has a medium to high resistance (e.g., R of about 0.10 to about 0.30 cmH2O). 0.5 L -1 In a particular embodiment, the DPI has a resistance of about 0.14 cmH2O 0.5 L -1 It is a variant of the RS01 DPI, which is a 2.5-minute inhalation DPI. In certain embodiments, the medium or higher resistance DPI limits extrathoracic deposition while also limiting the likelihood of coughing after inhalation.

[0245] In some embodiments, the compositions described herein are administered using a Handihaler (Boehringer Ingelheim), RS00 (Plastiape), or Aerolizer® (Novartis) (U.S. Pat. No. 3,991,761; incorporated herein by reference), Breezhaler® (U.S. Patent Application Publication No. 2007 / 0295332; incorporated herein by reference) (Novartis). In certain embodiments, the compositions described herein are administered using a Turbospin (PH&T) or a variant thereof, such as T-326 (Podhaler™) (Novartis), as described in U.S. Pat. Nos. 8,069,851 and 7,559,325, both of which are incorporated herein by reference, or an AIR inhaler (Acorda Therapeutics).

[0246] In certain embodiments, the compositions described herein are administered using a single-use disposable inhaler. In some embodiments, the single-use disposable inhaler is TwinCaps (Hovione), Dose1® (Micro Engineering Solutions), ICOone™ (Iconovo), or Cyclops® (Pure IMS).

[0247] In some embodiments, the dry powder composition of the present disclosure is administered intranasally to increase the pH of the ASL in the nose and increase the nasal transepithelial potential. Potential devices include Aptar's Unidose system.

[0248] manufacturing The mixture of cholesterol and phospholipids can be prepared by dissolving in organic solvent. In a particular embodiment, the lipid mixture is prepared with HSPC / DSPG / Chol in a w / w / w ratio of 54 / 24 / 22. Depending on the desired composition, the lipid mixture can be supplemented with additional PL and calcium chloride.

[0249] In some embodiments, the method of preparing the compositions described herein comprises dispersing a lipid mixture in water to form multilamellar liposomes. In certain embodiments, this step comprises dispersing the lipid mixture in water to form multilamellar liposomes. m It involves adding the lipid to warm water at a higher temperature. In some embodiments, the temperature of the water is between 65° C. and 90° C. In some embodiments, the dispersion is achieved using a high shear mixer such as an UltraTurrax® T-50.

[0250] In some embodiments, the method further comprises forming a coarse FC-in-water emulsion composed of micron-sized emulsion droplets by adding a fluorinated (FC) blowing agent (e.g., perfluorooctyl bromide (PFOB, perflubron), perfluorooctylethane (PFOE), or perfluorodecalin (PFD)) with mixing. In certain embodiments, the coarse emulsion is homogenized under high pressure using a MicroFluidizer® or a piston gap homogenizer (e.g., Avestin Emulsiflex®) to form a submicron emulsion. In some embodiments, the pressure during homogenization is about 10,000 to about 20,000 psi, and homogenization is performed for a set period of time (depending on the batch size) or for a specified number of individual passes. In some embodiments, one individual pass is performed at the end of the process for a set period of time to ensure that all droplets have passed through the homogenizer at least once.

[0251] In some embodiments, the method further comprises adding a drug substance to the submicron emulsion while mixing. In certain embodiments, this addition is performed in a one-pot process. In some embodiments, the resulting composite dispersion of emulsion droplets and suspended drug particles is mixed in a high shear mixer or passed through a homogenizer.

[0252] In some embodiments, emulsion preparation and drug wet milling are carried out in separate tanks in a two-pot process. In certain embodiments, this allows for in-process size measurement of the micronized drug particles by laser diffraction or dynamic light scattering. In some embodiments, the two pots are then combined with mixing.

[0253] In certain embodiments, the emulsion preparation and wet-milling steps are performed with a concentrated emulsion / suspension to limit the amount of liquid processed by the homogenizer, and in some embodiments, water is added in a final step to achieve the target solids content and PFOB volume fraction in the final liquid feed.

[0254] In some embodiments, the feedstock is sprayed into a stream of warm filtered air which evaporates the solvent and conveys the dried product to a cyclone separator or baghouse. In certain embodiments, the spent air is then exhausted along with the vaporized solvent. In some embodiments, the operating conditions of the spray dryer, such as inlet and outlet temperatures, feed rate, atomization pressure, drying air flow rate, and nozzle configuration, can be adjusted to obtain the desired particle size, moisture content, and production yield of the resulting dried particles. In certain embodiments, the method further includes setting a Niro PSD-1 scale spray dryer to have an inlet air temperature of 80° C. to 200° C., an outlet air temperature of about 40° C. to 120° C., a liquid feed rate of 30 g / min to 120 g / min, a total air flow rate of 50 scfm to 230 scfm, and an atomization air flow rate of 30 to 90 scfm. The solids content of the spray dried feedstock will typically be 0.5% to 10% w / v, and the blowing agent concentration will typically be 3% to 30% v / v. In some embodiments, the desired settings depend, at least in part, on the scale and type of equipment used.

[0255] As the water in the mist droplets evaporates, the diameter of the aqueous mist droplets is reduced and the slowly diffusing emulsion droplets and AmB crystals concentrate at the air / water interface, revealing hollow droplet cores. As the high boiling oil phase evaporates, pores remain in the spray-dried particles. The final particle morphology consists of porous lipid particles with drug particles embedded in them.

[0256] In certain embodiments, the outlet temperature and the collector jacket temperature (if utilized to control RH in the collector) should be below the lowest lipid main transition temperature. In certain embodiments where the Chol / PL ratio is less than 0.05 w / w, the lowest lipid main transition temperature is above 80° C., and the outlet temperature and collector jacket temperature will be 70° C. or higher. Decreasing the Chol / PL ratio and increasing the HSPC / DSPG ratio (Examples 9 and 12) can eliminate the Chol-rich phase, which can lead to higher outlet temperatures, improved production rates, and reduced residual solvent in the spray-dried powder.

[0257] If the atomized droplets dry too quickly, the advantage of the fine emulsion droplets can be lost, so controlling the drying rate can be beneficial.

[0258] definition As used herein, the term "treat" refers to performing an intervention that results in: (a) preventing the occurrence of a condition or disease in a subject who may be at risk for developing or who may be predisposed to a condition or disease, but who has not yet been diagnosed with the condition or disease; (b) preventing the condition or disease, e.g., slowing or halting its onset; or (c) alleviating or relieving 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) preventing the condition or disease, e.g., slowing or halting its onset; or (b) alleviating or relieving the condition or disease, e.g., causing regression of the condition or disease.

[0259] As used herein, a "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.

[0260] As used herein, "effective amount" refers to any amount sufficient to achieve a desired biological effect.

[0261] As used herein, "therapeutically effective amount" refers to any amount sufficient to achieve a desired therapeutic effect, eg, treatment of CF.

[0262] As used herein, "active ingredient," "therapeutically active ingredient," "active agent," "drug," or "drug substance" refers to the active ingredient of a drug, which is also known as the "active pharmaceutical ingredient (API)."

[0263] As used herein, "amorphous" refers to a state in which a material lacks long-range order at the molecular level and can exhibit the physical properties of a solid or a liquid, depending on temperature. Typically, such materials do not exhibit distinct X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, the change from solid-like to liquid-like properties occurs in a "glass transition", typically defined as a second-order phase transition.

[0264] As used herein, "crystalline" refers to a solid phase in which the material has an internal structure with regular order at the molecular level and exhibits a distinctive X-ray diffraction pattern with well-defined peaks. Such materials also exhibit the properties of a liquid when heated sufficiently, but this change from solid to liquid is characterized by a phase change, typically a first order phase transition ("melting point"). In the context of this disclosure, a crystalline active ingredient means an active ingredient with a crystallinity of greater than 75%. In certain embodiments, the crystallinity is preferably 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%.

[0265] As used herein, "drug loading" refers to the percentage of one or more active ingredients on a weight basis, of the total weight of a composition.

[0266] As used herein, "mass median diameter" or "MMD" or "X 50 " is reported herein to mean the median diameter of particles within a population of particles that is typically polydisperse, i.e., a population of particles that consists of a range of particle sizes. 50 Values ​​are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany) unless the context indicates otherwise.

[0267] As used herein, "tapped density" or "ρ tapped " is the USP <616> It was measured in a manner similar to Method I described in Bulk Density and Tapped Density of Powders. Tapped density represents a closer approximation of particle density than injected bulk density, and measurements are approximately 20% lower than the actual particle density.

[0268] As used herein, "mass median aerodynamic diameter" or "MMAD" refers to the average aerodynamic size of a plurality of particles, typically in a polydisperse population. "Aerodynamic diameter" is generally the diameter of a unit density sphere that has the same settling velocity in air as a powder, and is therefore a useful method for characterizing the settling behavior of an aerosolized powder or other dispersed particle or particle composition. Aerodynamic particle size distribution (APSD) and MMAD are determined herein by cascade impaction using a NEXT GENERATION IMPACTOR™ (Copley Scientific). In general, if a particle is too aerodynamically large, fewer particles will reach a particular region of the lung. If a particle is too small, a larger percentage of the particles may be exhaled. In contrast, d a represents the aerodynamic diameter of a single particle.

[0269] As used herein, "nominal dose" or "ND" refers to the mass of drug loaded into a receptacle (e.g., a capsule or blister) in a non-reservoir-based dry powder inhaler. ND is sometimes also referred to as a metered dose.

[0270] As used herein, "emitted dose" or "ED" refers to a measure of the delivery of dry powder from an inhaler device after an actuation or dispersion event from a powder unit. ED is defined as the ratio of the dose delivered by the inhaler to the nominal or metered dose. ED is an experimentally determined parameter and can be determined using an in vitro device set-up that mimics patient dosing. ED is sometimes also referred to as delivered dose (DD).

[0271] As used herein, "fine particle fraction" (FPF) refers to the percentage of active ingredient in an emitted dose that has an aerodynamic size of less than 5 μm. Aerodynamic particle size distribution (APSD) is determined herein by cascade impaction using a NEXT GENERATION IMPACTOR™.

[0272] As used herein, "Solids Content" refers to the concentration of one or more active ingredients and excipients dissolved or dispersed in the liquid solution or dispersion that is to be spray dried.

[0273] As used herein, the "airway of a subject" refers to any or all of the following lung structures: the trachea, bronchi, and bronchioles of the lower respiratory tract.

[0274] The term "about" refers to the variation in numerical values ​​typically encountered by one of ordinary skill in the art of respiratory 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 ​​set forth herein.

[0275] Throughout this specification and in the claims which follow, unless the context indicates otherwise, the word "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of a stated integer or step or group of steps, and not to mean the exclusion of any other integer or step or group of integers or steps.

[0276] Unless otherwise stated or apparent from the context, numerical ranges include the endpoints and any value therebetween. EXAMPLES

[0277] Various aspects and embodiments of the present disclosure will be further elucidated with reference to the following examples, which are illustrative in nature and should not be construed as limiting the subject matter of the present disclosure.

[0278] Characterization method Amphotericin B (AmB) content and purity. AmB content and purity of formulated bulk powder or aerodynamic particle size distribution (APSD) samples were determined by reverse phase high performance liquid chromatography (RP-HPLC) with detection at 383 nm. Samples were analyzed using an Agilent 1260 Infinity II HPLC system (Wilmington, Delaware, USA). Separation was achieved on an Agilent InfinityLab Poroshell 120 EC-C18, 3.0 x 150 mm, (2.7 μm) column using gradient elution (solvent A = 10 mM acetate buffer, pH 4.2; solvent B = acetonitrile / methanol, 1.0 v / v). AmB was quantified using a one-point calibration with USP certified reference standards.

[0279] 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, Delaware, USA). Separation was achieved on a Haisil Clipeus™ C18 column (5 μm) using an isocratic elution method (acetonitrile / isopropanol, 1.0 v / v). Cholesterol was quantified using a one-point calibration with Cholesterol HP, Ph.Eur / USP-NF raw material (Carbogen Amcis, Barbery-la-Forêt, France).

[0280] 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 microdosing unit, and a RODOS / M dry powder dispersion unit. Approximately 2 mg to 5 mg of powder was filled into an ASPIROS tube and fed at 5 mm / s into the RODOS, which operated at a dispersion pressure of 4 bar and a vacuum of 65 mbar. Powder was introduced at an optical density of approximately 1% to 5% and data were collected over a measurement period of up to 15 seconds. Particle size distribution was calculated by the instrument software using the Fraunhofer model. Values ​​reported represent the average of three independent measurements for each collector.

[0281] Tap density. Tap density (ρ tapped ) is the known volume (0.593 cm 3 The tap density was determined using a cylindrical cavity having a diameter of 1 / 4" (1 mm). Powder was filled into the sample holder using a microspatula. The sample cell was then lightly tapped on the platform. When the sample volume decreased, more powder was added to the cell. The tapping and powder addition steps were repeated until the cavity was filled and further tapping did not further compact the powder bed. The tap density is defined as the mass of this tapped powder bed divided by the volume of the cavity.

[0282] Bulk density. Bulk density (ρ bulk ) represents the mass of powder loaded into the sample holder to the required volume without tapping.

[0283] Carr's Flowability Index. The Carr's Flowability Index C provides an indication of the compressibility of a powder. It is expressed by the following formula:

[0284]

number

[0285] As the Carr flowability index increases, the flowability of the powder is considered to decrease. Values ​​below 10% are indicative of powders with the best free flowing properties, values ​​between 11 and 15% are indicative of powders with good free flowing properties, values ​​between 16 and 20% are associated with fair powder flowability, values ​​between 21 and 25% are associated with acceptable powder flowability, values ​​between 26 and 31% are associated with poor powder flowability, values ​​between 32 and 37% are associated with very poor flowability, and values ​​above 38% are associated with almost no flowability.

[0286] Moisture content. Moisture content was determined by Karl Fischer coulometric titration using a Nitto Seiko Analytech Moisture Analyzer CA-310 equipped with a fritless cathode and a vaporizer model VA-300.

[0287] Dynamic water vapour sorption. Moisture sorption isotherms at 25°C were measured using a dynamic vapor sorption (DVS) instrument manufactured by Surface Measurement Systems (UK). This instrument gravimetrically measures the uptake and loss of water vapour by a material. The DVS system is equipped with a recording microbalance with a resolution of ±0.1 μg and a drift of approximately ±1 μg per day. The first step in the experimental run was to dry the samples at 25°C and 0% RH for 24 h to bring the samples to constant mass. The instrument was then programmed from 0-2% RH to 5% RH, after which the RH was increased in 5% RH increments up to 90% RH and decreased in 5% RH increments from 90% to 0% RH. dm / dt=0.005% / min was chosen as the equilibrium criterion that the system would reach at each RH step before automatically progressing to the next RH step. The mass of the samples used in this study was between 10 and 15 mg.

[0288] Differential Scanning Calorimetry. The DSC thermograms of a given sample were measured using a TA Instruments Model Q2000 Differential Scanning Calorimeter (New Castle, Del.) equipped with a refrigerated cooling system. The sample cell was fitted with a flow rate of 50 cm. 3The refrigeration control system (RCS) was purged with dry nitrogen at a flow rate of 110 cm 3 Nitrogen was used at 5°C / min. Tzero aluminum pans containing approximately 5-10 mg of powder were sealed using a sample encapsulation press. Samples were equilibrated at -40°C and then heated to 200°C at 5°C / min.

[0289] Aerodynamic particle size distribution. Aerodynamic particle size distribution (APSD) was determined using an RS01 dry powder inhaler (Mod. 7 Ultra High Resistance 2 model), a USP induction port (IP), and a Next Generation Impactor™ or NGI™ and was calculated according to the USP <601> and Ph.Eur.2.9.18. The flow control device was adjusted to operate with a pressure drop of 4 kPa and a total volume of 4 L passing through the inhaler. The resistance of the RS01 DPI variant used was 0.143 cmH2O. 0.5 L -1 min (0.045kPa 0.5 L -1 This is 44.2 L min at a pressure drop of 4 kPa. -1 Approximately 10 mg was hand-filled into size #3 inhalation grade HPLC capsules (VCaps, Qualicaps). The aerosol powder emitted from the inhaler was drawn through a USP IP and sized in the NGI. At each stage of the NGI, the emptied capsules and devices were extracted with a sample dissolution solution containing methanol. Further dilutions were performed to reduce the AmB concentration within the linearity of the detection range. The mass of AmB at each stage was determined using the HPLC method described above and the <5 μm fine particle dose (FPD) was calculated. <5μm ) and mass median aerodynamic diameter (MMAD) were calculated.

[0290] Cell lines and growth conditions. NuLi, CuFi-1, and CuFi-4 cells (Welsh lab, University of Iowa) were grown in Thermo Scientific BioLite cell culture-treated 75 cm 2Cells were grown from frozen stocks in flasks. The flasks were pre-coated with 4 mL of 60 μg / mL human placental collagen type IV (Sigma-Aldrich) for a minimum of 1 h at 37°C prior to seeding, rinsed twice with PBS and allowed to dry. Cells were cultured in 12 mL of Bronchial Epithelial Cell Growth Medium (BEGM) BulletKit (Lonza CC-3170) containing basal medium and eight SingleQuots supplements (bovine pituitary extract (BPE), 2 mL; hydrocortisone, 0.5 mL; hEGF, 0.5 mL; adrenaline, 0.5 mL; transferrin, 0.5 mL; insulin, 0.5 mL; retinoic acid, 0.5 mL; triiodothyronine, 0.5 mL). The Gentamicin-AmB aliquot was discarded and instead the 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). The original CF transplant donor was genotyped by Integrated Genetics. The cell line was secondarily confirmed by the ATCC repository to have the correct genotype and to be free of mycoplasma contamination. The MycoAlert Mycoplasma Detection Kit (Lonza LT07-418) was used to detect any RNA transcripts common to a wide range of mycoplasmas. The cell line was confirmed to be free of mycoplasma. Cells were grown to >90% confluence at 37°C, 5% CO2 and then trypsinized with 4mL of 0.25% trypsin (Gibco 25200-056) containing 1mM EDTA. Trypsin was inactivated with 10mL of HEPES-buffered saline (Lonza CC-5024) containing 1% fetal bovine serum. Cells were centrifuged at 1,500 rpm for 5 minutes at room temperature in an Eppendorf centrifuge 5430R and resuspended in BEGM medium for passaging.For culture on differentiation membrane supports, cells were resuspended after centrifugation in Ultroser G medium, which consisted of 1:1 DMEM:Ham's F-12 supplemented with 4 v / v% Ultroser G (Crescent Chemical) and 50 μg / mL penicillin-streptomycin (Corning Cellgro), 50 μg / mL gentamicin (Sigma-Aldrich G1397), and 2 μg / mL fluconazole (Sigma-Aldrich). For all studies, the membrane supports used were Corning Costar 0.4 μm 24-well plates, Transwell clear polyester membrane inserts (0.33 cm). 2 ) (Corning 3470). These membranes were collagen coated in an identical manner to the flasks detailed above, except that 100 mL of collagen was used and only one rinse with PBS was used. The inserts were seeded with 115,000 cells each. The membranes were matured at the air-liquid interface for a minimum of 14 days until full differentiation was achieved, with Ultroser G medium changed at least once a week as needed. After maturation, medium was changed every 7 days. For covariate controls, the membranes used in the experiments were as old and mature as possible.

[0291] For primary cultured airway epithelial cells, embryonic cells were obtained from individuals with CF undergoing embryo transfer or organ donation. Tissue was dissociated and the dissociated cells were seeded directly onto Transwell filters and cultured at the air-liquid interface. Cultures were used ≥3 weeks after seeding, when epithelial cells had differentiated into multiple cell types typical of the airway and the electrical properties of the cells reflected those of the excised tissue.

[0292] Fluorescence microscopy assay for measurement of airway surface liquid pH. Fresh suspensions of ABCI were prepared for each experiment by dispersing approximately 2 mg of ABCI in approximately 100 μL of perfluorohexane, PFH (FC-72, Sigma-Aldrich) to reach a final AmB concentration of approximately 1 mM. After dissolution in methanol, AmB concentrations in stock suspensions were measured in triplicate by absorbance spectroscopy. Concentrations were determined by measuring the absorbance at 406 nm (ε 406 =164,000M -1 cm -1 ) was calculated using Beer's law. The stock suspension was then diluted with PFH to give suspensions with AmB concentrations ranging from approximately 0.5 to 50 μM.

[0293] Small diameter NuLi and CuFi cells were used to measure ASL pH. ASL pH was measured using SNARF-conjugated dextran (Molecular Probes), a proportional pH indicator. SNARF powder was suspended in PFH by sonication and diffused onto the apical surface of the cells. After 2 hours, ASL pH was measured. SNARF was excited at 514 nm and emission was recorded at 580 nm and 640 nm using a Zeiss LSM 800 microscope equipped with a water immersion lens for cell line cultures at 40x magnification. 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 powders tested in this assay were suspended in an appropriate volume of PFH, which were sonicated for 1 min to aid in suspension. AmBisome was suspended in PFH by vortexing. 20 μL of the suspension was then applied to the surface of cultured airway epithelium (A = 0.33 cm 2 ) at concentrations ranging from 0.5 to 50 μM. In all experiments, the pH of the ASL of compound-treated epithelia was measured and compared to results obtained from vehicle-treated epithelia. For apical compound administration, cultured airway epithelia were incubated at 37°C for approximately 22 hours before ASL pH measurement.

[0294] Preparation of red blood cell stock suspension. 1 mL of human whole blood (Na-heparin preparation; BioIVT, Westbury, NY) was centrifuged at 10,000xg for 2 min at room temperature. The supernatant was removed and the pellet was resuspended in 1 mL of 0.9% (m / v) saline with gentle inversion (red blood cells are lysed by pipetting and vortexing). The resulting suspension was centrifuged at 10,000g for 2 min. The supernatant was then removed and the saline wash was repeated twice. After the final wash, the supernatant was removed and the red blood cell pellet was resuspended in 1 mL of resuspension buffer (10 mM Na2HPO4·7H2O, 10 mM NaH2PO4·H2O, 150 mM NaCl, 1 mM MgCl2·6H2O, pH 7.4) to create the red blood cell stock suspension.

[0295] Minimum hemolytic concentration (MHC) assay. Compounds to be tested were prepared in a dilution series in DMSO (D6-99.9%; Cambridge Isotope Laboratories) such that each concentration was 25.63 times the final concentration. Compound dilutions were diluted 1:25 in suspension buffer for a total of 100 μl in 0.2 mL microcentrifuge tubes and the solutions mixed by vortexing. The negative control (0% lysis) contained DMSO in suspension buffer only, and the positive control (100% lysis) contained DMSO in water only, since the latter would completely lyse the red blood cells due to osmotic pressure. A volume of 2.52 μL of red blood cell suspension was added to each tube (including the control), each tube was mixed by gentle inversion, and incubated stationary for 2 hours at 37°C. After incubation, each sample was mixed again by gentle inversion and centrifuged at 3,214×g for 6 minutes. After centrifugation, 60 μL of supernatant was removed and added to a 96-well plate and absorbance was read at 540 nm. Data was normalized to the negative control and treated as % total hemolysis relative to the positive control.

[0296] Example 1: Lipid-Coated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004) The nominal anhydrous compositions of four compositions of lipid-coated AmB (ABCI-001, ABCI-002, ABCI-003, ABCI-004) are shown in Table 4. Three controls are also shown: AmBisome® (i.e., liposomal amphotericin B, L-AmB); a lyophilized AmB / Chol complex prepared by Burke et al. (US Pat. No. 5,326,691; US ​​Pat. No. 5,404,217; US Patent Publication No. 2012 / 0128728); and Amphotericin B Inhalation Powder (ABIP), a dry powder composition of AmB developed by Nektar Therapeutics for the treatment of invasive pulmonary aspergillosis (US Pat. No. 7,326,691; US ​​Pat. No. 8,404,217; US Patent Publication No. 2012 / 0128728).

[0297] [Table 4]

[0298] The AmB / lipid compositions detailed in Table 4 are plotted on the pseudo-ternary phase diagram in Figure 2. The PL component at the apex of the ternary phase diagram represents the sum of the masses of the various PL components (e.g., HSPC, DSPC, DSPG).

[0299] Commercially available L-AMB contains the same lipid components (i.e., HSPC, DSPG, Chol) as the ABCI composition, but they are present in different ratios. Furthermore, L-AMB has a different drug and lipid composition compared to the ABCI composition. The drug substance in L-AMB is encapsulated in small unilamellar vesicles (liposomes). The composition has a Chol / AmB ratio of 2.5 mol / mol and a HSPC / DSPG ratio of 2.3 (i.e., 7 / 3 w / w). The lipid particles are lyophilized to a dry powder in the presence of a high percentage of sucrose, which acts as a cryoprotectant to maintain the integrity of the liposomes during lyophilization. 4-Head CFTR - / - 1cm of pig 2When 60 mg of reconstituted L-AMB was administered to the apical side of airway epithelial cells via the tracheal window, the pH of the ASL increased by approximately 0.2 pH units, from pH 6.8 to pH 7.0 (Patent Document 2).

[0300] The lyophilized AmB:Chol complex studied by Burke et al. (Patent Document 1; Patent Document 2) has a higher Chol / AmB ratio of 5.0 mol / mol, but no PL is added. The AmB:Chol complex is formed by flash nanoprecipitation from a solution of material in dimethylsulfoxide / chloroform when the solution is rapidly injected into a non-solvent (water). The resulting suspension is lyophilized to form a dry powder. The AmB:Chol complex showed a large increase in bicarbonate secretion and a large increase in the pH of the ASL (about 0.1-0.2 pH units) in CuFi-1 cells.

[0301] ABIP is a spray-dried composition containing wet-milled AmB crystals coated with DSPC and calcium chloride in a molar ratio of 2:1. This composition does not contain cholesterol (i.e., Chol / AmB=0 mol / mol). It also contains only saturated phosphatidylcholine (DSPC) and no added DSPG. ABIP improves the pH of the ASL of CuFi-1 cells at low doses (2 μM with FC-70), but not at high doses (50 μM) (Examples 10 and 12). This result is consistent with previous studies with pure AmB, suggesting that some Chol is required to maintain ion channel activity over a wide range of AmB concentrations. ABIP compositions containing highly crystalline drug substance have been well tolerated in early clinical development.

[0302] Compositions are also shown for the three ABCI compositions (ABCI-001, ABCI-002, and ABCI-003) used in the nonclinical toxicology studies. The Chol / AmB ratios for these powders range from 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, since the presence of large amounts of Chol may lead to disordered packing of PL acyl chains and an unacceptable increase in cohesive forces between particles, resulting in "sticky" powders. Both ABCI-003 and ABCI-004 are encompassed within the shaded triangle in FIG. 2, whereas ABCI-001 and ABCI-002 are not.

[0303] As mentioned above, ABCI-001 and ABCI-002 are outside the shaded triangle in Figure 2 because the lipids in these compositions 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 studied to date (i.e., ABIP, L-AMB (AmBisome), and AmB:Chol complex) are significantly outside the shaded triangle in Figure 2.

[0304] The DSC thermogram of ABCI-001 (30 w / w% AmB, Chol / AmB=1.2 mol / mol) contains a significant proportion of Chol-rich Lo phase (Example 4). For ABCI-002, the drug content is reduced to 14 w / w% while the Chol / AmB molar ratio is kept constant, which results in a decrease in the Chol / PL ratio from 0.29 w / w to 0.09 w / w. Nevertheless, a small proportion of Chol-rich Lo phase still remains. Dry powders with phase-separated Lo phases may exhibit high interparticle cohesion forces and increased hygroscopicity and deliquescence at high relative humidity (Example 5). These characteristics may adversely affect powder yield during spray drying and may also result in a relatively large mass median aerodynamic diameter (MMAD=about 4 μm) (Example 9).

[0305] ABCI-003 maintains the 14 w / w% drug loading of ABCI-002, but the nominal Chol / AmB ratio is reduced to 0.4 mol / mol. Overall, the Chol content is reduced from 14.6% in ABCI-001 to 2.4% in ABCI-003, and the Chol / PL ratio is reduced to 0.03 w / w. At this Chol / PL ratio, the Lo phase is solubilized in the So phase (Example 4). ABCI-003 also increases the HSPC / DSPG ratio from 2.3 w / w to 9.0 w / w, which improves the solubility of Chol in the PL phase, reduces NaCl formation, and reduces the hygroscopicity of the powder (Example 5).

[0306] Unlike ABIP, all ABCI compositions containing added Chol show significant improvement in ASL pH at both low and high AmB concentrations (Example 10). Furthermore, the presence of DSPG has been shown to enhance the degree of ASL pH improvement observed (Example 12).

[0307] Many of the constraints on Chol / AmB, Chol / PL, and HSPC / DSPG ratios are specific to the development of dry powder compositions for inhalation and may not be applicable to liquid-based aerosols such as inhaled L-AMB, nor may they be directly applicable to compositions that do not contain PL.

[0308] Example 2: Preparation of ABCI-001 by spray drying Preparation of raw materials. First, lipids and calcium chloride are dispersed in warm water using a high shear mixer (UltraTurrax T-50) to form multilamellar vesicles (MLVs). To promote the formation of MLVs, the aqueous phase is heated to the lipid main transition temperature (T m) requires higher temperatures (T>65°C). The MLV dispersion was cooled (T<30°C) and perfluorooctyl bromide (PFOB) was filtered and added with mixing using a Watson-Marlow peristaltic pump to form crude PFOB-in-water emulsion droplets stabilized by a lipid monolayer. The emulsion droplets act as pore formers to form a porous coating on the crystalline drug particles. The crude emulsion was homogenized in a Model M-100 microfluidizer in one separate pass under high pressure to form nanoemulsion droplets (diameter approx. 200-500 nm). The drug substance was then added to the nanoemulsion under high shear mixing. The composite dispersion consisting of suspended drug and nanoemulsion droplets was passed through the homogenizer for two more separate passes. The homogenization process wet-mills the AmB particles to a size suitable for pulmonary delivery. On a number basis, most of the wet-milled AmB crystals have diameters less than 1000 nm. In one embodiment, the final feed composition had a solids content of 3.0 w / w%, a PFOB content of 20 v / v%, and a theoretical batch size of 318 g (9 liters of feed) or 424.2 g (12 liters of feed).

[0309] Production of dry powders 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 internal diameter), a DorrClone cyclone, and a lower end geometry with a straight pipe and a Brewer valve. A 1 L Eagle collector mounted below the Brewer valve was jacketed and maintained at 50°C.

[0310] The liquid feed was pumped to the spray dryer by 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 was adjusted to maintain the dryer outlet temperature at 55±3°C. The total gas flow rate was approximately 140 Nm 3 / hour (approximately 85 scfm).

[0311] In suspension-based feed, each atomized droplet (mass median diameter=about 10 μm) contains dispersed drug crystals and approximately 1000 submicron-range emulsion droplets. At the beginning of the drying process, the relatively highly volatile aqueous phase begins to evaporate. The rapid retreat of the atomized droplet interface enriches the interface with slowly diffused drug and emulsion particles. This creates a void at the center of the droplet being dried. As the drying process continues, the relatively less volatile oil phase in the emulsion droplet evaporates, forming a hollow pore at the location of the oil phase. Overall, the resulting hollow spray-dried composite particles contain drug crystals embedded in the interphase of the porous lipid matrix.

[0312] ABCI-002, ABCI-003, and ABCI-004 were manufactured using the same overall process, but differed in the composition of the liquid feed.

[0313] Example 3: Wet-milling of AmB and its effect on the properties of ABCI-002 The physical form of AmB (crystalline or amorphous) may affect its wet-grinding 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).

[0314] In fact, the low crystallinity of batch '202 had a detrimental effect on the wet-milling process, resulting in the X-ray diffraction pattern of the wet-milled drug. 50 and X 90 (determined by laser diffraction using a Malvern Mastersizer on a suspension of the drug) was significantly increased (Table 5).

[0315] [Table 5]

[0316] Multiple batches of ABCI-002 were produced in the Niro Mobile Minor using the process described in Example 2 with AmB batches '202 (ABCI-002 batch FP21060) and '203 (ABCI-002 batch FP21059). The total solids content and %PFOB in the liquid feed were 2 w / w% and 10 v / v%, respectively. The liquid feed rate was approximately 44.5 g / min. The atomizer had a gas flow rate of 11.5 ± 1.0 Nm 3 The gas was operated at 7.0 scfm / hr with an inlet temperature of 104±5°C. This corresponded to approximately 44.5 g / min. The total gas flow rate was approximately 140 Nm 3 / hr (approximately 85 scfm). The target batch size was 40 g.

[0317] The physicochemical properties and aerosol performance of the two batches are detailed in Table 6. No significant differences were observed in the powder properties and aerosol performance, suggesting that even a crystallinity as low as 77% remains suitable for the preparation of ABCI-002 compositions.

[0318] [Table 6]

[0319] Example 4: Effect of lipids on the phase behavior of lipid-coated crystalline compositions of AmB A comparison of the DSC thermograms for ABCI-001, ABCI-002, ABCI-003, and ABIP is shown in FIG.

[0320] ABIP is a single steep and coordinated T m with an extrapolated onset temperature of 96.2° C. The addition of Chol and DSPG to the ABCI composition results in a more complex thermogram with a broad transition and multiple peaks.

[0321] At a Chol / PL ratio of 0.29 w / w (ABCI-001), two phase-separated domains are observed: a Chol-rich Lo phase with an onset temperature of 62.5 °C and a broad PL-rich So phase with an onset temperature of 83.9 °C. As demonstrated for hydrated DPPC bilayers (Fig. 1), the addition of Chol to the So phase leads to increased disorder and ultimately to phase separation with the coexisting Lo phase.

[0322] The Chol-rich phase transition is very broad and contains overlapping features. The presence of Chol crystallites in the Lo phase is supported by the powder X-ray diffraction (XRPD) patterns, which show a diffraction peak at approximately 5.2° 2θ for the ABCI variants. The phase separation of cholesterol crystallites is more easily distinguished in PLCI-001 (i.e., fewer diffraction peaks in the same region and no interference from amphotericin B peaks) (Figure 4). The PLCI composition is a placebo composition that does not contain AmB but does contain the other components of the ABCI composition.

[0323] As the Chol content of 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 for Chol / (Chol+PL)>10 w / w%. The data is 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 about 9.4 mol% Chol. Thus, compositions with less than about 9.4 mol% Chol can be expected to have an undetectable low temperature peak.

[0324] In the dehydrated state, the onset of the So-Lo2 phase region tends to occur at much higher Chol concentrations (i.e., relative to the hydrated state in Figure 1). Thus, the ordered So phase of the ABCI composition is maintained at much higher Chol contents (about 9.4 mol% versus 6 mol% for the hydrated DPPC bilayer). Also, the So phase is eliminated in hydrated DPPC / Chol mixtures above about 20 mol% Chol (Figure 1), whereas in dehydrated ABCI-001 the So-Lo2 phase region is extended beyond 36.5 mol% Chol. Thus, compared to the phase diagram of the hydrated DPPC-Chol mixture in Figure 1, the phase diagram of ABCI-001 shows a higher T m values ​​and to the right to higher cholesterol content.

[0325] ABCI-001 High T m The peaks are also very broad and contain overlapping features. m The enthalpy of the peak tends to increase with increasing PL content and to decrease with increasing Chol content, which is due to the T m This suggests that this peak below is related to the So phase.

[0326] The binary mixture of HSPC / DSPG with 20% AmB composition (without Chol) showed similar overlapping features, suggesting the coexistence of an immiscible So phase. The phase separation of PC and PG domains in the presence of calcium ions is consistent with observations in other studies. The high T m The onset temperature and enthalpy for the peak are 83.9° C. and 19.74 J / g, respectively.

[0327] By transition from ABCI-001 to ABCI-002, the drug loading was decreased from 30 w / w% to 14 w / w%, while the Chol / AmB ratio was maintained at 1.2 mol / mol. The decrease in AmB and Chol led to an increase in PL content and a decrease in the Chol / PL ratio from 0.28 w / w to 0.092 w / w. A small amount of low T mIf the peak remains visible in ABCI-002, T m The T increased from 62.5 to 64.6 °C for the Chol-rich domain, while ΔH decreased from 7.73 to 1.93 J / g (Fig. 3). m The peaks remain broad with overlapping features. Compared to ABCI-001, the onset temperature increases from 83.9 to 88.5 °C, suggesting increased order in the acyl chains with increasing Chol content.

[0328] In ABCI-003, the drug loading remains at 14 w / w%, but the Chol / AmB ratio is reduced from 1.2 mol / mol to 0.4 mol / mol. This allows the Chol / PL ratio to be reduced from 0.092 w / w to 0.030 w / w compared to ABCI-002. The low T of ABCI-003 thermogram m The expansion of the peak area (Figure 3) shows that the Chol-rich peak has been eliminated. m The peak is also much steeper, with an onset temperature of 91.1°C, an increase of 7.2°C compared to ABCI-001.

[0329] For long-term stability of an amorphous solid, the glass transition temperature T g is the storage temperature T s It may be beneficial to have a T of at least 50°C higher than the T of the PL. m represents the order-disorder transition. T m is 91.1℃, the acceleration Ts is 40℃, T m This means that the temperature exceeds 50°C.

[0330] As mentioned above, decreasing the Chol / PL ratio has a significant effect on the powder properties, increasing the production yield from 74.5% to 82.4%, decreasing the Carr flowability index from 41.4 to 27.0, and also dramatically decreasing the interparticle cohesion as shown by the approximately half-reduction in the MMAD.

[0331] A linear decrease in the enthalpy of the Lo phase transition is observed with increasing Chol content of the powder composition (Figure 5). A multipoint regression suggests that the x-intercept (i.e., the point where the enthalpy becomes zero) occurs at a Chol / PL ratio of 0.05 w / w. This ratio is used to define the shaded region in Figure 2.

[0332] Example 5: Effect of lipid composition on hygroscopicity of AmB compositions at high relative humidity FIG. 6 shows the dynamic water vapor sorption profile for various compositions. A significant increase in water sorption is observed for the composition containing Chol and DSPG compared to ABIP. Without intending to be bound by theory, it is believed that the increase in water sorption is due to the presence of NaCl in the composition, which occurs through the interaction of DSPG with calcium chloride. Divalent calcium ions can bind strongly to the anionic DSPG Na, resulting in the displacement of sodium ions, which can then interact with chloride ions from CaCl2 to form NaCl. At high RH, compositions containing DSPG / CaCl2 deliquesce, and the extent of water sorption and deliquescence is directly proportional to the amount of NaCl formed.

[0333] ABCI-003, which has an HSPC / DSPG ratio of 9.0 w / w in the PL component, is significantly less hygroscopic than ABCI-001 and ABCI-002, which have an HSPC / DSPG ratio of 2.3 (i.e., 7 / 3) w / w.

[0334] Example 6: Physicochemical properties of ABCI-001 Spray-dried powder of ABCI-001 was produced in 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, tapped density, Carr's flowability index, and moisture content) are detailed in Table 7.

[0335] Characterized by powder spray drying from a drug suspension, the ABCI-001 powder is enriched in AmB by approximately 10% of the nominal drug content. This enrichment reduces the Chol / AmB molar ratio to 1.0 mol / mol. Despite this enrichment, the assay values ​​for AmB and Chol were consistent across the five batches with relative standard deviations (RSDs) of 5% and 1%, respectively. The purity of the input 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 the five ABCI-001 lots. Furthermore, no new degradant peaks were observed in the RP-HPLC chromatograms.

[0336] [Table 7]

[0337] The primary particle size distribution obtained by laser diffraction is typical of spray-dried microparticles from an emulsion-based feedstock and is the average X of five batches. 50 The value was 1.9±0.1 μm, with an average X 90 is 4.7±0.5μm. Within a batch, X 50 The RSDs for varied between 4.7% and 9.6%, with most of this variation coming from the first collection before equilibrium was established in the spray dryer.

[0338] The observed low bulk density (0.071 ± 0.008 g / cm 3 ) and tap density (0.121±0.015g / cm 3 ) are also characteristic of powders made from emulsion-based liquid feedstocks (Table 7). The high compressibility of the fine, low-density particles was demonstrated by the average Carr flowability index of 41.5 ± 2.0%, suggesting that the ABCI-001 powder has little or no flowability. The average residual moisture content of the powders was 3.2 ± 0.2%.

[0339] Example 7: Physicochemical properties of ABCI-002 The physicochemical properties of ABCI-002 are shown in Table 8. These results are somewhat similar to those shown for ABCI-001. This is not surprising considering that ABCI-002 (14 w / w% AmB) represents ABCI-001 (30 w / w% AmB) diluted with additional PL while maintaining a constant Chol / AmB ratio of 1.16 mol / mol.

[0340] [Table 8]

[0341] Example 8: Physicochemical properties of ABCI-003 The physicochemical properties of ABCI-003 are detailed in Table 9. Compared to ABCI-001, the drug loading was reduced from 30 w / w% to 14 w / w%, and the nominal Chol / AmB ratio was reduced from 1.2 mol / mol to 0.4 mol / mol.

[0342] [Table 9]

[0343] Example 9: Comparison of three ABCI compositions A comparison of the physicochemical properties and aerosol performance of the three ABCI compositions is shown in Table 10.

[0344] [Table 10]

[0345] There are several clear trends when increasing PL / Chol from 3.5 w / w to 10.9 w / w to 33.1 w / w (ABCI-001 to ABCI-003): first, the yield increases from 74.5% to 82.4% (+10.6%), and second, the Carr flowability index decreases from 41.4 to 27.0, indicating a significant improvement in powder flowability.

[0346] There is also a significant change in aerosol performance. This may reflect a significant decrease in interparticle cohesion and an increase in powder dispersibility as the Chol content decreases. The percentage of drug in the coarse fraction decreases from 36.0% to 6.1%, while the percentage in the respiratory tract fraction increases from 49.3% to 71.8%. FPF <5μm The ED increased from 56% to 92% and the MMAD decreased from 3.9 to 2.1 μm. The significant reduction in MMAD also allows for reduced deposition in the nasal cavity of rodents when delivering aerosols to the nose only.

[0347] Example 10: Improvement of ASL pH following apical administration to cultured airway epithelial cells with respect to various compositions of AmB A cell-based assay was used to investigate the efficacy of various lipid-coated crystal compositions of AmB. The pH of ASL observed in normal lung epithelial cells (NuLi) is approximately 7.6. In contrast, the pH of ASL observed in CuFi-1 cells (ΔF508 / ΔF508 CF mutant) is approximately 6.85. A decrease in the pH of ASL has a significant impact on host defense in CF lungs. ASL pH values ​​higher than 7.0 improve the efficacy of defensins, peptides with antibacterial activity in the lung that are important for host defense. Thus, measurements of the pH of ASL are a useful surrogate for evaluating the efficacy of various inhaled AmB compositions.

[0348] Table 11 compares the improvement in ASL pH observed for various AmB compositions using immortalized primary airway epithelial cells. The ASL pH observed in a normal immortalized airway epithelial cell line (NuLi) developed at the University of Iowa is approximately pH 7.5. Two immortalized CF airway epithelial cell lines were studied: CuFi-1 cells with a homozygous ΔF508 / ΔF508 mutation, and CuFi-4 cells with a heterozygous G551D / ΔF508 mutation. The ASL pH measured in these cells varied from approximately pH 6.7 to pH 7.0 in the various studies. Primary cell cultures of airway epithelial cells from individuals affected with CF, including those with nonsense mutations, were also studied. The pH of ASL measured in primary airway epithelial cells from control subjects without CF had a pH of approximately 6.9 (Shah et al. Airway acidification initiates host defense abnormalities in cystic fibrosis mice. Science 2016, 351: 503-507).

[0349] Findings from these studies include: AmB compositions without added Chol (e.g., pure AmB, ABIP) are effective at a concentration of 2 μM in perfluorohexane vehicle, but not at an AmB concentration of 50 μM (Figure 7b); AmB / Chol complexes show approximately a 0.1 pH unit improvement over vehicle controls at AmB concentrations of 2 μM and 50 μM (Table 11); following administration of 2 μM AmB to the apical side of primary cultured airway epithelial cells from subjects with CF, there is an approximately 0.1 pH unit improvement in the apical side of primary cultured airway epithelial cells from subjects with CF, regardless of CFTR genotype. An improvement in ASL pH of ~0.2 pH units is observed (Table 11); L-AMB supplemented with PL (HSPC / DSPG=7 / 3 w / w) shows an improvement in ASL pH of about 0.4 pH units, which is maintained at 2 μM and 50 μM AmB (Table 11); Ivacaftor increases ASL pH by about 0.2 pH units in CuFi-4 cells, but as expected, only minimally in CuFi-1 cells harboring a homozygous F508 mutation (Table 11); Trikafta increases ASL pH by only about 0.2 pH units in CuFi-4 cells, but only minimally in CuFi-1 cells harboring a homozygous F508 mutation (Table 11); In CuFi-1 cells, ABCI compositions with Chol / AmB ratios of 0.4 mol / mol or greater provide a similar increase in ASL pH as ABCI-003 (Table 11); while ABCI compositions with Chol / AmB ratios of 0.4 mol / mol or greater provide a maximal 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, the extent of improvement in ASL pH decreases at Chol / AmB ratios below 0.4 mol / mol (Figure 7a; Figure 7c); improvements of more than 0.2 pH units were observed for Chol / AmB ratios as low as 0.05 mol / mol. is observed (Figure 7b; Figure 7c); the improvement in ASL pH is enhanced by the presence of DSPG in the composition compared to HSPCs alone (Figure 7b; Figure 7c); no significant difference in ASL pH was observed following administration of ABCI-001, ABCI-002, and ABCI-003 (Figure 7a); the average improvement in ASL pH in primary cultured airway epithelium from individuals with CF was approximately 0.7 pH units, from pH 6.3 to pH 7.0; six of seven primary cultured airway epithelium samples from individuals with CF improved to pH 6.A pH of 9 or higher (i.e., complete restoration of the pH of ASL compared to healthy participants) was achieved, and the above samples included two cell cultures from individuals carrying nonsense mutations (Figure 8). Based on these results, the optimal Chol / AmB ratio for the ABCI composition could be 0.4-1.2 mol / mol (Figure 2).

[0350] [Table 11] TIFF2025510096000016.tif206170

[0351] Example 11: Dose Response Observed with ABCI-001 The dose-dependence of ASL pH improvement for ABCI-003 is shown in Figure 9. The concentration on the horizontal axis represents the concentration of AmB suspended in perfluorohexane vehicle for administration to CuFi-1 cells, not the actual ASL concentration. EC based on perfluorohexane concentration 50 and E.C. 90 were approximately 0.5 μM and 2.0 μM, respectively.

[0352] Example 12: ABCI compositions with various Chol / AmB ratios and DSPG contents Burke et al. demonstrated that an AmB:Chol complex with a Chol / AmB ratio of 5.0 mol / mol maintained improved ASL pH in CuFi-1 cells over a suspension concentration range of 2 μM to 50 μM in perfluorohexane (Patent Document 1; Patent Document 2; both of which are incorporated herein by reference).

[0353] Similar results were observed with the lipid-coated AmB composition of the present disclosure, provided that Chol was added. At an AmB concentration of 50 μM, ABIP (without added Chol) did not improve the pH of ASL.

[0354] In contrast, ABCI compositions with theoretical Chol / AmB ratios of approximately 1.2 (e.g., ABCI-001, ABCI-002) show strong improvement in the pH of ASL (e.g., 0.35-0.45 pH units) at 50 μM.

[0355] Previous studies have suggested that the benefits achieved by the addition of Chol may be lost at Chol / AmB ratios below 1.0 mol / mol (Patent Document 1; Patent Document 2). Surprisingly, it was discovered 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 an AmB concentration of 50 μM.

[0356] Thus, even a Chol / AmB ratio as low as 0.4 mol / mol remains effective, although Chol may be required to maintain effective improvement of ASL pH over a wide range of delivered doses. The observed efficacy for this low Chol / AmB ratio was established in a small-scale design of experiments (DoE) study (Figure 7A). The study also investigated the effect of reducing the concentration of DSPG in the composition. The theoretical anhydrous composition of the spray-dried powder produced with the Buchi B-191 spray drying base is detailed in Table 12. The physicochemical properties of the ABCI variants are shown in Table 13. The aerosol performance of selected ABCI variants is shown in Table 14.

[0357] [Table 12]

[0358] [Table 13]

[0359] [Table 14]

[0360] As the Chol and DSPG content in the powder decreases, the interparticle cohesion tends to decrease. This is reflected in the dramatic decrease in the coarse particle fraction, as indicated by deposition in stages 1 and 2 of the USP throat and impactor. 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 change in the pattern of stage deposition. The APSD of ABCI-001 is centered in stages 2 and 3, with an MMAD of 3.9 μm. ABCI-002 shows predominant deposition in stages 3 and 4, with a decrease in the MMAD to 2.6-2.7 μm. Finally, for batch R21058, the Chol / AmB ratio decreases to 0.4 mol / mol and the HSPC content in the PL increases to 90 w / w%. Here, the main deposition peaks at impactor stages 4 and 5, with an MMAD of 2.1 μm.

[0361] Data for batch R21058 expressed as a percentage of emitted dose: FPF <5μm Considering that 98% of the emitted dose of batch R21058 is deposited in multiple stages of the impactor (i.e., only 2% is deposited in the induction port or USP throat), the impaction parameters for the overall unimodal particle size distribution are

number

[0362] The significant reduction in MMAD not only reduces deposition in the URT in humans, but also reduces nasopharyngeal deposition in rats in non-clinical trials. The improvement in ASL pH achieved with the various ABCI variants is plotted in Figure 7. Data was collected at an AmB concentration of 50 μM in a fluorocarbon vehicle after 22 hours of incubation (N=12 replicates per composition). Data was also collected at 2 μM for batch R21058 (N=6 replicates). The ASL pH for the ABCI variants using CuFi-1 cells was approximately 7.25-7.35 (ΔpH=approximately 0.4-0.5). Thus, all batches showed a significant increase in ASL pH (p<0.0001 compared to CuFi-1 control). The degree of ΔpH improvement observed with the lipid-coated crystal compositions is significantly greater than the approximately 0.15-0.2 pH unit improvement observed with the AmB:Chol complex used by Burke et al. (Patent Document 1; Patent Document 2).

[0363] Example 13: Evaluation of the relative viscosity of ASL fluid in primary epithelial cells from humans with CF following administration of ABCI-003 Absence or dysfunction of the CFTR protein leads to defective ion transport, the underlying defect that causes ASL depletion (i.e., increased ASL thickness), increased ASL viscosity, and impaired mucociliary clearance.

[0364] The ability of ABCI to reduce the viscosity of the ASL fluid of primary cultures of CF airway epithelial cells, compared to the FC-72 delivery vehicle alone, was evaluated. Addition of ABCI to the apical side of primary cultures of CF airway epithelium reduced the mean ASL viscosity (τ ASL / τ saline ) values ​​were reduced from 3.3-fold compared to saline to approximately 1.9-2.2-fold, and similar activity was observed regardless of CFTR genotype (Figure 10).

[0365] The relative viscosity of ASL in non-CF airway epithelium is 2.5 times that of saline. Thus, these data indicate that ABCI can reduce the relative viscosity of ASL in CF airway epithelium to the level observed in airway epithelial cells from individuals not affected by CF. Because increased viscosity of ASL is associated with impaired mucociliary clearance and increased risk of infection / inflammation in subjects affected by CF, these data suggest that compositions according to embodiments described herein may alleviate one of the most serious sequelae of CF in the airways.

[0366] Example 14: Antibacterial and bactericidal activity of ASL fluid in primary epithelial cells from humans with CF following administration of ABCI Many aspects of the airway innate host defense system are pH sensitive, including the rheological properties of secreted mucins, mucociliary clearance, activity of proteases, and activity of antimicrobial peptides (e.g., defensins). The antibacterial activity of ASL against S. aureus in coated gold grids is reduced by approximately 50% in primary airway epithelium from subjects without CF.

[0367] The effect of apical administration of ABCI-003 on ASL antibacterial activity was evaluated in primary airway epithelial cells from six CF subjects with different CFTR mutations. Exposure of CF-derived airway epithelial cells to ABCI-003 increased the percentage of S. aureus killed from 22% in the vehicle control to 40% in the high-dose ABCI-003 group (i.e., nearly doubling the antibacterial activity of ASL to levels similar to those measured in non-CF cells) (Figure 11).

[0368] The CFTR mutation status of the CF subjects from whom cells were harvested is shown in Figure 11. Of note, the response in cells from two individuals with two nonsense mutations (refractory to modulator drugs) was at least as high as the individual with the more common ΔF508 mutation, which is a candidate for modulator therapy. Furthermore, the improvement in ASL killing for the 50 μM ABCI-003 dose was similar to non-CF controls.

[0369] Example 15: Effect of lipid composition and AmB crystallinity on hemolysis Toxicity of AmB. Intravenous administration of Fungizone results in a number of acute systemic reactions, including fever, shivering, chills, hypotension, hypertension, anorexia, nausea, vomiting, headache, and tachypnea, leading to the nickname "amphoterrible." Its main chronic side effect is nephrotoxicity. Although lipid-based compositions (e.g., L-AMB) reduce these acute systemic effects, nephrotoxicity remains the dose-limiting toxicity.

[0370] Recent work from Burke's laboratory suggests that the toxic effects of AmB in mammalian cells are due to the extraction of cholesterol from the plasma membrane into the extramembrane sterol sponge phase, rather than the formation of ion channels as previously assumed (Anderson et al. Nature Chem Biol. 2014; 10:400-406; Gray et al. PNAS. 2012; 109:2234-2239). Chol extraction toxicity was previously thought to occur only when the sterol:AmB ratio was less than about 1.0 mol / mol.

[0371] The physical form of the API (i.e., amorphous or crystalline) and particle size can affect the "apparent" solubility of AmB in water, which can 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 oral inhalation (US Patent Application Publication No. 2012 / 0128728). In the context of this disclosure, a hemolysis assay was used to evaluate the effect of API physical form and compositional attributes on Chol extraction from red blood cell membranes.

[0372] Table 15 compares the hemolysis after administration of various compositions containing AmB. Fungizone®, a micellar composition of AmB solubilized in sodium deoxycholate without Chol, serves as a positive control, and L-AMB serves as a negative control. ABCI-001, ABCI-002, and ABCI-003, all of which contain highly crystalline AmB and Chol, show similar hemolysis to the negative control. Surprisingly, this is true even when the Chol / AmB ratio is significantly less than 1.0. For example, ABCI-003, which has a theoretical Chol / AmB ratio of only 0.4 mol / mol (actual value = 0.33 mol / mol), shows hemolysis comparable to L-AMB.

[0373] [Table 15]

[0374] The example of ABIP composition is instructive: ABIP formulated with completely amorphous AmB is highly hemolytic, whereas ABIP formulated with crystalline drug is not, indicating that the physical form or "apparent solubility" of the amorphous phase can enhance the above effects.

[0375] Example 16: Drug Loading and Target Density of Dry Powder Compositions Nominal dose (ND), tap density (ρ tapped ), and the container volume of the dry powder inhaler (V r ) can be used to calculate drug burden according to the following formula:

number

[0376] This formula assumes that 50% of the container volume is filled. Optimal performance of the capsule may occur when the fill volume is 25%-75% of the capsule volume, but fill volumes as low as about 10% and as high as 90% may be used. If the fill volume is too low, the emitted dose (ED) value may be reduced since the capsule and device losses during aerosol administration are fixed.

[0377] To achieve a nominal dose (ND) of 2 mg with a powder with a tap density of 0.1 g / mL (size 3 capsule), the %AmB is approximately 13.4 w / w% when the capsule is half filled, and ranges from 6.7 w / w% to 20.1 w / w% at 25% to 75% of the fill volume. Thus, ABCI-003 powder with a tap density of 0.11 g / mL may be a suitable design to achieve the target dose of 2 mg.

[0378] To obtain a tap density of 0.1 g / mL and an ND of 0.67 mg, the %AmB is 4.5%, which ranges from 2.25 to 6.75 w / w% for 25% to 75% of the volume of the size 3 capsules to be filled.

[0379] To obtain a nominal dose of 0.1 mg, the drug loading required to achieve 50% fill volume is approximately 0.7% (tap density = 0.1 g / mL).

[0380] FIG. 12 provides an estimate of drug loading requirements based on the nominal dose and tap density of the powder.

[0381] In general, for nominal doses less than about 10 mg, the %AmB varies from 0.1% to 50%, e.g., from 0.5% to 20%. For nominal doses of about 10 mg, the tap density is set to 0.2 g / cm 3 It may be advantageous to adjust the dose to about 0.1 g / cm for lower doses of less than 3 mg. 3 It is advantageous to use a lower tap density, on the order of 1. The tap density in the spray dried powder can be controlled within the composition by varying the spray drying conditions, varying the solids content, and varying the concentration of the pore forming process aid PFOB.

[0382] Example 17: Clearance of compositions from airway surface liquids Administration of the compositions according to the present disclosure by oral inhalation deposits AmB in the ASL on the apical side of airway epithelial cells, where ion channel formation in the epithelial cell membrane occurs. Given that only drugs that dissolve in the ASL can contribute to ion channel formation, it is desirable to select a dose and regimen that maintains a trough ASL concentration that is sufficiently higher than the equilibrium solubility of AmB in the ASL (i.e., greater than 0.2 μg / mL).

[0383] Drug particles deposited in the airway lumen after inhalation (IH) administration are distributed from the ASL to other compartments by multiple clearance pathways, including: (1) absorption of dissolved AmB from the ASL into the systemic circulation, (2) particulate clearance in the mucociliary escalator or by cough clearance, (3) phagocytosis of particles by circulating and tissue-resident alveolar macrophages, and (4) binding or association of AmB with pulmonary matter.

[0384] Due to the low solubility of AmB, systemic absorption is limited by dissolution, and most of the drug not cleared by the mucociliary escalator is distributed to lung tissue, where AmB accumulates over time due to its slow clearance. Similar to intravenous administration, intravenous administration of AmB results in biphasic elimination kinetics from the ASL and plasma due to slow redistribution of the drug from lung tissue.

[0385] The clearance of ABCI-001 from the ASL was evaluated in bronchoalveolar lavage fluid (BALF) samples obtained from Sprague-Dawley rats after both IT administration (Study FY21-211A) and IH administration (Study FY21-211B). Both FY21-211A and FY21-211B were non-GLP single dose studies. Study FY21-211A evaluated IT administration of ABCI-001 and ABIP at a delivered dose of 5 mg / kg. Study FY21-211B evaluated three delivered doses of ABCI-001 (6.8, 13.6, and 23 mg / kg) by inhalation via the nose only. In both studies, BALF collections were evaluated at six time points (0.5, 1, 2, 4, 8, and 16 hours after administration). The bronchoalveolar lavage procedure significantly dilutes the concentration of AmB in the ASL. Urea diffuses rapidly in the body to reach identical concentrations in plasma and ASL, so it can be used as an endogenous marker of ASL dilution. This allows the volume of ASL in BALF samples to be calculated from a simple dilution principle. AmB concentrations in ASL were quantified using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method, and urea concentrations in ASL and plasma were quantified using a colorimetric blood urea nitrogen assay.

[0386] The AmB concentration of ASL versus time profiles for the three delivered doses of ABCI-001 administered in FY21-211B (6.8, 13.6, and 23.0 mg / kg) were combined on one graph with the results obtained after intratracheal administration of ABCI-001 at a dose of 5 mg / kg and ABIP in study FY21-211A. To do this, the ASL concentrations were normalized to the 12 mg / kg dose level. Because the dose-normalized ASL concentration versus time profiles for each of the five treatments largely overlapped and appeared to share similar rates of decline, the pooled data were fitted to a single exponential decline equation to estimate the initial rate of this ASL clearance in rats. The results indicate that exposure to ASL and the initial deposition of AmB in ASL are essentially independent of the administered ABCI-001 dose and are comparable to ABIP. Furthermore, the half-life of the initial decline of AmB in ASL after administration of ABCI-001 and ABIP to rats was approximately 16 hours, such a clearance rate of AmB from ASL that can support once-daily dosing of compositions according to embodiments described herein.

[0387] Example 18: Pulmonary tissue distribution and clearance of AmB in rats and dogs after IH administration of the composition Following IH administration, a significant proportion of AmB present in ASL is distributed to lung tissue. Lung tissue concentrations were measured in 28-day GLP toxicology studies with ABCI-001 in Sprague-Dawley rats (Study FY21-235), with ABCI-003 in rats (Study FY22-071), and with ABCI-003 in beagle dogs (Study FY21-234). Lung tissue concentrations were also measured in non-GLP studies after administration of a single dose of ABCI-001 (Study FY21-211B) and after administration of multiple doses of ABCI-003 (Study FY22-070) in rats. Mean lung tissue concentrations at necropsy are detailed in Table 16.

[0388] [Table 16]

[0389] After repeated doses of 1.8 mg / kg / day delivered over 14 or 28 days, mean lung concentrations of AmB were generally greater than 1.0 mg / g in both rats and dogs. Lung concentrations in both species increased with increasing dose, but not in a dose-proportional manner. At the end of repeated dosing, mean lung tissue concentrations of AmB from ABCI-001 and ABCI-003 in both species were approximately 80-100 times higher than those reported for AmB in lung tissue after repeated IV administration of liposomal AmB. Lung tissue concentrations observed with ABCI-001 and ABCI-003 are comparable to those measured for L-AMB in the liver and spleen after IV administration (1-10 mg / g). Lung tissue concentrations observed with repeated dosing of ABCI-001 and ABCI-003 (>1.0 mg / g) were much higher than those determined after administration of single doses of ABCI-001 and ABCI-003 (<0.1 mg / g), even when the single dose was as high as 23.0 mg / kg (Study FY21-211B). The large increase in lung tissue levels with repeated dosing is due to accumulation of substantially insoluble drug substance in the lung. The accumulation rate, AR, describes the relationship between dosing interval and the rate of elimination of the drug at steady state, i.e.: AR=1 / 1-e -kτ where k is the terminal elimination rate constant and τ is the dosing interval.

[0390] The t1 / 2 measured for ABCI-003 in lung tissue in dogs in FY21-234 was approximately 10 days. The lung tissue t1 / 2 measured during washout of ABCI-003 in rats (study FY22-071) was 20-25 days in the medium and high dose groups. Substantial accumulation in lung tissue was also observed for ABIP in a 14-day GLP study in dogs. After cessation of dosing, the t1 / 2 for redistribution of AmB from lung tissue was 19 days. In a corresponding 14-day GLP study in rats, the t1 / 2 for redistribution of AmB from lung tissue was 22 and 34 days for the delivered doses of 1.1 mg / kg and 12.4 mg / kg, respectively. These results are consistent with the t1 / 2 of 1-4 weeks measured for clearance of AmB from the liver after IV administration. Slow redistribution of drug from lung tissue results in biphasic clearance of drug from the ASL and plasma due to flip-flop PK, where the rate at which a drug enters the ASL or plasma compartment is slower than its absorption or excretion rates.

[0391] Assuming that the terminal t1 / 2 of AmB is about 20 days, k is about 0.035 days. -1 For once-daily administration, the AR is equal to 29.4, indicating that a daily AmB dose accumulates up to 30-fold in lung tissue at steady state. The extent of accumulation is reflected in the high lung tissue concentrations measured in rats and dogs for ABIP, ABCI-001, and ABCI-003.

[0392] The low lung tissue AmB exposure observed in the FY22-071 study is due to the low delivered dose and the weekly dosing regimen. With weekly dosing of ABCI-003, the AR is equal to 4.6. A loading dose 5 times higher than the maintenance dose can be used to maintain a constant lung tissue concentration over the duration of dosing. Given that our therapeutic target is ASL, achieving equilibrium concentrations of AmB in lung tissue quickly is not as important as doing so in ASL.

[0393] Maintaining a trough concentration of greater than 0.2 μg / mL in the ASL can be important for the efficacy of the compositions described herein. As mentioned above, the measured t1 / 2 of AmB in the ASL is about 16 hours. This results in an AR of about 2 at steady state. Thus, a loading dose 2.5-3 times higher than the maintenance dose can allow consistent ASL peak and trough AmB concentrations over the dosing period of the compositions described herein.

[0394] Example 19: Plasma toxicokinetics of AmB in rats and dogs after IH administration In a 28-day GLP toxicology study (Study FY21-235), the elimination of AmB from plasma was evaluated in male and female Sprague-Dawley rats after inhalation of single or repeated doses of ABCI-001. The study included three treatment groups, with group means of achieved delivered doses: 4.7 mg / kg (low dose), 9.3 mg / kg (medium dose), and 17.3 mg / kg (high dose). Plasma samples were collected pre-dose and at 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and 24.0 hours after dosing, and plasma AmB concentrations were quantified using a validated LC-MS / MS method with a lower limit of quantification of 1 ng / mL. Toxicokinetic parameters were determined using Phoenix WinNonlin version 8.3 software.

[0395] Females consistently showed higher exposure than males, but there was limited evidence of gender differences. Thus, combined male and female means are presented in Table 17. Due to AmB's low solubility, systemic absorption of AmB was slow after single or repeated inhalations, with median time to maximum concentration (tmax) generally observed to be between 4 and 8 hours, independent of dose, sex of the animal, and day of dosing. Minimal accumulation of AmB in plasma was observed over the 28-day dosing period, with mean accumulation ratios of the area under the dose-normalized time-concentration curve to the last measurable concentration (AUClast) and maximum concentration (Cmax) ranging from 0.699 to 1.51. On day 1, low-, medium-, and high-dose males met reporting criteria, and therefore their plasma ABCI-001 elimination t1 / 2 could be estimated with values ​​ranging from 5.28 to 8.55 hours. On day 28, elimination t1 / 2 for low- and medium-dose females ranged from 7.16 to 9.33 hours. These values ​​are consistent with the 8 hour t1 / 2 value reported for a 4 mg / kg IV dose of AmBisome in rats with a 24 hour sampling period.

[0396] [Table 17]

[0397] Mean plasma C in rats max The values ​​were approximately two orders of magnitude higher for IV liposomal AmB (AmBisome) compared with IH ABCI-001 at a comparable dose (Table 18), while lung tissue C max The absolute bioavailability of ABCI absorbed systemically over 24 hours on day 1 is approximately 3%, assuming a lung deposition fraction of 0.1 in rats, providing further evidence that the drug is highly distributed in tissues.

[0398] IH administration provides significant targeting of the drug to the lung, resulting in lung tissue concentrations nearly two orders of magnitude higher and plasma concentrations two orders of magnitude lower than those achieved following IV administration.

[0399] [Table 18]

[0400] Plasma TK was also evaluated in male and female beagle dogs following inhalation of single or multiple doses of ABCI-003 in a 28-day GLP toxicology study (Study FY21-234). The study included three treatment groups with mean delivered doses: 0.51 mg / kg (low dose), 1.8 mg / kg (medium dose), and 4.9 mg / kg (high dose). Sample collection time points, AmB quantification, and noncompartmental data analysis were performed in the same manner as described above for rats.

[0401] There was limited evidence of gender differences, so combined male and female means are presented in Table 19. As in rats, plasma exposures increased non-proportionally with dose on days 1 and 28, with AmB concentrations remaining elevated at or near Cmax levels through the 24 hour time point, particularly for the mid and high dose groups. Concentrations remained quantifiable in all animals at necropsy on day 28 and after 14 days of recovery from inhalation on day 42.

[0402] [Table 19]

[0403] In contrast to rats, there was consistent evidence of plasma AmB accumulation in dogs over the 28-day dosing period, with mean accumulation ratios for the area under the dose-normalized time concentration curve from 0 to 24 hours (AUC0-24hr) and Cmax values ​​ranging from 1.54 to 3.58. These differences in drug deposition may reflect differences in pulmonary anatomy and physiology between the two species. Plasma concentrations measured with ABCI-003 and the magnitude of accumulation observed were consistent with previous studies using ABIP.

[0404] Plasma ABCI-003 elimination t1 / 2 was only estimable and could meet reporting criteria for the two high-dose female animals in the recovery study, with values ​​of 103 and 105 h. As noted above for the IV composition, t1 / 2 measured using non-compartmental methods is strongly influenced by sampling time due to slow clearance of AmB from plasma due to redistribution from tissues. The longer t1 / 2 observed in dogs compared to rats (approximately 100 h versus 5-10 h) simply reflects the longer sampling period (i.e., up to day 42 in recovery animals).

[0405] Mean plasma Cmax values ​​in dogs were approximately two orders of magnitude greater for IV AmB deoxycholate (Fungizone®) and approximately three orders of magnitude greater for IV liposomal AmB (AmBisome®) compared to comparable doses of IH ABCI-003 (Table 20). Lung tissue concentrations were 19-100 times higher for ABCI-003. Absolute bioavailability over 24 hours was 0.6%, assuming a deposition fraction of 0.25 in dogs. The low bioavailability observed in rats and dogs is consistent with the low systemic toxicity observed with IH administration of Fungizone® and AmBisome®, and expected with IH compositions according to embodiments described herein.

[0406] [Table 20]

[0407] Example 20: Toxicological studies in rats and dogs Toxicity observed in Study FY21-211B (single dose study in rats). In Study FY21-211B, single doses of 6.8, 13.6, and 23.0 mg / kg of ABCI-001 were administered to 54 Sprague-Dawley rats by inhalation through the nose only in a non-GLP study. The ABCI-001 composition contained 30 w / w% AmB with a Chol / AmB ratio of 1.2 mol / mol. ABCI-001 was well tolerated with no abnormal clinical findings or gross findings at necropsy. AmB exposure in plasma, ASL, and lungs tended to increase with increasing dose, but was not dose-proportional. Measured lung tissue concentrations were 0.010, 0.014, and 0.016 mg / g, respectively. A dose-dependent increase in alveolar macrophages was observed, but no adverse changes (e.g., increased inflammatory cells) were noted on histopathology. The increase in macrophages was due to normal particulate clearance of the inhaled dry powder. Although a non-GLP study, the 23.0 mg / kg dose supports a proportional human dose of ABCI of approximately 46 mg with a 10-fold safety margin.

[0408] Toxicity observed in Study FY21-235 (a 28-day GLP study in rats). Following the success of the single-dose study, a 28-day GLP repeat-dose toxicology study with 14 days of recovery was conducted in rats with ABCI-001 (Study FY21-235). Slow clearance of the virtually insoluble AmB drug substance led to significant accumulation of AmB in lung tissue following administration of daily doses of 4.7, 9.3, and 17.3 mg / kg / day. Lung tissue concentrations of AmB in the low, mid, and high dose groups were 1.2, 2.0, and 2.9 mg / g, respectively.

[0409] Peak plasma drug concentrations remained below 55 ng / mL, 2 to 3 orders of magnitude lower than those observed after intravenous administration of comparable concentrations of AmB and significantly below the 1000 ng / mL threshold for systemic toxicity.

[0410] Early deaths occurred in 4 / 38, 3 / 38, and 13 / 47 animals in the low, mid, and high dose groups, respectively. This was presumed to be due to obstruction of the large airways due to excessive secretion of mucus material. Some of the animals in the ABCI-001-treated groups showed intermittent respiratory-related symptoms (wheezing, perceptible bubbling sounds, rapid breathing) during the dosing phase. The first symptoms appeared on day 4 in the high dose group and became more frequent with continued exposure. However, there was a poor correlation between clinical signs and mortality.

[0411] No significant differences were observed in body weight, food consumption, hematology, clinical chemistry, and coagulation.

[0412] Histopathological evaluation revealed lesions in the larynx, trachea, and lungs of males and females in all dose groups. Findings consisted of: a minimal to moderate increase in yellow / gold pigmented macrophages; minimal to moderate mixed cell inflammation in the larynx, trachea, and lungs; minimal to moderate accumulation of mucus material; and a minimal to moderate increase in respiratory epithelial cell hyperplasia. In Study FY21-235, significant morbidity and mortality were observed, so a no observable adverse effect level (NOAEL) could not be established. The lowest adverse effect level (LOAEL) was the lowest dose studied (4.7 mg / kg).

[0413] Study FY22-071 (28-day GLP study in rats). A second 28-day GLP study with 14 days of recovery was conducted in Sprague-Dawley rats (Study FY22-071). This study used ABCI-003 and significantly reduced AmB burden in lung tissue. The ABCI-003 composition contained 14 w / w% AmB with a Chol / AmB ratio of 0.4 mol / mol. Compared to ABCI-001, ABCI-003 was able to reduce interparticle cohesion by maintaining lipids in a highly ordered So phase. Three dose groups were used, a loading dose followed by a weekly maintenance dose. The doses for low, medium, and high were: 1.08 / 0.17 mg / kg, 2.54 / 0.41 mg / kg, and 5.74 / 1.06 mg / kg, respectively. Lung tissue concentrations were 0.019 mg / g, 0.050 mg / g, and 0.088 mg / g for the low, mid, and high doses, respectively.

[0414] In contrast to Study FY21-235, the survival portion of Study FY22-071 showed no evidence of morbidity or mortality. Gross pathology findings were also unremarkable. Although a minimal increase in macrophages was observed, these findings were not accompanied by the adverse findings identified in Study FY21-235 (mucus material, epithelial hyperplasia, or inflammation). No significant differences were observed in body weight, food consumption, hematology, clinical chemistry, and aggregation. The NOAEL was the maximum dose administered, i.e., 5.74 / 1.06 mg / kg.

[0415] Study FY21-234 (28-day GLP study in dogs). In study FY21-234, ABCI-003 was administered to beagle dogs at daily doses of 0.5, 1.8, and 4.9 mg / kg. This high dose in dogs was comparable to the low dose in rat study FY21-235, where a NOAEL was not established. Furthermore, lung tissue exposure was similarly high (0.25, 1.38, and 3.54 mg / g) in rats. However, in contrast to the rat study, no morbidity or mortality was observed at any dose in dogs. None of the clinical signs, body weight, food consumption, clinical pathological characteristics (hematological characteristics, aggregation, urinalysis), ophthalmological characteristics, or ECG parameters were affected by treatment with ABCI-003. Clinical pathological characteristics at day 29 for the low dose showed no effects. Unfortunately, due to a clinical chemistry analyzer failure, data could not be evaluated for the mid- and high-dose groups and the recovery groups (air, placebo control, and high dose) on day 29. Given the known clinical profile of the effects on clinical chemistry of systemic AmB administration at plasma exposures orders of magnitude greater, 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), missed clinical pathology findings are unlikely.

[0416] Additionally, although an increase in the number of pigmented macrophages was observed, there was no evidence of increased inflammation, mucus hypersecretion, or epithelial cell hyperplasia. Histopathological findings were not adverse and were considered consistent with normal particulate clearance from the lung. The NOAEL was the high dose group, i.e., 4.9 mg / kg / day.

[0417] 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. The presence of large amounts of undissolved particulate material of any kind in the lungs can result in adaptive, and ultimately deleterious, pulmonary changes that are directly related to the physical burden of foreign matter present in the respiratory tract. These changes may be independent of the chemical or pharmacologically mediated toxicity of the drug.

[0418] Increased amounts of insoluble material can lead to increased numbers of macrophages, and excessive macrophage stimulation can lead to recruitment of other inflammatory cells, ultimately generating secondary tissue damage. This response is often referred to as "lung overload". It is important to distinguish this lung overload, due to reduced macrophage lung clearance from a volumetric overload of low-toxicity particulates, from direct macrophage toxicity from compounds such as crystalline quartz, which can kill macrophages or impair their function.

[0419] Adverse observations related to lung overload occur when concentrations of poorly soluble particles (e.g., titanium dioxide, carbon black) in lung tissue exceed approximately 1 mg / g. An adaptive response (i.e., an increase in macrophages without an inflammatory response) is typically observed at concentrations below approximately 0.1 mg / g.

[0420] 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, which are above the threshold concentration for lung overload in rats. Consistent with the phenomenon of lung overload, these lung burdens resulted in substantial morbidity and mortality in all dose groups. This is consistent with other studies that have demonstrated that rat macrophages are particularly sensitive to overload.

[0421] In study FY22-071, doses and regimens were adjusted to maintain AmB lung tissue concentrations below the threshold of 0.1 mg / g, below which only adaptive macrophage responses are expected. Indeed, only minimal macrophage increases were observed, and there was no evidence of toxicity associated with lung overload.

[0422] It is also noteworthy that a single dose of ABCI-001 as high as 23 mg / kg (study FY21-211B) also produced low lung burden (0.016 mg / g) and adaptive macrophage responses in rats, suggesting that the adverse effects may be due to accumulation of the drug in the lung over time, rather than an acute response to high AmB concentrations in the lung epithelium after dosing.

[0423] The adaptive and adverse responses observed with the composition in rats 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 contention that the toxicity observed with ABCI-001 in study FY21-235 was due to macrophage responses associated with pulmonary overload of substantially insoluble particles, rather than a chemically or pharmacologically mediated drug effect.

[0424] In general, rats are much more sensitive to the effects of lung overload than other species under similar dosing conditions. This is evident in the case of ABCI-003, where, despite the establishment of a NOAEL in dogs at a deposited dose of 1.23 mg / kg, lung overload was still observed in rats at a deposited dose about three times lower (0.47 mg / kg). Forbes and colleagues summarize the species differences related to macrophage overload and report 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 slower clearance rates of less soluble particles (e.g. titanium dioxide, lime dust, silica) that are deposited in higher fractions, lung overload is not common in humans. The reduced susceptibility may be related to anatomical (i.e., the absence of bronchioles in rats) and physiological (i.e., greater uptake of particles by interstitial macrophages and transport to lymphatics) differences. Therefore, the toxicity observed in rats is not predictive of toxicity in humans, and the dog is considered the most appropriate species for the assessment of human risk.

[0425] Although rats are a sensitive species, dogs are considered a more relevant species for the assessment of risk in humans. The NOAEL in dogs (4.9 mg / kg) provides a 12.6-fold safety margin compared to the maximum nominal dose (i.e., 10 mg) proposed in a Phase I single ascending dose clinical study in healthy participants. At the maximum daily dose proposed in a Phase I multiple dose study in healthy participants, the margin increases to 31.5-fold.

[0426] Another method for estimating the safety margin is detailed below based on AmB exposure in lung tissue. The expected lung tissue concentration in human subjects after a 10 mg loading dose for 28 days followed by a daily maintenance dose of 4 mg is 0.08 mg / g. This is 12.5 times lower than the lung tissue concentration in rats known to cause lung overload. Furthermore, this is lower than the lung tissue concentration of 0.1 mg / g below which only adaptive responses are observed in rats.

[0427] 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 Comparable XRPD diffraction patterns (Figure 13) and main transition temperatures determined by differential scanning calorimetry (Figure 14) were observed for the development batch used in the GLP toxicology studies and the GMP batch used in early clinical development. The results suggest that wet-milled amphotericin B crystals retain crystallinity while embedded within a shell containing a mixture of phospholipids and cholesterol, where the main transition temperature (i.e., gel to liquid crystalline phase transition temperature) of the dehydrated powder exceeds 90°C.

[0428] Example 22: Aerosol performance of GMP batches of ABCI-003 Spray dried ABCI-003 powder was filled into size 3 HPMC capsules using a drum filling process (Harro Hoefliger) with good precision (RSD<3%) and accuracy (2 mg dose).

[0429] Capsule retention, device retention, and delivered dose of AmB from the RS01 dry powder inhaler were determined at a pressure drop of 4 kPa and an inhalation volume of 4 L. The mean delivered dose was 83.1 ± 2.4%, with capsule and device retentions of 6.3 ± 0.27% and 7.0 ± 2.0%, respectively (N = 5). Drug concentrations were quantified by RP-HPLC method.

[0430] The aerodynamic particle size distribution (APSD) of ABCI-003 was determined with a Next Generation Impactor operating under identical conditions detailed above (Figure 15). Batch MSV-ENG-129 represents a fill development run with a GMP spray dried batch intended for use in Phase I. Results for this batch are compared to development lots after one month of stability at refrigerated conditions.

[0431] Key features of the APSD include less than 5% deposition in the USP throat and in stage 1 of the impactor. A unimodal particle size distribution peaks at stage 4 with significant deposition in stages 3 and 5. The MMAD for the development batch was 2.5 μm and for two runs with clinical material the MMAD was 2.8 μm. The <5 μm fine particle dose was 69.6% of the nominal dose for the development batch and 66.8% of the nominal dose for the clinical batch. The fine particle dose at stage 3-filter, a surrogate for total lung dose, was 71.4% of the nominal dose for the development batch and 70.6% of the nominal dose for the clinical batch. The development batch had more deposition in the device and capsule. Overall, the clinical batches are expected to achieve a total lung dose of 60-70% of the nominal dose and greater than 75-85% of the delivered dose.

[0432] Incorporation by Reference All patents and publications mentioned in the above description are incorporated by reference in their entirety into this application.

[0433] Equivalent Although the present invention has been described in some detail by way of explanation and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that the present invention can be practiced by modifying or altering the present invention within a wide range of equivalent conditions, formulations, and other parameters without affecting the scope of the present invention or any specific embodiment thereof, and that such modifications or alterations are intended to be encompassed by the appended claims.

Claims

1. (i) Amphotericin B (AmB), or any pharmaceutically acceptable salt or hydrate thereof; (ii) Cholesterol (Chol); (iii) Phospholipids comprising hydrogenated soybean phosphatidylcholine (HSPC) and distearoyl phosphatidylglycerol (DSPG); and (iv) Calcium chloride (CaCl ２ ) A pharmaceutical composition containing the following:

2. The amount of AmB is approximately 0.5% to approximately 25 w / w%, and / or The amount of chlor is approximately 0.1% to approximately 8 w / w%, and / or The amount of CaCl₂ is approximately 1% to approximately 10 w / w%, and / or The pharmaceutical composition according to claim 1, wherein the amount of phospholipid is approximately 60% to approximately 95% w / w%.

3. The pharmaceutical composition according to claim 1, wherein the weight ratio of chol to phospholipid is about 0.001:1 to about 0.1:

1.

4. The pharmaceutical composition according to claim 1, wherein the weight ratio of hydrogenated soybean phosphatidylcholine (HSPC) to distearoyl phosphatidylglycerol (DSPG) is about 2:1 to about 19:

1.

5. The pharmaceutical composition according to claim 1, wherein the molar ratio of chol to amB is about 0.05:1 to about 1.2:

1.

6. Phospholipids and CaCl ２ The pharmaceutical composition according to claim 1, wherein the molar ratio of is approximately 4:1 to approximately 2:

1.

7. The pharmaceutical composition according to claim 1, wherein AmB has a degree of crystallinity higher than approximately 75%.

8. (i) Approximately 14.0 w / w% amphotericin B (AmB); (ii) Approximately 2.3 w / w% cholesterol; (iii-a) Approximately 70.3 w / w% hydrogenated soy phosphatidylcholine (HSPC); (iii-b) Approximately 7.8 w / w% distearoyl phosphatidylglycerol (DSPG); and (iv) Approximately 5.52 w / w% calcium chloride (CaCl ２ ) To include, essentially be from, or consist of, (i) Approximately 14.0 w / w% amphotericin B (AmB); (ii) Approximately 6.81 w / w% cholesterol; (iii-a) Approximately 51.2 w / w% hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 22.8 w / w% distearoyl phosphatidylglycerol (DSPG); and (iv) Approximately 5.2 w / w% calcium chloride (CaCl₂) To include, essentially be from, or consist of, (i) Approximately 3.4 w / w% amphotericin B (AmB); (ii) Approximately 0.57 w / w% cholesterol; (iii-a) Approximately 80.72 w / w% hydrogenated soybean phosphatidylcholine (HSPC); (iii-b) Approximately 8.97 w / w% distearoyl phosphatidylglycerol (DSPG); and (iv) Approximately 6.34 w / w% calcium chloride (CaCl₂) The pharmaceutical composition according to claim 1, comprising, essentially consisting of, or comprising.

9. (i) The chol / amB ratio is approximately 0.4 to 1.2 mol / mol; (ii) The chlor / PL ratio is less than approximately 0.05 w / w; (iii) The HSPC / DSPG ratio is approximately 2.3 to approximately 9.0 w / w; and (iv) PL / Ca ２＋ The ratio is approximately 2:1 mol / mol. A pharmaceutical composition according to claim 1, comprising the following:

10. The pharmaceutical composition according to claim 1, wherein AmB and Chol are not complexed, and AmB is not encapsulated in liposomes.

11. The pharmaceutical composition according to claim 1, wherein AmB is coated with a porous shell of phospholipids and chol.

12. The pharmaceutical composition according to claim 1, which is formulated as a dried powder.

13. Median diameter of powder particles X ５０ The thickness is approximately 1.0 to approximately 4.0 μm, and / or The tap density of the powder particles is approximately 0.03 to approximately 0.4 g / mL, and / or The Carr fluidity index of the powder particles is approximately 20 to approximately 32, and / or AmB is coated with a porous shell of phospholipids and chol, the main transition temperature (T m) of the shell is at least 80°C, and / or The pharmaceutical composition according to claim 12, wherein the water content of the powder is approximately 1.5 to approximately 6 w / w%.

14. The pharmaceutical composition according to claim 1, formulated for administration to the lungs or airways.

15. The pharmaceutical composition according to claim 14, formulated for aerosol administration.

16. The pharmaceutical composition according to claim 12, wherein the aerodynamic median mass of the powder particles is about 1.5 μm to about 4.0 μm.

17. A pharmaceutical composition according to any one of claims 1 to 16, for use in a method of treating a disease or disorder, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to a subject requiring treatment, and the administration is pulmonary administration.

18. The pharmaceutical composition according to claim 17, wherein the disease or disorder is cystic fibrosis, non-cystic fibrotic bronchiectasis, or chronic obstructive pulmonary disease.

19. A pharmaceutical composition according to any one of claims 1 to 16, for use in a method of increasing the pH of airway surface fluid, or a method of increasing bicarbonate secretion into airway surface fluid, or a method of increasing forced expiratory volume in one second (FEV1), wherein the method comprises administering an effective amount of the pharmaceutical composition to a subject of interest, and the administration is pulmonary administration.

20. The pharmaceutical composition according to claim 19, which increases the target FEV1 by approximately 3% to approximately 20%.

21. The pharmaceutical composition according to claim 19, wherein the subject has cystic fibrosis, non-cystic fibrotic bronchiectasis, or chronic obstructive pulmonary disease.

22. A pharmaceutical composition according to claim 17, which is administered to the target airway.

23. The pharmaceutical composition according to claim 17, which is administered as an aerosol.

24. The pharmaceutical composition according to claim 17, wherein the subject is a human being under 12 years of age, or the subject is a human being at least 12 years of age.

25. The disease or disorder is cystic fibrosis, Cystic fibrosis is refractory to treatment with Ivacaftor, or The pharmaceutical composition according to claim 17, wherein cystic fibrosis is refractory to treatment with a combination of Elexacaftor, Tezacaftor, and Ivacaftor.

26. The nominal dose or quantitative dose is 0.01 mg to 10 mg, and / or The pharmaceutical composition according to claim 17, which is administered once a day.

27. The pharmaceutical composition according to claim 17, wherein the administration comprises first delivering a loading dose of the pharmaceutical composition, and subsequently delivering a maintenance dose of the pharmaceutical composition, and the mass ratio of the loading dose to the maintenance dose is about 2:1 to about 5:

1.

28. The pharmaceutical composition according to claim 27, wherein the mass ratio of the loading dose to the maintenance dose is approximately 2.5:1 to approximately 3:

1.

29. The pharmaceutical composition according to claim 17, wherein the absolute bioavailability of AmB is about 0.1% to about 5%.

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