Pharmaceutical compositions of therapeutic polyene macrolides and methods for their use

Abstract

A pharmaceutical composition comprising a plurality of nanoparticles comprising a compound of the following structure (1), or a pharmaceutically acceptable salt thereof, is disclosed. Methods for its use and preparation are also disclosed. [Formula 1] TIFF2023516288000061.tif62125

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition and a method for using the same. [Background technology]

[0002] Macrolide antibiotics contain a macrocyclic lactone ring bonded to one or more deoxy sugars. The pharmaceutical applications of macrolide antibiotics are often limited by their limited shelf life and the difficulty in achieving efficient delivery.

[0003] The following compounds are therapeutic polyene macrolides: [ka]

[0004] A novel formulation containing compound 1 or a pharmaceutically acceptable salt thereof is needed. [Overview of the project] [Means for solving the problem]

[0005] In one embodiment, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles containing a pharmaceutical active ingredient which is a compound having the following structure or a pharmaceutically acceptable salt thereof: [ka]

[0006] In some embodiments, the active pharmaceutical ingredient is, [ka] or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable polymer excipient. In some embodiments, a plurality of nanoparticles comprise a pharmaceutically acceptable polymer excipient. In some embodiments, the active pharmaceutical ingredient is nanoencapsulated. In some embodiments, the pharmaceutically acceptable polymer excipient is poly(alkylcyanoacrylate) or polyphosphazene. In some embodiments, the pharmaceutically acceptable polymer excipient is poly(alkylcyanoacrylate). In some embodiments, the pharmaceutically acceptable polymer excipient is poly(ethylhexylcyanoacrylate), poly(ethylcyanoacrylate), poly(n-hexylcyanoacrylate), poly(4-methylpentylcyanoacrylate), poly(ethylbutylcyanoacrylate), poly(butylcyanoacrylate), or poly(octylcyanoacrylate). In some embodiments, the pharmaceutically acceptable polymer excipient is poly(ethylhexylcyanoacrylate). In some embodiments, the pharmaceutically acceptable polymer excipient is a lactic-glycolic acid copolymer (Poly(lactic-co-glycolic acid)). In some embodiments, the pharmaceutically acceptable polymer excipient is a protein (e.g., casein, albumin (e.g., human serum albumin, bovine serum albumin, or egg albumin), fibroin, gelatin, or a combination thereof). In some embodiments, the weight ratio of protein to pharmaceutically active ingredient is 1:1 to 20:1 (e.g., 5:1 to 20:1, 1:1 to 5:1, 5:1 to 10:1, or 10:1 to 20:1).

[0008] In some embodiments, the present invention relates to poly(ethylhexyl cyanoacrylate) and the following structure: [ka] The present invention provides a pharmaceutical composition comprising multiple nanoparticles containing a pharmaceutical active ingredient which is a compound or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles comprising a lactic acid-glycolic acid copolymer and a pharmaceutical active ingredient which is a compound having the following structure:

Chemical formula

[0010] In some embodiments, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles comprising casein, albumin, fibroin, gelatin, or a combination thereof and a pharmaceutical active ingredient which is a compound having the following structure:

Chemical formula

[0011] In some embodiments, the pharmaceutically acceptable polymeric excipient is albumin (e.g., human serum albumin, bovine serum albumin, or ovalbumin). In some embodiments, the pharmaceutically acceptable polymeric excipient is fibroin. In some embodiments, the pharmaceutically acceptable polymeric excipient is gelatin. In some embodiments, the pharmaceutically acceptable polymeric excipient is casein.

[0012] In some embodiments, the pharmaceutical composition is a lyophilized composition. In some embodiments, the pharmaceutical composition further comprises a plurality of microbubbles.

[0013] In another aspect, the present invention provides a pharmaceutical composition comprising a plurality of microbubbles and a plurality of nanoparticles comprising a compound having the following structure:

Chemical formula

[0014] In some embodiments, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the nanoparticles include poly(alkylcyanoacrylate), polyphosphazene, or lactic acid-glycolic acid copolymers.

[0016] In some embodiments, the microbubbles include perfluorocarbons, hydrocarbons, sulfur fluoride gas, air, components of air, or mixtures thereof. In some embodiments, the microbubbles include nitrogen (N2), oxygen (O2), argon (Ar), carbon dioxide (CO2), helium (He), neon (Ne), methane (CH4), or mixtures thereof. In some embodiments, the microbubbles include perfluorocarbons. In some embodiments, the microbubbles include air or components thereof. In some embodiments, at least a portion of the multiple nanoparticles associate with the microbubble surface (for example, the pharmaceutical composition is a Pickering emulsion).

[0017] In some embodiments, the pharmaceutical composition further comprises a surface-active protein (e.g., albumin (e.g., human serum albumin or bovine serum albumin)). In some embodiments, the pharmaceutical composition comprises 0.1% to 2% (e.g., 0.4% to 0.6%, e.g., 0.5%) (w / w) of a surface-active protein (e.g., albumin (e.g., human serum albumin or bovine serum albumin)).

[0018] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable surfactant. In some embodiments, the pharmaceutically acceptable surfactant is a nonionic surfactant. In some embodiments, the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester, sorbitan ester, polysorbate, polyethoxylated castor oil, polyoxyethylene / polyoxypropylene block copolymer, or a combination thereof. In some embodiments, the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester, or a combination thereof. In some embodiments, the polyoxyethylene fatty acid ester is polyoxyethylated 12-hydroxystearic acid. In some embodiments, the polyoxyethylene ether is polyoxyethylene lauryl ether.

[0019] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable stabilizer. In some embodiments, the pharmaceutically acceptable stabilizer is vanillin, butylated hydroxytoluene, butylated hydroxyanisole, or vitamin E. In some embodiments, the pharmaceutically acceptable stabilizer is vanillin. In some embodiments, the pharmaceutical composition contains 0.1 to 10% (preferably 0.5 to 8%, more preferably 1 to 5%) (w / w) of the pharmaceutically acceptable stabilizer based on the particle mass.

[0020] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable oil. In some embodiments, the pharmaceutically acceptable oil is selected from the group consisting of medium-chain triglycerides, long-chain triglycerides, and combinations thereof. In some embodiments, the pharmaceutically acceptable oil is one or more medium-chain triglycerides. In some embodiments, one or more medium-chain triglycerides are selected from the group consisting of Miglyol, Captex, and Kollisolv. In some embodiments, the pharmaceutical composition contains 0.5 to 5% (w / w) of the pharmaceutically acceptable oil relative to the particle mass.

[0021] In some embodiments, the nanoparticles have a mean number average diameter of 20-200 nm (preferably 40-100 nm), as measured by dynamic light scattering. In some embodiments, the nanoparticles have a mean number average diameter of 30-150 nm (preferably 80-100 nm), as measured by nanoparticle tracking analysis. In some embodiments, for example, in a pharmaceutical composition formulated for oral administration, the polydispersity index of the nanoparticles is 0.5 or less (e.g., 0.3 or less). In some embodiments, for example, in a pharmaceutical composition formulated for parenteral administration (e.g., intravenous administration), the polydispersity index of the nanoparticles is 0.3 or less (e.g., 0.2 or less).

[0022] In some embodiments, the pharmaceutical composition is an aqueous composition. In some embodiments, the pH of the pharmaceutical composition is 4.0 to 8.0 (for example, pH 5.0 to 7.0).

[0023] In some embodiments, the pharmaceutical composition includes a cosolvent (e.g., a polar organic solvent). In some embodiments, the polar organic solvent is dimethyl sulfoxide, N-methyl-2-pyrrolidone, N,N-dimethylformamide, or a combination thereof.

[0024] In certain preferred embodiments, the pharmaceutical composition is an aqueous composition comprising N-methylpyrrolidone and a pharmaceutically acceptable polymer excipient, which is a lactic acid-glycolic acid copolymer.

[0025] In certain preferred embodiments, the pharmaceutical composition is an aqueous composition comprising poly(ethylhexyl cyanoacrylate), vanillin, and 6-O-palmitoyl-L-ascorbic acid.

[0026] In some embodiments, the pharmaceutical composition is an aqueous composition comprising poly(ethylhexyl cyanoacrylate); vanillin; 6-O-palmitoyl-L-ascorbic acid; polyoxyethylated 12-hydroxystearic acid (e.g., Kolliphor HS 15); polyoxyethylene lauryl ether (e.g., Brij L23); and a medium-chain triglyceride (e.g., Miglyol).

[0027] In some embodiments, the pharmaceutical composition is an aqueous composition comprising a lactic acid-glycolic acid copolymer, N-methyl-2-pyrrolidone, and polysorbate.

[0028] In some embodiments, the pharmaceutical composition is an aqueous composition comprising a lactic acid-glycolic acid copolymer, N,N-dimethylformamide, and a polyoxyethylene / polyoxypropylene block copolymer.

[0029] In some embodiments, the pharmaceutical composition contains 1 to 15% (e.g., 2 to 15%; preferably 3 to 10%; more preferably 3.5 to 10%; or more preferably 3 to 6%) of dry (w / w) pharmaceutical active ingredient, as measured by liquid chromatography. In some embodiments, the pharmaceutical composition contains 4.5% to 5.5% of dry (w / w) pharmaceutical active ingredient, as measured by liquid chromatography. In some embodiments, the pharmaceutical composition contains 1.5% to 5.5% (e.g., 1.5% to 3.0%) of dry (w / w) pharmaceutical active ingredient, as measured by liquid chromatography. In some embodiments, the pharmaceutical composition contains 5% to 10% of dry (w / w) pharmaceutical active ingredient, as measured by liquid chromatography.

[0030] In another aspect, the present invention provides a method for treating a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject. In another aspect, the present invention provides the use of a plurality of nanoparticles described herein or the pharmaceutical composition described herein in the manufacture of a pharmacopoeia for the treatment of a subject in need thereof. In another aspect, the present invention provides the pharmaceutical composition described herein for use in the treatment of a subject in need thereof.

[0031] In some embodiments, the subjects are genera Candida, Cryptococcus, Aspergillus, Colletotrichum, Geotrichum, Hormonema, Lecythophora, Paecilomyces, Penicillium, and Rhodotorula. In some embodiments, the subjects suffer from fungal infections caused by species of the genera *Torula*, *Fusarium*, *Saccharomyces*, *Trichoderma*, *Trichophyton*, *Scopularilopsis*, *Histoplasma*, *Blastomyces*, or *Cocciodioides*. In some embodiments, the subjects suffer from fungal infections caused by species of the genera *Candida*, *Aspergillus*, or *Cryptococcus*. In some embodiments, the subjects suffer from fungal infections caused by azole-resistant *Aspergillus* species.

[0032] In some embodiments, the pharmaceutical composition is administered intravenously, by inhalation, intranasally, orally, sublingually, buccally, percutaneously, intradermally, intramuscularly, vaginally, parenterally, intra-arterially, intracranially, intrathecally, subcutaneously, intraorbitally, intraventricularly, intraspinally, intraperitoneally, or topically.

[0033] In yet another aspect, the present invention provides a method for delivering a therapeutically effective amount of compound 1, compound 1A, or a pharmaceutically acceptable salt thereof to a target site, the method comprising administering the pharmaceutical composition described herein to the target. In yet another aspect, the present invention provides the use of the pharmaceutical composition described herein in the manufacture of a pharmacopoeia for delivering a therapeutically effective amount of compound 1, compound 1A, or a pharmaceutically acceptable salt thereof to a target site. In yet another aspect, the present invention provides the pharmaceutical composition described herein for use in delivering a therapeutically effective amount of compound 1, compound 1A, or a pharmaceutically acceptable salt thereof to a target site.

[0034] In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the target site is the lung of the subject.

[0035] In yet another embodiment, the present invention relates to a pharmaceutically acceptable polymer excipient and the following structure: [ka] The present invention provides a method for producing a plurality of nanoparticles comprising a compound or a pharmaceutically acceptable salt thereof, the method comprising the step of polymerizing a monomer precursor of a pharmaceutically acceptable polymer excipient in a liquid containing the monomer precursor and the compound or a pharmaceutically acceptable salt thereof, wherein the polymerization step produces a plurality of nanoparticles.

[0036] In some embodiments, the compound has the following structure: [ka] It is a compound of or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, the liquid further comprises a pharmaceutically acceptable surfactant. In some embodiments, the pharmaceutically acceptable surfactant is a nonionic surfactant.

[0038] In some embodiments, the liquid further comprises a pharmaceutically acceptable stabilizer. In some embodiments, the liquid further comprises a pharmaceutically acceptable oil.

[0039] In some embodiments, the monomer precursor is an alkylcyanoacrylate, and the pharmaceutically acceptable polymer excipient is a poly(alkylcyanoacrylate).

[0040] In some embodiments, the nanoparticles have a number-average diameter of 20-200 nm (preferably 40-100 nm) as measured by dynamic light scattering. In some embodiments, the nanoparticles have a number-average diameter of 30-150 nm (preferably 80-100 nm) as measured by nanoparticle tracking analysis (NTA).

[0041] In some embodiments, the liquid is an aqueous composition. In some embodiments, the pH of the liquid is 0.5 to 8.0 (for example, pH 0.5 to 3.0). In some embodiments, the pH of the liquid is 2.0 to 8.0 (preferably pH 3.0 to 7.0).

[0042] In some embodiments, the method further includes adding a plurality of microbubbles. In some embodiments, the method further includes freeze-drying a plurality of nanoparticles. In some embodiments, the method further includes dialysis of a plurality of nanoparticles against deionized water. In some embodiments, the method further includes adjusting the pH of the liquid to a range of 4.0 to 8.0 (preferably a range of 5.0 to 7.0).

[0043] In some embodiments, the step of adjusting the pH is performed during the polymerization step.

[0044] definition When used herein, the term "approximately" refers to a value within ±10% of the value that follows it.

[0045] The term “dry (w / w)” percentage, as used herein, refers to the weight percentage of a component in a composition excluding a pharmaceutically acceptable liquid carrier. Dry (w / w) percentages may be measured, for example, using liquid chromatography.

[0046] As used herein, the term "nanoparticles" refers to a group of particles having a Z-average diameter of less than 1000 nm as measured by dynamic light scattering.

[0047] The term "pharmaceutical composition," as used herein, refers to a composition formulated with pharmaceutically acceptable excipients and used as part of a therapeutic regimen for the treatment of a disease in a mammal.

[0048] As used herein, the term "pharmaceutical dosage form" refers to a pharmaceutical composition intended to be administered to a subject as is, without further modification (e.g., without dilution in a liquid solvent, suspension in a liquid solvent, or dissolution in a liquid solvent).

[0049] The term “pharmaceutically acceptable excipient” as used herein means any component other than the active agent described herein (e.g., a vehicle capable of suspending or dissolving the active agent) that is substantially non-toxic and substantially non-inflammatory in the patient. Excipients may include, for example, antioxidants, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), flavorings, fragrances, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, liquid solvents, and buffers.

[0050] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, etc., within the bounds of normal medical judgment, and that is commensurate with a reasonable benefit-risk ratio. pharmaceutically acceptable salts are known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. PH Stahl and CG Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting free base groups with suitable organic acids. Examples of acids suitable for forming such salts include acetic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bicarbonate, bisulfate, tartaric acid, butyric acid, calcium edetate, camsilicic acid, carbonic acid, chlorobenzoic acid, citric acid, edetate, edicyl acid, estolic acid, esyl acid, esylic acid, formic acid, fumaric acid, and gluceptic acid. Examples include hydroxy acids, gluconic acid, glutamic acid, glycolyl arsanilic acid, hexamic acid, hexyl resorcinic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, methyl nitrate, methyl sulfuric acid, mucinic acid, muconic acid, napsylic acid, nitric acid, oxalic acid, p-nitromethanesulfonic acid, pamoic acid, pantothenic acid, phosphoric acid, monohydrogen phosphoric acid, dihydrogen phosphoric acid, phthalic acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfonic acid, sulfuric acid, tannic acid, tartaric acid, theoclic acid, and toluenesulfonic acid. Glutamate salts are particularly preferred.

[0051] As used herein, the term "subject" refers to a human or non-human animal (e.g., mammal) that is suffering from or at risk of suffering from a disease, disorder, or condition, as determined by a qualified professional (e.g., a physician or nurse), with or without known clinical tests in the art on a sample from which the subject is derived. Non-limiting examples of diseases, disorders, or conditions include fungal infections caused by species of the genera Candida, Cryptococcus, Aspergillus, Colletotrichum, Geotrichum, Hormonema, Lecythophora, Paecilomyces, Penicillium, Rhodotorula, Fusarium, Saccharomyces, Trichoderma, Trichophyton, and Scopularilopsis. Preferably, the fungal infection is caused by a species of Candida, Aspergillus, or Cryptococcus. More preferably, the fungal infection is caused by an azole-resistant Aspergillus species.

[0052] As used herein, “treatment” and “treating” refer to the medical management of a subject with the intention of improving, recovering from, stabilizing, preventing, or curing a disease, disorder, or condition. The terms include active treatment (treatment aimed at improving a disease, disorder, or condition); causal treatment (treatment targeting the cause of the associated disease, disorder, or condition); symptomatic (or palliative) treatment (treatment designed to alleviate the symptoms of a disease, disorder, or condition); preventive treatment (treatment aimed at minimizing, partially or completely preventing, the onset of the associated disease, disorder, or condition); and supportive care (treatment used to complement another treatment). [Brief explanation of the drawing]

[0053] [Figure 1] This chart shows the survival analysis of mice challenged with Aspergillus fumigatus AF91, either untreated or treated with compound 1A 1.0 mg / kg, compound 1A 0.5 mg / kg, AmBisome 7.5 mg / kg, AmBisome 3.5 mg / kg, voriconazole 7.5 mg / kg, caspofungin 1.0 mg / kg, or a vehicle (a 5% glucose aqueous solution containing 5% DMSO). [Figure 2] This chart shows the survival analysis of mice challenged with Aspergillus fumigatus AF91, either untreated or treated with compound 1A 1.0 mg / kg, compound 1A 0.5 mg / kg, ambisome 1.0 mg / kg, or ambisome 0.5 mg / kg. [Figure 3]This chart shows the survival analysis of mice challenged with Candida albicans SC5314, either untreated or treated with compound 1A 0.7 mg / kg, compound 1A 0.35 mg / kg, ambisome 5.4 mg / kg, ambisome 2.7 mg / kg, voriconazole 4 mg / kg, caspofungin 0.35 mg / kg, fluconazole 6 mg / kg, or vehicle (5% glucose aqueous solution containing 5% DMSO). [Figure 4] This chart shows the survival analysis of mice challenged with Candida albicans SC5314, either untreated or treated with compound 1A 0.7 mg / kg, compound 1A 0.35 mg / kg, ambisome 0.7 mg / kg, or ambisome 0.35 mg / kg. [Figure 5] This chart shows the LC-UV trace of formulation 45 containing 1.8% (w / w) of compound 1A. The LC-UV trace shows an undesirable reaction between compound 1A and the poly(alkylcyanoacrylate) (PACA) polymer at a retention time of 17-19 minutes. [Figure 6] This chart shows the LC-UV trace of formulation 49 containing 2.4% (w / w) of compound 1A. The LC-UV trace indicates that no degradation or reaction of compound 1A occurred during the formulation process. The impurity of 19.5 minutes is also present in the materials used in the formulation. [Figure 7] This chart shows the LC-UV trace of formulation 73 containing 0.68% (w / w) of compound 1A. The LC-UV trace indicates that no degradation or reaction of compound 1A occurred during the formulation process. [Modes for carrying out the invention]

[0054] In general, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles and a method of using the same. The pharmaceutical composition of the present invention has the following structure: [ka] The material comprises multiple nanoparticles containing a pharmaceutical active ingredient, which is a compound or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the active pharmaceutical ingredient has the following structure: [ka] It is a compound of or a pharmaceutically acceptable salt thereof.

[0056] The nanoparticles described herein may contain polymer excipients (e.g., polymer nanoparticles) or lipids (e.g., lipid nanoparticles such as liposomes and micelles).

[0057] The nanoparticles described herein may include lipids, such as phospholipids (e.g., phosphatidylcholine, phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol). In some embodiments, the nanoparticles described herein include phospholipids that are phosphatidylcholine (e.g., dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, egg phosphatidylcholine, and soy phosphatidylcholine) or phosphatidylglycerol (e.g., dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dilaurylphosphatidylglycerol, or dimyristoylphosphatidylglycerol). For example, lipids (e.g., phospholipids) can encapsulate (enclose) pharmaceutical active ingredients in vesicles or micelles.

[0058] Preferably, the pharmaceutical compositions described herein include a pharmaceutically acceptable polymer excipient (e.g., poly(alkylcyanoacrylate), lactic acid-glycolic acid copolymer, or protein (e.g., albumin, fibroin, gelatin, casein, or a combination thereof)). Advantageously, the pharmaceutical compositions described herein may exhibit a commercially acceptable shelf life. While not wishing to be bound by theory, encapsulation (encapsulation) of compound 1 (e.g., compound 1A) or its pharmaceutically acceptable salt in a pharmaceutically acceptable polymer excipient may provide sufficient stability for compound 1 (e.g., compound 1A) or its pharmaceutically acceptable salt to have a commercially acceptable shelf life. For example, the pharmaceutical compositions described herein may retain 90% to 110% of the labeled dose of compound 1 (e.g., compound 1A) or its pharmaceutically acceptable salt after storage at 4°C for two weeks.

[0059] The pharmaceutical compositions described herein may contain, as measured by liquid chromatography, at least 2%, preferably at least 3%, and particularly at least 5% dry (w / w) of compound 1 or a pharmaceutically acceptable salt thereof (e.g., compound 1A or a pharmaceutically acceptable salt thereof). The pharmaceutical compositions described herein may contain, as measured by liquid chromatography, at least 2% (e.g., at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, or at least 9.5%) dry (w / w) of compound 1 or a pharmaceutically acceptable salt thereof (e.g., compound 1A or a pharmaceutically acceptable salt thereof). The pharmaceutical compositions described herein may contain up to 15%, preferably up to 12%, and particularly up to 10% dry (w / w) of compound 1 or a pharmaceutically acceptable salt thereof (e.g., compound 1A or a pharmaceutically acceptable salt thereof), as measured by liquid chromatography. Non-limiting examples of the range include 2-15%, preferably 3-10%, and particularly 3-6%, as measured by liquid chromatography (e.g., 3.5-15%, 4-15%, 4.5-15%, 5-15%, 5.5-15%, 6-15%, 6.5-15%, 7-15%, 7.5-15%, 8-15%, 8.5-15%, 9-15%, 9.5-15%, 3-10%, 3.5-10%, 4-10%, 4.5-10%, 5-10%, 5.5-10%, 6-10%, 6.5%). Examples include compound 1 or a pharmaceutically acceptable salt thereof (e.g., compound 1A or a pharmaceutically acceptable salt thereof) in dry (w / w) form, in concentrations of ~10%, 7~10%, 7.5~10%, 8~10%, 8.5~10%, 9~10%, 9.5~10%, 3~7.5%, 3.5~7.5%, 4~7.5%, 4.5~7.5%, 5~7.5%, 5.5~7.5%, 6~7.5%, 6.5~7.5%, 7~7.5%, 3~5%, 3.5~5%, 4~5%, or 4.5~5%).

[0060] The pharmaceutical compositions described herein contain a plurality of nanoparticles. The plurality of nanoparticles may have a number-average diameter of, for example, 20 to 200 nm (preferably 40 to 100 nm) as measured by dynamic light scattering. Preferably, the plurality of nanoparticles have a number-average diameter of 30 to 150 nm as measured by nanoparticle tracking analysis (NTA) (more preferably 80 to 100 nm).

[0061] The pharmaceutical compositions described herein include one or more pharmaceutically acceptable excipients, such as pharmaceutically acceptable polymer excipients, surfactants (e.g., nonionic surfactants), stabilizers, carriers (e.g., oils), and / or flavoring agents.

[0062] In the pharmaceutical compositions described herein, polymer excipients may be used, for example, to encapsulate (encase) the active pharmaceutical ingredient. Non-limiting examples of pharmaceutically acceptable polymer excipients include poly(alkylcyanoacrylate), lactic acid-glycolic acid copolymers, amphiphilic polyphosphazenes, and proteins. Preferably, pharmaceutically acceptable polymer excipients are poly(alkylcyanoacrylate) (e.g., poly(ethylhexylcyanoacrylate), poly(ethylcyanoacrylate), poly(n-hexylcyanoacrylate), poly(4-methylpentylcyanoacrylate), poly(ethylbutylcyanoacrylate), poly(butylcyanoacrylate), or poly(octylcyanoacrylate)). More preferably, pharmaceutically acceptable polymer excipients are poly(ethylhexylcyanoacrylate) (e.g., poly(2-ethylhexylcyanoacrylate)). Preferred proteins include albumin, fibroin, gelatin, casein, and combinations thereof.

[0063] Nanoparticles containing poly(alkylcyanoacrylate) can be prepared in situ by polymerization of alkylcyanoacrylate monomers in a composition containing compound 1, 1A or pharmaceutically acceptable salts thereof. The process for preparing poly(alkylcyanoacrylate) nanoparticles may include customizing the nanoparticle surface by introducing a hydrophilic polymer (e.g., a pharmaceutically acceptable polymer excipient, and a surfactant having a reactive moiety that can react with, for example, a pharmaceutically acceptable polymer excipient precursor (e.g., a monomer that makes up a pharmaceutically acceptable polymer excipient)). Polymerization of the monomer can be initiated using such a surfactant. Alternatively, polymerization of monomers can be initiated using a polymerization initiator such as an azo initiator (e.g., 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylpropionic acid)dimethyl, 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile, 1,1'-azobis(cyclohexane-l-carbonitride), or 2,2'-azobis(N-butyl-2-methylpropionamide)). In some embodiments, the pharmaceutical compositions described herein include nanoparticles comprising a pharmaceutically acceptable polymer excipient and a pharmaceutically active ingredient which is compound 1, 1A or a pharmaceutically acceptable salt thereof. Preferably, the nanoparticles are coated with polyethylene glycol (PEG). Advantageously, the PEG coating on the nanoparticles may reduce clearance by, for example, the immune system.

[0064] Surfactants may be used, for example, to stabilize pharmaceutical compositions against crystallization and mechanical stress (such as stirring and / or shearing). Surfactants may be nonionic or ionic. Non-limiting examples of nonionic surfactants include polyoxyethylene ethers, polyoxyethylene esters (e.g., polyoxyethylene fatty acid esters), sorbitan esters, polysorbates, sorbitol, ethoxylated phenols, ethoxylated diphenols, polyethoxylated castor oil, polyoxyethylene / polyoxypropylene block copolymers (e.g., poloxamer), poloxamine, fatty acid monoglycerides, fatty acid diglycerides, polysaccharides (e.g., hyaluronic acid or sialic acid), proteins (e.g., albumin or casein), and combinations thereof. Preferably, the surfactant is a polyoxyethylene ether or polysorbate. More preferably, the surfactant is polyoxyethylene-modified 12-hydroxystearic acid (e.g., Kolliphor HS 15), polyoxyethylene lauryl ether (e.g., Brij L23), or a combination thereof. Non-limiting examples of ionic surfactants include, for example, sodium dodecyl sulfate, sodium lauryl sulfate, sulfosuccinates, and fatty acid salts.

[0065] Surfactants can be covalently bonded to polymer excipients. In non-limiting examples, the surfactants described herein may be contained in a mixture of a solvent, an active agent, and a monomer of the polymer excipient. Thus, a surfactant (e.g., a free -OH group in the surfactant) can initiate polymerization of a monomer (e.g., alkylcyanoacrylate) to encapsulate the active agent within surfactant-coated nanoparticles (e.g., PEG-coated nanoparticles).

[0066] For example, stabilizers may be used to stabilize pharmaceutical compositions against oxidative stress. Non-limiting examples of stabilizers include vanillin, butylated hydroxytoluene, butylated hydroxyanisole, vitamin E, and 6-O-palmitoyl-L-ascorbic acid. Preferably, the stabilizer is vanillin. The pharmaceutical composition contains, for example, 0.1 to 10%, preferably 0.5 to 8%, particularly 1 to 5% (for example, 0.1 to 9%, 0.1 to 8%, 0.1 to 7%, 0.1 to 6%, 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2%, 0.1 to 1%, 0.1 to 0.5%, 0.2 to 10%, 0.2 to 9%, 0.2 to 8%, 0.2 to 7%, 0.2 to 6%, 0.2 to 5%, 0.2 to 4%, 0.2 to 3%, 0.2 to 2%, 0.2 to 1%, 0.2 to 0.5%, 0.5 to 10%, 0.5 to 9%, 0.5 to 8%, 0.5 to 7%, 0.5 to 6%) relative to the particle mass. It may contain stabilizers in amounts of 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, 0.5-1%, 1-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1-2%, 2-10%, 2-9%, 2-8%, 2-7%, 2-6%, 2-5%, 2-4%, 2-3%, 3-10%, 3-9%, 3-8%, 3-7%, 3-6%, 3-5%, 3-4%, 4-10%, 4-9%, 4-8%, 4-7%, 4-6%, 4-5%, 5-10%, 5-9%, 5-8%, 5-7%, 5-6%, or 5% (w / w).

[0067] A carrier may be used to suspend or solubilize the active pharmaceutical ingredient in a pharmaceutical composition. The carrier may also be used to prevent Ostwald ripening during formulation preparation. The suspension or solubilization carrier may be an aqueous carrier, e.g., water or saline solution (e.g., isotonic saline solution). Further non-limiting examples of pharmaceutically acceptable carriers include pharmaceutically acceptable oils (e.g., medium-chain triglycerides, long-chain triglycerides, or combinations thereof). Preferably, the pharmaceutically acceptable oil is one or more medium-chain triglycerides (e.g., Miglyol, Captex, and Kollisolv). The pharmaceutical composition is, for example, 0.5-5%, preferably 1-5%, particularly 2-3% relative to the particle mass (e.g., 0.5-5%, 0.5-4.5%, 0.5-4%, 0.5-3.5%, 0.5-3%, 0.5-2.5%, 0.5-2%, 0.5-1.5%, 0.5-1%, 1-5%, 1-4.5%, 1-4%, 1-3.5%, 1-3%, 1-2.5%, 1-2%, 1-1.5%, 1.5-5%, 1.5-4.5%, 1.5-4%, 1.5- It may contain pharmaceutically acceptable oils in the following amounts: 3.5%, 1.5-3%, 1.5-2.5%, 1.5-2%, 2-5%, 2-4.5%, 2-4%, 2-3.5%, 2-3%, 2-2.5%, 2.5-5%, 2.5-4.5%, 2.5-4%, 2.5-3.5%, 2.5-3%, 3-5%, 3-4.5%, 3-4%, 3-3.5%, 3.5-5%, 3.5-4.5%, 3.5-4%, 4-5%, 4-4.5%, or 4.5-5%) (w / w).

[0068] For orally administered pharmaceutical compositions, flavorings can be included to make them more palatable. Any effective flavoring can be used. Flavorings may be natural, artificial, or mixtures thereof. Flavorings provide a taste that helps to mitigate the undesirable taste of the active ingredient. In one embodiment, flavorings may provide the taste of mint, menthol, honey lemon, orange, lemon lime, grape, cranberry, vanilla berry, bubblegum, or cherry. Flavorings may be natural or artificial sweeteners, such as sucrose, magnasweet, sucralose, xylitol, sodium saccharin, cyclamate, aspartame, acesulfame, and salts thereof.

[0069] The pharmaceutical compositions described herein may be aqueous compositions (e.g., suspensions). The pH of the pharmaceutical composition may be, for example, 4.0 to 8.0 (preferably 5.0 to 7.0). Alternatively, the pharmaceutical composition may be a lyophilized composition. The lyophilized composition may be reconstituted to prepare an aqueous composition before use.

[0070] The pharmaceutical compositions described herein may be used to treat subjects that require them. A method of treating a subject includes administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject. Subjects include, for example, Candida, Cryptococcus, Aspergillus, Colletotrichum, Geotrichum, Hormonema, Lecythophora, Paecilomyces, Penicillium, and Rhodotorula. a) The subject may have a fungal infection caused by a species of the genera Fusarium, Saccharomyces, Trichoderma, Trichophyton, Scopularilopsis, Histoplasma, Blastomyces, or Cocciodioides. The subject may have one or more of the following conditions: chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, chronic pulmonary aspergillosis, recovery from solid organ transplantation, recovery from blood transplantation, and immunosuppression after cancer chemotherapy, in addition to a fungal infection (e.g., invasive mycosis). Preferably, the fungal infection is caused by a species of Candida, Aspergillus, or Cryptococcus. More preferably, the fungal infection is caused by an azole-resistant Aspergillus species.

[0071] The pharmaceutical compositions described herein may be administered to a subject in single or multiple doses. If administered in multiple doses, the doses may be spaced apart, for example, by 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months. The pharmaceutical compositions may be administered according to a schedule, or they may be administered without a predetermined schedule. It should be understood that for any particular subject, a specific administration regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the pharmaceutical composition.

[0072] The attending physician will ultimately determine the appropriate dose and administration regimen, but the effective dose of the compound of the present invention may be, for example, the total daily dose of the pharmacokinetic ingredient (API) described herein, between 0.05 mg and 3000 mg. Alternatively, the dose may be calculated using the patient's body weight.

[0073] In the method of the present invention, the duration of administration of the multi-dose pharmaceutical composition to the subject can vary. In some embodiments, the pharmaceutical composition is administered to the subject over a period of 1 to 7 days; 1 to 12 weeks; or 1 to 3 months. In some embodiments, the pharmaceutical composition is administered to the subject over a period of, for example, 4 to 11 months or 1 to 30 years. In some embodiments, the pharmaceutical composition is administered to the subject at the onset of symptoms. In any of these embodiments, the amount of pharmaceutical composition administered may vary during the administration period. If the pharmaceutical composition is administered daily, administration can be carried out, for example, 1 to 12 times per day.

[0074] Exemplary routes of administration for the pharmaceutical compositions described herein include intravenous, inhalation, intranasal, oral, sublingual, buccal, transdermal, intradermal, intramuscular, vaginal, parenteral, intra-arterial, intracranial, intrathecal, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, and topical administration. Preferably, the route of administration is intravenous.

[0075] The pharmaceutical compositions described herein include those formulated for intravenous or intra-arterial administration. The pharmaceutical compositions described herein may include the microbubbles and nanoparticles described herein. Preferably, the pharmaceutical compositions comprising the microbubbles and nanoparticles are formulated for intravenous administration. Advantageously, the pharmaceutical compositions comprising the microbubbles and nanoparticles described herein can facilitate the targeted delivery of compound 1 or 1A to target tissues (e.g., the lungs and / or the heart).

[0076] The pharmaceutical composition may include any common, commercially available contrast agent microbubbles, such as Albunex (GE Healthcare), Optison (GE Healthcare), Sonazoid (GE Healthcare), Sonovue (Bracco), or other common contrast agent microbubbles known to those skilled in the art. In some pharmaceutical compositions, the microbubble surface may associate with nanoparticles. Such microbubbles may be produced, for example, in a solution of nanoparticles, as described herein. Advantageously, the nanoparticles may have a stabilizing effect on the microbubble surface. The microbubbles may be, for example, microbubbles filled with a gas or its precursor. The gas may include, for example, perfluorocarbons, hydrocarbons (e.g., methane), sulfur fluoride (e.g., SF6), halogens, air, air components (e.g., nitrogen (N2), oxygen (O2), argon (Ar), carbon dioxide (CO2), helium (He), or neon (Ne)), or mixtures thereof. Preferably, the gas is a perfluorocarbon, air, air components (e.g., nitrogen (N2), oxygen (O2), argon (Ar), carbon dioxide (CO2), helium (He), or neon (Ne)), or a mixture thereof, and more preferably, the gas is a perfluorocarbon.

[0077] While we do not wish to be bound by theory, the solubility of gas in microbubbles may affect the ability of microbubbles to circulate in the blood and to accumulate in the respiratory system. For example, microbubbles filled with perfluorocarbon gas may have an extended circulation time. While we do not wish to be bound by theory, the extended circulation time may be due to the low solubility of perfluorocarbon in the blood. The pharmaceutical compositions described herein may include microbubbles containing a gas, for example, perfluorocarbon. Alternatively, the gas may be, for example, air or a component thereof. Alternatively, the gas may be, for example, sulfur fluoride gas, preferably sulfur hexafluoride (SF6) gas.

[0078] Commercially available microbubbles are typically provided as a suspension of gaseous microbubbles stabilized by a shell of lipids, proteins, and / or other surfactants. Microbubbles can be produced using surface-active compounds, such as proteins, polymers, lipids, surfactants, or mixtures thereof. Surface-active compounds can stabilize the microbubbles. Preferred non-limiting examples of surface-active proteins are albumin (e.g., human or bovine serum albumin, or albumin from other suitable biocompatible albumin sources, including synthetic albumin) and casein. Preferred non-limiting examples of surface-active lipids are phospholipids. Microbubbles may also contain additional stabilizers and excipients, such as cholesterol or polyoxyethylene-polyoxypropylene.

[0079] The pharmaceutical compositions described herein may include, for example, a modifying agent. The modifying agent can modify the interactions between the components of the pharmaceutical composition. The modifying agent can form complexes or crosslinks between microbubbles and / or surface-active compounds and nanoparticles, thereby, for example, increasing the stability of the pharmaceutical composition. The modifying agent can introduce interactions between, for example, surface-active compounds and nanoparticles. The modifying agent may be, for example, urea (H2N-CO-NH2). Preferably, the pharmaceutical composition includes a protein (preferably casein) as the surface-active compound and urea as the modifying agent. Urea can act as a protein denaturant. While we do not wish to be bound by theory, it is thought that urea may interfere with hydrogen bonds involved in protein folding. Urea may also form complexes with acidic groups on the surface of nanoparticles, modifying the hydrophilicity of the nanoparticles.

[0080] In pharmaceutical compositions containing a surface-active compound (e.g., a protein) and urea, the amount of active agent delivered to the target tissue is enhanced. By introducing a stronger interaction between the surface-active compound and nanoparticles, urea can stabilize microbubbles. While not bound by theory, it is thought that modifications of microbubbles, surface-active compounds, and / or nanoparticles may enhance the stability of the association between microbubbles and nanoparticles in the pharmaceutical composition. Because the association between microbubbles and nanoparticles is enhanced, the number of nanoparticles delivered to the target lung tissue may be greater than in compositions lacking agents that enhance the association between microbubbles and nanoparticles.

[0081] The pharmaceutical compositions described herein contain microbubbles, and the nanoparticles may form a Pickering emulsion. Hydrophobic solid particles can be strongly adsorbed at the interface between immiscible fluids (e.g., oil-water), thus forming a Pickering emulsion (an emulsion stabilized by solid nanoparticles or fine particles). Therefore, nanoparticles associated with the microbubble surface can stabilize the composition as a Pickering emulsion. Advantageously, the pharmaceutical compositions described herein can be further stabilized by formulation as a Pickering emulsion. The average diameter of the microbubbles associated with the nanoparticles may be, for example, 0.5 to 30 μm (e.g., 1 to 10 μm). The diameter of the microbubbles can be measured, for example, by two-dimensional analysis of images of the microbubbles using an ImageJ image analyzer.

[0082] Furthermore, the pharmaceutical compositions described herein may include, for example, free nanoparticles in addition to microbubble surface-associated nanoparticles. The pharmaceutical compositions described herein may include nanoparticles associated with microbubbles as described herein, as well as free nanoparticles as described herein.

[0083] Pharmaceutical compositions comprising microbubbles and nanoparticles as described herein can be prepared, for example, by methods comprising combining gases or microbubbles with nanoparticles as described herein. For example, nanoparticles may be in solution. Nanoparticles can be prepared in situ as described herein, or they may be reconstituted from a dry composition.

[0084] Pharmaceutical compositions comprising microbubbles and nanoparticles as described herein include, for example, a. Adding the microbubbles and nanoparticles described herein to a solution, and b. Mixing solutions to manufacture a pharmaceutical composition. It may be prepared according to a method that includes [a certain component].

[0085] Alternatively, a pharmaceutical composition comprising microbubbles and nanoparticles as described herein may include, for example, a. To synthesize nanoparticles described herein, b. Combining nanoparticles with surface-active compounds, c. Adding gas to the solution, and d. Mixing solutions to manufacture a pharmaceutical composition. It may be prepared according to a method that includes [a certain component].

[0086] In some embodiments, the solution is mixed (e.g., stirred) for 2 seconds to 60 minutes (e.g., 1 to 10 minutes). Methods for mixing the solution are known in the art and include, for example, sonication, mechanical stirring, microfluidics, and shaking. In some methods for preparing microbubble-containing formulations, the composition may be degassed before adding the microbubble gas. Methods for degassing are known in the art; non-limiting examples of degassing methods include, for example, sonication and freeze-pump-thaw degassing.

[0087] The pharmaceutical compositions described herein include those formulated for oral administration ("oral dosage forms"). Oral dosage forms may be, for example, tablets, capsules, liquid suspensions, powders, granules, or pellets, and these contain a pharmaceutically active ingredient and one or more pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers; granulators and disintegrants; binders; and lubricants, anti-adhesives, colorants, flavorings, plasticizers, wetting agents, and buffering agents.

[0088] Controlled-release compositions for oral use can be constructed to release an active drug by controlling the dissolution and / or diffusion of the pharmacokinetic active ingredient. Dissolution or diffusion-controlled release can be achieved by appropriate coating of tablets, capsules, pellets, granules, or particles containing the API, or by incorporating the API-containing particles into a suitable matrix. In some embodiments, the composition includes a biodegradable, pH, and / or temperature-sensitive polymer coating. For example, an oral dosage form may contain a pharmacokinetic active ingredient (e.g., nanoparticles as described herein).

[0089] The pharmaceutical compositions described herein may be prepared using the techniques and methods described herein as well as those known in the art.

[0090] The present invention further has the following structure: [ka] The present invention relates to a method for producing multiple nanoparticles containing a pharmaceutical active ingredient, which is a compound or a pharmaceutically acceptable salt thereof.

[0091] In particular, multiple nanoparticles may include, for example, pharmaceutically acceptable polymer excipients.

[0092] Therefore, this method involves polymerizing a monomer precursor of a pharmaceutically acceptable polymer excipient in a liquid containing the monomer precursor and the compound or a pharmaceutically acceptable salt thereof. The polymerization step produces a plurality of nanoparticles.

[0093] In some embodiments, the active pharmaceutical ingredient has the following structure: [ka] It is a compound of or a pharmaceutically acceptable salt thereof.

[0094] In the manufacturing method described herein, the liquid may further contain a pharmaceutically acceptable surfactant (e.g., a nonionic surfactant, e.g., polyoxyethylene ether, polyoxyethylene fatty acid ester, sorbitan ester, polysorbate, polyethoxylated castor oil, polyoxyethylene / polyoxypropylene block copolymer, or a combination thereof). Preferably, the pharmaceutically acceptable surfactant is polyoxyethylene ether (e.g., polyoxyethylene lauryl ether), polyoxyethylene fatty acid ester (e.g., polyoxyethylated 12-hydroxystearic acid), or a combination thereof.

[0095] In the manufacturing method described herein, the liquid may further contain a pharmaceutically acceptable stabilizer (e.g., vanillin, butylated hydroxytoluene, butylated hydroxyanisole, or vitamin E). Preferably, the pharmaceutically acceptable stabilizer is vanillin.

[0096] In the manufacturing method described herein, the liquid may further contain a pharmaceutically acceptable oil (for example, an oil selected from the group consisting of medium-chain triglycerides, long-chain triglycerides, and combinations thereof). Preferably, the pharmaceutically acceptable oil is one or more medium-chain triglycerides (for example, Miglyol, Captex, and Kollisolv).

[0097] In the manufacturing method described herein, the monomer precursor may be, for example, an alkylcyanoacrylate (e.g., ethylhexylcyanoacrylate), and the pharmaceutically acceptable polymer excipient may be, for example, a poly(alkylcyanoacrylate) (e.g., poly(ethylhexylcyanoacrylate)).

[0098] In the manufacturing method described herein, the nanoparticles may have a number-average diameter of 20 to 200 nm (e.g., 40 to 100 nm) as measured by dynamic light scattering. In the manufacturing method described herein, the nanoparticles may have a number-average diameter of 30 to 150 nm (e.g., 80 to 100 nm) as measured by nanoparticle tracking analysis.

[0099] The liquid may be, for example, an aqueous composition (for example, an aqueous composition having a pH of 0.5 to 8.0 (for example, a pH of 0.5 to 3.0, 2.0 to 8.0, or 3.0 to 7.0)).

[0100] The manufacturing method may further include the step of freeze-drying multiple nanoparticles. Additionally or alternatively, the manufacturing method may further include the step of dialyzing multiple nanoparticles against deionized water.

[0101] In the manufacturing methods described herein, the pH of the liquid may be adjusted as needed. For example, the pH of the liquid may be adjusted to a range of 4.0 to 8.0 (e.g., 5.0 to 7.0). In some embodiments, the step of adjusting the pH is performed during the polymerization step. In other embodiments, the step of adjusting the pH is performed before or after the polymerization step.

[0102] The following examples are intended to illustrate the present invention. They are not intended to limit the invention in any way. [Examples]

[0103] Example 1. Preparation of polyphosphazene nanoparticle composition Ethyl 4-aminobenzoate (CAS: 94-09-7) and polyethylene glycol-substituted (8-13 repeating units) amphiphilic polyphosphazene (POPZ) polymer (SINTEF) with a molecular weight of 7-11 kDa were used. 11 mg of compound 1A (custom-made) was dissolved in 11 mL of DMF and 100 mg of POPZ. The solution was added dropwise to 11 mL of distilled water with vigorous stirring at room temperature. The sample was dialyzed in one shift against distilled water. The particle solution was lyophilized to produce dried nanoparticle powder with a theoretical loading of 10% (w / w) compound 1A.

[0104] Example 2. Preparation of poly(2-ethylhexyl cyanoacrylate) nanoparticle composition at pH=2 An aqueous solution containing PEG stabilizers Kolliphor HS 15 (0.12 g) and Brij L23 (0.12 g) was prepared in 12 mL of 0.01 M HCl (pH 2). A solution containing 0.096 g of compound 1A (custom-made), dissolved in 2-ethylhexyl cyanoacrylate (0.8 g) together with stabilizers (0.05 g vanillin and 15 μL Miglyol 812), was prepared and stirred at room temperature for 2 hours.

[0105] The two solutions were mixed on ice and homogenized for 3 minutes using an ultrasonic device (50% amplitude) with 10-second pauses every 30 seconds. The nanoemulsion was polymerized on a rotator at room temperature for 3 hours. The pH was adjusted to 6 with 0.1 M NaOH, and further polymerization was carried out overnight at room temperature (on the rotator). The particulate sample was dialyzed against distilled water. The final product was a liquid suspension. The theoretical loading was 10% (w / w).

[0106] Example 3. Preparation of poly(2-ethylhexyl cyanoacrylate) nanoparticle composition at pH=4 The same procedure as described in Example 2 was used in this example, except that the aqueous solution contained 0.1 mM HCl (pH=4) instead of 0.01 M HCl.

[0107] Example 4. Preparation of poly(2-ethylhexyl cyanoacrylate) nanoparticle composition at pH=1 The same procedure as described in Example 2 was used in this example, except that the aqueous solution contained 0.1 M HCl (pH=1) instead of 0.01 M HCl.

[0108] Example 5. Preparation of poly(2-ethylhexyl cyanoacrylate) nanoparticle composition without Brij L23 The same procedures as those described in Examples 2-4 were used in this example, except that the aqueous solution did not contain Brij L23 and the amount of Kolliphor HS 15 was doubled.

[0109] Example 6. Preparation of a poly(2-ethylhexyl cyanoacrylate) nanoparticle composition containing more vanillin. The same procedures as those described in Examples 2-5 were used in this example, except that the amount of vanillin was increased to 0.1 g.

[0110] Example 7. Preparation of a poly(2-ethylhexyl cyanoacrylate) nanoparticle composition containing less vanillin. The same procedures as those described in Examples 2-5 were used in this example, except that the amount of vanillin was reduced to 0.025 g.

[0111] Example 8. Preparation of a poly(2-ethylhexyl cyanoacrylate) nanoparticle composition at pH=2 and theoretical loading=14.8% The same procedure as described in Example 2 was used in this example, except that the theoretical loading of compound 1A was 14.8% (w / w). The measured final loading of compound 1A was 3.3% (w / w).

[0112] Example 9. Preparation of poly(2-ethylhexyl cyanoacrylate) nanoparticle composition at pH=3 and theoretical loading=14.8%: The same procedure as described in Example 2 was used in this example, except that (1) the aqueous solution contained 1 mM HCl (pH=3) instead of 0.01 M HCl, and (2) the theoretical loading of compound 1A was 14.8% (w / w). The measured final loading of compound 1A was 3.6% (w / w).

[0113] Example 10. Preparation of a poly(2-ethylhexyl cyanoacrylate) nanoparticle composition at pH=4 and theoretical loading=14.8% The same procedure as described in Example 3 was used in this example, except that the theoretical loading of compound 1A was 14.8% (w / w). The measured final loading of compound 1A was 3.8% (w / w).

[0114] Example 11. Physicochemical characterization of nanoparticle composition Size and size distribution were measured using dynamic light scattering (Malvern Zetasizer and Nanoparticles Tracking Analyzer (NTA)) in phosphate buffer (pH 7). Dry weight was determined by drying three sample aliquots overnight at 50°C. For LC-DAD-QTOF analysis, three sample aliquots were weighed / pipetted, dissolved in DMSO, and further diluted to determine drug loading and stability. The concentration of compound 1A was determined using three samples of amphotericin B USP as standards. The LC-QTOF method was as follows: Mobile phase: 0.1% formic acid [A] and acetonitrile [B] HPLC system: Agilent 1290 HPLC with 1290 DAD connected to QTOF Column: Polaris 3 C18, 150 x 2 mm, 3 μm (Varian) Column thermostat: 30℃ Flow rate: 0.3ml / min Injection volume: 2 μL Wavelength: 385 nm for measuring compound 1A / AMB, scan 190-600 nm Software: MassHunter Qualitative Analysis B.0.600 Post time: 5 minutes The QTOF was operated in negative electrospray mode (ESI-). The HPLC gradient is shown in Table 1. [Table 1]

[0115] Size and size distribution Table 2 summarizes the nanoparticle size, size distribution, dry weight, and drug loading. [Table 2]

[0116] Table 3 provides details of the particle size distribution measured by NTA. [Table 3]

[0117] Drug loading and stability In the composition from Example 1, 42 mol% of the polyene in the particles was compound 1A. The loading of compound 1A was 1.1% (w / w), and the total polyene loading was 2.5% (w / w).

[0118] The composition from Example 2 contained the same polyene impurities as Example 1, but at a lower level, as 10% of the total polyenes corresponded to the same major polyene impurities observed in Example 1. The concentration of compound 1A in the liquid sample was 0.59 mg / mL, giving a loading of 2.8% (w / w) of compound 1A in the particles.

[0119] In the composition of Example 3, no significant degradation of compound 1A was observed. The concentration of compound 1A in the liquid sample was 0.40 mg / mL, resulting in a 3.9% (w / w) loading of compound 1A in the particles. Small amounts of minor polyene impurities were observable, but their concentrations were too low to determine their mass.

[0120] Long-term stability of compound 1A in poly(2-ethylhexyl cyanoacrylate) nanoparticles The composition of Example 3 was re-analyzed two weeks after preparation. No degradation was observed by UV at λ=385nm after storage at 4°C for two weeks.

[0121] The shelf life of a composition can be tested over longer periods and using different temperature and humidity levels.

[0122] Example 12. Efficacy of the composition of Example 3 The efficacy of the compositions was determined using a minimum inhibitory concentration assay, and the concentration of the active compound that provided 50% inhibition of indicator organism growth was reported (MIC50). The assay was performed in well plates containing two (Mueller-Hinton medium) or three (M19 medium) parallel cell cultures for each condition.

[0123] Cell culture medium: Mueller-Hinton and M19 (NaCl-free) Indicator organism: Candida albicans ATCC 10231 Stock solutions of the active compound and the control: • Amphotericin B (USP): Vacuum-dried powder was dissolved in DMSO to a concentration of 2.5 mg / mL, and the final concentration was obtained after inoculation of 2.5 μg / mL in the well with the highest concentration. • Compound 1A (custom-made): The powder was dissolved in DMSO to a concentration of 2.5 mg / mL, and the final concentration was obtained by inoculating 2.5 μg / mL of Compound 1A in the well with the highest concentration. The batch is assumed to be 70% pure. • Composition of Example 3: The formulation was a suspension containing 18 mg / mL of nanoparticles. The suspension was diluted in culture medium and Candida albicans inoculum to final concentrations of 5 μg / mL of compound 1A and 130 μg / mL of poly(2-ethylhexyl cyanoacrylate). Ten dilutions were prepared from this solution to give the lowest concentration of compound 1A at 0.009 μg / mL. • Empty poly(ethylbutylcyanoacrylate) particles: Empty poly(ethylbutylcyanoacrylate) particles were used as a reference. The empty poly(ethylbutylcyanoacrylate) was prepared in the same manner as in Examples 2 and 3, except that it was prepared at a 10% (w / w) vanillin concentration and pH 1. The particles were diluted in the same manner as in Example 3. The highest poly(ethylbutylcyanoacrylate) concentration tested was 130 μg / mL. Growth measurements were performed using an OD600.

[0124] As described in Example 11, compound 1A was found to be stable in the composition of Example 3. This substance was tested in an in vitro efficacy assay against Candida albicans (C. albicans) to verify that the active compound was released from the particles at a rate sufficient to inhibit Candida albicans (C. albicans) to the same extent as pure compound 1A.

[0125] The measured MIC50 values ​​were higher in M19 medium than in Mueller-Hinton medium because the strain grew faster in M19 medium. Plates were manually measured overnight. Assays in both M19 medium and Mueller-Hinton medium showed that the MIC50 of the composition of Example 3 was 2 to 2.5 times higher than the MIC50 of pure compound 1A. As a control, inhibition of empty poly(ethylbutylcyanoacrylate) particles was tested in M19 medium. Inhibition of Candida albicans ATCC 10231 growth was not observed up to the highest concentration of poly(ethylbutylcyanoacrylate) tested, which was 130 μg / mL. The highest concentration of the composition of Example 3 tested (5 μg / mL) contained 130 μg / mL of poly(2-ethylhexylcyanoacrylate). [Table 4]

[0126] Example 13 Preparation and Characterization of Dye-Loaded Nanoparticles: Prepare other nanoparticles containing compound 1 or a pharmaceutically acceptable salt thereof (e.g., compound 1A or a pharmaceutically acceptable salt thereof). For example, PEG-coated and dye-loaded polymer or lipid nanoparticles are prepared by a miniemulsion process as follows:

[0127] Polymer nanoparticles: An oil phase is prepared consisting of a mixture of alkyl cyanoacrylate (e.g., n-butyl cyanoacrylate) and a pharmaceutically acceptable oil (e.g., Miglyol) and an added near-infrared dye (e.g., DIR). Then, an aqueous phase containing one or more surfactants (e.g., Brij L23 and / or Kolliphor HS 15) is added to the oil phase. Certain surfactants such as Kolliphor HS 15 can be used as polymerization initiators to produce polymer excipients (e.g., poly(alkylcyanoacrylate)) covalently bonded to polyethylene glycol. An oil-in-water emulsion is prepared by mixing the oil phase and the aqueous phase. The dispersion is dialyzed against an aqueous fluid (e.g., using a Spectra / Por dialysis membrane MWCO 100,000 Da) to remove surfactants not incorporated into the nanoparticles.

[0128] Lipid nanoparticles: A lipid phase consisting of a mixture of lipids (e.g., stearic acid and isopropyl palmitate) and a mixture of pharmaceutically acceptable oil (e.g., Miglyol) and an added near-infrared dye (e.g., DIR) is preheated until melted. An aqueous phase is prepared consisting of distilled water and additives (e.g., surfactants (e.g., lecithin 8OH and Andean QDP Ultra)). The lipid phase and the aqueous phase are mixed.

[0129] Exemplary preparation and characterization of nanoparticles carrying compound 1A: PEG-coated poly(alkylcyanoacrylate) nanoparticles carrying compound 1A are prepared by a miniemulsion method as follows: Prepare an oil phase containing alkylcyanoacrylate (e.g., 2-ethyl-butylcyanoacrylate), a pharmaceutically acceptable oil (e.g., Miglyol), and compound 1A. Prepare an aqueous phase containing surfactants (e.g., Brij L23 and Kolliphor HS 15). Kolliphor HS 15 may also function as a polymerization initiator. Prepare an oil-in-water emulsion by mixing the oil phase and the aqueous phase. Remove unbound surfactants from the particles by large-scale dialysis of the dispersion against an aqueous fluid (e.g., using a dialysis membrane, MWCO 100,000 Da).

[0130] Dynamic light scattering (e.g., Zetasizer) can be used to determine the hydrodynamic diameter, hydrodynamic diameter distribution, and zeta potential. The dry weight content (nanoparticle concentration) of the final solution can be determined after sufficient drying. To calculate the amount of encapsulated drug, the drug contents are extracted from the particles, and the amount of extracted compound 1A is quantified using LC-MS / MS. Dynamic light scattering typically reveals the nanoparticle size (z-mean) of the drug-loaded nanoparticles.

[0131] Manufacturing and characterization of nanoparticle-stabilized microbubbles: Gas-filled microbubbles that associate with nanoparticles are prepared as follows: Prepare a solution containing a surface-active compound (e.g., 2% (w / w) casein). Mix PEGylated nanoparticles carrying compound 1A (e.g., those described in Examples 2, 3, and 8-10 or this example) with the casein solution. Saturate the solution with gas (e.g., air or perfluoropropane). Seal the vial using a septum under a gas-filled atmosphere. In some methods for preparing microbubble-containing formulations, the composition may be degassed before adding the microbubble gas.

[0132] The average size and concentration of the obtained nanoparticle-stabilized microbubbles are determined from optical microscope images using a 20x phase-contrast objective lens and a cell counter (hemocytometer). The microbubbles are counted, and their size is calculated by analyzing the images using an ImageJ image analysis system.

[0133] Fluorescence microscopy (using the same type of nanoparticles encapsulated only with a fluorescent dye instead of a drug) and electron microscopy are used to confirm that the nanoparticles associate with microbubbles that form a stabilized (mono) layer.

[0134] Example 14 This study evaluates the potential of nanoparticle-associated microbubbles for specific drug delivery targeting the lungs, for example, in healthy animals (e.g., mice). High local concentrations can be achieved with gas-filled microbubbles stabilized by nanoparticles.

[0135] method This study is designed with one animal per group. Nanoparticles labeled with near-infrared fluorescent dyes are developed and used according to the procedure described in Example 13. These nanoparticles are localized into the bodies of small animals using a whole-body imager.

[0136] If it is necessary to slaughter test animals in the experiment, the animals may be anesthetized before use and slaughtered before they wake up. During storage at the animal facility, the animals' welfare was monitored, and they were given free access to food and water.

[0137] Animal experiments: 1. Randomly select animals, weigh them, and subcutaneously inject them with a solution that provides complete anesthesia (e.g., fentanyl / medetomidine / midazolam / water (2:1:2:5)). 2. Insert a catheter to enable intravenous injection. 3. Inject the desired bubbles. 4. Put the animal to sleep for the desired amount of time before euthanizing it. 5. Next, the lungs, liver, kidneys, and spleen can be collected. 6. Fluorescence from organs can be imaged using a fully animal-specific imager (e.g., Pearl).

[0138] Nanoparticles: Near-infrared labeled nanoparticles are used for animal experiments.

[0139] Following a control containing only nanoparticles, a pharmaceutical composition containing nanoparticles associated with microbubbles (e.g., as described in Example 13), where the microbubbles contain a gas (e.g., perfluoropropane).

[0140] To directly compare the results, lungs from multiple animals (e.g., three animals) are imaged together.

[0141] Microbubbles containing lipid nanoparticles: Microbubbles will be manufactured and tested using lipid nanoparticles.

[0142] The stability of lung accumulation is tested to evaluate whether the nanoparticles remain in the lungs or are redistributed to other organs. Then, the in vivo distribution in animals (e.g., two animals) is examined at desired time intervals (e.g., 1 and 2 hours after injection).

[0143] High local concentrations of nanoparticles in the lungs are beneficial for the targeted delivery of active pharmaceutical ingredients to lung tissue.

[0144] Example 15 This study will test gas-filled microbubbles stabilized by nanoparticles in targeted drug delivery to the lungs. This study will be conducted, for example, in healthy mice.

[0145] method Nanoparticles labeled with a fluorescent dye (e.g., a near-infrared dye) are prepared as described in Example 13. These nanoparticles are localized in the body of a small animal using a whole-body imager.

[0146] If it is necessary to slaughter test animals in an experiment, the animals may be anesthetized before being used in the experiment and then slaughtered before they wake up.

[0147] Manufacturing of nanoparticle-stabilized microbubbles: Gas-filled microbubbles that associate with nanoparticles are manufactured as follows:

[0148] Prepare a solution containing a surface-active compound (e.g., 2% (w / w) casein). Mix the dye-loaded PEGylated nanoparticles with the casein solution. Saturate the solution with a gas (e.g., sulfur hexafluoride or perfluoropropane). In some batches, add a modifier (e.g., urea) to further promote association between microbubbles and nanoparticles. Seal the vial using a septum under a gas-filled atmosphere. Animal experiments are carried out as described in Example 10. Each animal is intravenously injected with, for example, one of the following: A) Perfluoropropane-filled microbubbles stabilized by poly(2-ethylhexyl cyanoacrylate) nanoparticles. B) Perfluoropropane-filled microbubbles containing urea stabilized by poly(2-ethylhexyl cyanoacrylate) nanoparticles. C) Sulfur hexafluoride-filled microbubbles stabilized by poly(2-ethylhexyl cyanoacrylate) nanoparticles.

[0149] After injection, images are taken, and the in vivo distribution of drug-filled nanoparticles in the lungs of test animals is evaluated using fluorescence intensity.

[0150] Example 16 The efficacy of compound 1A was tested against a broad panel of yeast and filamentous fungal strains by measuring minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Amphotericin B (AmB), caspofungin (Cas), fluconazole (Flu), and voriconazole (Vor) were tested as comparators.

[0151] Materials: Isolates were obtained from the culture collection of the Center for Medical Mycology and included 20 strains each of Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Fusarium spp., and Mucormycetes (Rhizopus and Mucor spp.). The panel also included 10 strains each of Candida krusei, Aspergillus terreus, and Penicillium spp., as well as 5 strains of Paecilomyces spp. The panel included 17 Candida strains with known high MICs against other currently available antifungal agents; recent clinical strains were also included. The study also included 15 dimorphic fungal isolates.

[0152] Minimum inhibitory concentration (MIC) tests were performed according to Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M38-A2 standards for susceptibility testing of yeast and filamentous fungi, respectively (Cryptococcus isolates were incubated for 72 hours). For yeast strains, the incubation temperature and time were 35°C and 24–48 hours, respectively, and the inoculum size was 0.5–2.5 × 10⁶. 3 The concentration was CFU / ml. For filamentous fungal strains, the inoculation size was 0.4-5 × 10⁴. 4The CFU / ml values ​​were used, and incubation times were specific to the strain and the drug. Throughout, the test medium was RPMI 1640, except for the use of YNB for Cryptococcus. Inhibition endpoints for compound 1A were recorded at 50% and 100% after both 24-hour and 48-hour incubations; the results for caspofungin against Aspergillus strains were read as the minimum effective concentration (MEC), the lowest concentration resulting in small, rounded, compact growth compared to confluent growth in the control.

[0153] The determination of the minimum fungicidal concentration (MFC) was performed according to an improved method previously described by Canton et al., Diagn. Microbiol. Infect. Dis., 45:203-206, 2003, and Ghannoum and Isham, Infectious Diseases in Clinical Practice, 15(4):250-253, 2007. Specifically, the entire contents of each clear well from the MIC assay were subcultured on potato dextrose agar. To avoid antifungal carryover, aliquots were immersed in agar, then streaked for isolation after drying, and cells were removed from the drug source. Fungicidal activity was defined as a reduction of 99.9% or more in colony-forming units (CFUs) / ml from the initial inoculation, while fungicidal activity was defined as a reduction of less than 99.9%. A drug is considered fungicidal if its MFC / MIC ratio is 4 or less (≤4), and fungiostatic if the ratio is greater than 4 (>4). MIC and MFC values ​​are considered equivalent if they are within two dilutions.

[0154] Endpoint Determination: All MIC endpoints for compound 1A were recorded as both 50% and 100% inhibition compared to the growth control. Compound 1A showed both 50% and 100% inhibition against all Paecilomyces spp. and Candida parapsilosis strains; however, all MIC values ​​below are reported as 100% inhibition endpoints.

[0155] Determination of incubation time: Inhibition of growth after exposure to compound 1A was recorded after 24-hour and 48-hour incubation. Exceptions to this were mucormycetes, which were read only at 24 hours due to their rapid growth rate, and Cryptococcus strains, which were read at 72 hours to match the incubation times of all other control groups. No difference was observed between the MIC values ​​recorded at 24 hours and those recorded at 48 hours for any of the tested species. However, since some strains of Penicillium and Paecilomyces did not show visible growth in the MIC assay at 24 hours, all MIC values ​​below are reported at the 48-hour incubation time.

[0156] result Determination of MIC and MFC for Candida strains: Table 5 shows the MIC and MFC data for compound 1A and the comparator against Candida albicans (C. albicans), including both fluconazole-susceptible (n=13) and resistant (n=7) strains. MIC 50 MIC is defined as the minimum concentration that blocks 50% of the tested strain. 90 This is defined as the minimum concentration that blocks 90% of the tested strains. MFC 50 This is defined as the minimum concentration required to kill 50% of the tested strains, and is MFC 90This is defined as the minimum concentration required to kill 90% of the tested strains. [Table 5]

[0157] Table 6 shows the fungicidal activity of compound 1A and the comparative substance against Candida glabrata strains. [Table 6]

[0158] Table 7 shows the MIC and MFC data for all drugs against the Candida krusei strain. [Table 7]

[0159] Table 8 shows the fungicidal activity against Candida parapsilosis (C. parapsilosis). [Table 8]

[0160] Table 9 shows the fungicidal activity of compound 1A and the comparative substance against Candida tropicalis. [Table 9]

[0161] Table 10 summarizes the MIC and MFC data for compound 1A and the control substance against all Candida strains. [Table 10]

[0162] Determination of MIC and MFC for Cryptococcus strains Table 11 shows the activity of compound 1A and its comparative substances against the Cryptococcus neoformans (Cr. neoformans) strain. [Table 11]

[0163] Determination of MIC and MFC for Aspergillus strains Tables 12-15 show the MIC and MFC data for compound 1A and the control substance against individual Aspergillus species. Table 16 is a summary of the MIC and MFC data for all Aspergillus strains tested.

[0164] [Table 12]

[0165] [Table 13]

[0166] [Table 14]

[0167] [Table 15]

[0168] [Table 16]

[0169] Determination of MIC and MFC for difficult-to-treat / rare fungi. Table 17 shows the MIC and MFC data for Fusarium strains. [Table 17]

[0170] Table 18 shows the MIC and MFC data for Fusarium strains. [Table 18]

[0171] The results for strains of the genera Penicillium and Paecilomyces can be seen in Tables 19 and 20, respectively. Table 20 shows that the number of Paecilomyces strains (5) was too small for the MIC to be determined. 50 and MIC 90 Since the values ​​could not be calculated, only the concentration range is included.

[0172] [Table 19]

[0173] [Table 20]

[0174] Determination of MIC and MFC for dimorphic fungi Table 21 shows the MIC data for strains of Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum. MFC was not performed on these fungi because they are limited fungi for which there are no standardized methods for MFC. [Table 21]

[0175] Determination of MIC and MFC for other Candida strains with high MICs to other antifungal agents. Table 22 compares the MIC data of compound 1A against Candida strains with high MIC to non-polyene comparator substances (n = 17). [Table 22]

[0176] Example 17 The efficacy of compound 1A was tested in the treatment of disseminated aspergillosis in an immunocompromised mouse model and compared to amphotericin B, voriconazole, and caspofungin.

[0177] Female CD-1 mice (Charles River Laboratories, Wilmington, MA), each weighing approximately 30 g, were used as models. The environmental control of the animal room was set to maintain a temperature of 16 - 22°C, a relative humidity of 30 - 70%, and a 12:12 light-dark cycle.

[0178] Preparation of standard inoculum Organism: Aspergillus fumigatus AF91 was obtained from the Culture Collection of CWRU Center for Medical Mycology. Cells were subcultured from the frozen stock on potato dextrose agar (PDA) plates. Then, the cells were harvested using sterile saline containing 0.05% Tween80, centrifuged, and washed three times with normal saline (0.85% NaCl). A challenge inoculum of 1x10 7 was prepared using a hemacytometer.

[0179] Verification of inoculum count: To confirm the number of inoculum, 10-fold dilutions of the working conidial suspension of Aspergillus fumigatus (A. fumigatus) were plated on PDA medium. The plates were incubated at 37°C for 2 - 4 days, and the number of colonies was determined.

[0180] Immunosuppression: Mice were subcutaneously administered cyclophosphamide at the following doses: 150 mg / kg 4 days before infection, 100 mg / kg 1 day before infection, and 100 mg / kg 2 days after inoculation. On the day of the challenge, blood was collected from one mouse in each group, and the white blood cell count was measured to confirm immunosuppression.

[0181] Infection: Each mouse was given 1x10 in 0.1 ml of physiological saline. 7 Conidia were challenged (via the tail vein). Animals were considered infected after successful IV administration of the inoculum and confirmation of the inoculum (see Section 7d). The efficacy of the treatment and control groups was evaluated using tissue fungal burden and survival as indicators. Tissue fungal burden was assessed using 5 mice per group (randomly selected), and survival was assessed using 10 mice per group.

[0182] Test compound: The sponsor provided the test item, compound 1A (batch ELN EXP-11-AJ1675, potency 928 mg / g). It was administered intravenously in a 5% glucose aqueous solution containing 5% dimethyl sulfoxide (DMSO), prepared on the day of use from a stock solution of compound 1A in DMSO stored at -20°C. The comparator substances (Ambizome, voriconazole, and caspofungin) were purchased from a pharmacy by the Center for Medical Mycology. These were dissolved in sterile water according to the manufacturer's instructions to obtain aliquots containing the selected dose.

[0183] In this report, the dose and concentration of ambisome are expressed as the amphotericin B content.

[0184] Treatment groups: Infected mice were randomized into the following groups (5 mice per group for tissue fungal load and 10 mice per group for survival).

[0185] Experiment I consisted of the following treatment groups: Compound 1A 1.0 mg / kg, Compound 1A 0.5 mg / kg, Ambisome 7.5 mg / kg, Ambisome 3.5 mg / kg, Voriconazole 7.5 mg / kg, Caspofungin 1.0 mg / kg, Vehicle (5% glucose aqueous solution containing 5% DMSO), and an untreated control group.

[0186] Experiment II consisted of the following treatment groups: Compound 1A 1.0 mg / kg, Compound 1A 0.5 mg / kg, Ambisome 1.0 mg / kg, Ambisome 0.5 mg / kg, and an untreated control group. All treatments were administered intravenously.

[0187] Treatment schedule: Starting 2 hours after inoculation, animals were treated for 7 days. Compound 1A and caspofungin were administered once daily, while ambisome was administered every other day in Experiment I and daily in Experiment II. Voriconazole was administered twice daily at 8-hour intervals due to its rapid clearance.

[0188] Tissue fungal load: Mice were sacrificed one day after the final day of treatment; then the kidneys and lungs were aseptically removed and weighed. The tissues were homogenized and serially diluted with phosphate-buffered saline. The homogenates were cultured on PDA plates for 48 hours to determine the colony-forming units (CFU); the tissue fungal load was expressed as CFU / tissue (g).

[0189] Survival analysis: Infected mice were monitored, and signs of disease (i.e., lethargy, weight loss, generalized growth failure) or mortality were recorded twice daily for 28 days after inoculation. The mean body weight of each treatment group was also recorded daily. Dying animals unable to take food / water were euthanized.

[0190] Statistical analysis: Survival differences were compared using the Kaplan-Meier method, and mean CFU in the kidneys or lungs were compared using non-parametric independent Mann-Whitney tests. P-values ​​less than 0.05 (<0.05) were considered statistically significant.

[0191] result In vitro activity: Table 23 shows the in vitro activities of Compound 1A, the comparative agent, and amphotericin B against Aspergillus fumigatus AF91 (infection strain). [Table 23]

[0192] Experiment I Survival: In Figure 1, survival is given as a percentage of the total number of animals in the group on Day 1 of treatment.

[0193] Kidney tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 24). The fungal burden in the kidneys was analyzed and given as mean log CFU ± standard deviation.

[0194] Lung tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 24). The fungal burden in the lungs was analyzed and given as mean log CFU ± standard deviation.

[0195] [Table 24]

[0196] Experiment II Survival: As seen in Figure 2, survival was given as a percentage of the total number of animals in the group on Day 1 of treatment.

[0197] Kidney tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 25). The fungal burden in the kidneys was analyzed and given as mean log CFU ± standard deviation.

[0198] Lung tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 25). The fungal burden in the lungs was analyzed and given as mean log CFU ± standard deviation.

[0199]

Table 25

[0200] Example 18 Evaluate the efficacy of Compound 1A compared to amphotericin B, voriconazole, fluconazole and caspofungin in the treatment of disseminated candidiasis in an immunodeficient mouse model.

[0201] Female BALB / c mice (Charles River Laboratories, Wilmington, MA), each weighing approximately 20 g, were used as models. Environmental management of the animal room was set to maintain a temperature of 16 - 22°C, a relative humidity of 30 - 70%, and a 12:12 light-dark cycle.

[0202] Preparation of standard inoculum: Clinical Candida albicans SC5314 strain was obtained from the CMM Culture Collection and used as the infectious fungus. Candida albicans was plated on Sabouraud Dextrose Agar (SDA) and incubated at 37°C for 2 days. Candida albicans cells cells were recovered by centrifugation and washing with physiological saline (0.85% NaCl). A challenge inoculum of 5x10 5 was prepared using a hemocytometer.

[0203] Verification of inoculum number: To confirm the number of inoculum, 10-fold dilutions of the working conidia suspension of C. albicans were plated on SDA medium. The plates were incubated at 37°C for 2 days and the number of colonies was determined.

[0204] Immunosuppression: Mice were subcutaneously administered the following doses of cyclophosphamide: 150 mg / kg 4 days before infection, 100 mg / kg 1 day before infection, and 100 mg / kg 2 days after inoculation. On the day of the challenge, blood was collected from one mouse in each group, and the white blood cell count was measured to confirm immunosuppression.

[0205] Infection: Each mouse was given 1x10 in 0.1 ml of physiological saline. 4 Spore blasts were challenged (via the tail vein). Animals were considered infected after successful IV administration of the inoculum and confirmation of the inoculum. The efficacy of the treatment and control groups was evaluated using tissue fungal load and survival as indicators, with 5 mice per group for tissue fungal load and 10 mice per group for survival. The tissue load arm and survival arm of Experiment I were performed separately at two different occasions.

[0206] Test compound: The sponsor provided the test item, compound 1A (batch ELN Exp-11-AJ1675, potency 928 mg / g). It was administered intravenously in a 5% glucose aqueous solution containing 5% dimethyl sulfoxide (DMSO), prepared on the day of use from a stock solution of compound 1A in DMSO stored at -20°C. The comparator substances (Ambizome, voriconazole, caspofungin, and fluconazole) were purchased from a pharmacy by the Center for Medical Mycology. These were dissolved in sterile water according to the manufacturer's instructions to obtain aliquots containing the selected dose.

[0207] In this report, the dose and concentration of ambisome are expressed as the amphotericin B content.

[0208] Treatment groups: Infected mice were randomized into the following groups (5 mice per group for tissue fungal load and 10 mice per group for survival).

[0209] Experiment I consisted of the following treatment groups: Compound 1A 0.7 mg / kg, Compound 1A 0.35 mg / kg, Ambisome 5.4 mg / kg, Ambisome 2.7 mg / kg, Voriconazole 4 mg / kg, Caspofungin 0.35 mg / kg, Fluconazole 6 mg / kg, Vehicle (5% glucose aqueous solution containing 5% DMSO), and an untreated control group.

[0210] Experiment II consisted of the following treatment groups: Compound 1A 0.7 mg / kg, Compound 1A 0.35 mg / kg, Ambisome 0.7 mg / kg, Ambisome 0.35 mg / kg, and an untreated control group. All treatments were administered intravenously.

[0211] Treatment schedule: Treatment began 2 hours after vaccination and continued for 7 days. Treatment was administered once daily, except for voriconazole. Voriconazole was administered twice daily at 8-hour intervals due to its rapid clearance.

[0212] Tissue fungal load: Mice were sacrificed one day after the final day of treatment, and the kidneys and brains were aseptically removed and weighed. The tissues were homogenized and serially diluted with phosphate-buffered saline. The homogenates were cultured on SDA plates for 48 hours to determine the colony-forming units (CFU); the tissue fungal load was expressed as CFU / tissue (g).

[0213] Survival analysis: Infected mice were monitored, and signs of disease (i.e., lethargy, weight loss, generalized growth failure) or mortality were recorded twice daily for 28 days after inoculation. The mean body weight of each treatment group was also recorded daily. Dying animals unable to take food / water were euthanized.

[0214] Statistical analysis: The difference in mean log CFU in the kidney or brain was compared using a non-parametric independent Mann-Whitney test. The difference in survival was compared using the Kaplan-Meier test. A p-value of <0.05 was considered statistically significant.

[0215] result In vitro activity: Table 26 shows the in vitro activity of Compound 1A, the comparative agent, and amphotericin B against Candida albicans SC5314 (infected strain). [Table 26]

[0216] Experiment I Survival: In Figure 3, survival is given as a percentage of the total number of animals in the group on Day 1 of treatment.

[0217] Renal tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 27). The fungal burden in the kidneys was analyzed and given as mean log CFU ± standard deviation.

[0218] [[ID=二十]]Brain tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 27). The fungal burden in the brain was analyzed and given as mean log CFU ± standard deviation. [Table 27]

[0219] Experiment II Survival: In Figure 4, survival is given as a percentage of the total number of animals in the group on Day 1 of treatment.

[0220] Renal tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 28). The fungal burden in the kidneys was analyzed and given as mean log CFU ± standard deviation.

[0221] Brain tissue fungal burden: The tissue fungal burden was evaluated 1 day after the last treatment or, in the case of moribund animals, immediately after death (Table 28). The fungal burden in the brain was analyzed and given as mean log CFU ± standard deviation. [Table 28]

[0222] Example 19 A total of 74 batches of particles were produced; of these batches, 26 were produced using PACA and 48 were produced using lactic acid-glycolic acid copolymer (PLGA).

[0223] Particle quality was evaluated using diameter (particle size), polydispersity index (PDI), and stability in suspension as criteria. In particular, the prepared particles were characterized as well-behaving, acceptable, and poorly behaving, as follows: The particle formulation exhibited good behavior and was a stable suspension with a z-average diameter of <200 nm, a PDI of <0.3, and no visible aggregates. The acceptable particle formulation was a stable suspension with a PDI of >0.31; The particulate formulations exhibiting poor behavior were unstable suspensions prone to phase separation and sedimentation.

[0224] Table 29 shows an overview of the formulation produced in this example. [Table 29-1] [Table 29-2] [Table 29-3] [Table 29-4]

[0225] Formulation 45 was prepared using the following components. TIFF0007897797000047.tif100120

[0226] Formulation 49 was prepared using the following components. TIFF0007897797000048.tif73119

[0227] Formulation 73 was prepared using the following components. TIFF0007897797000049.tif73119

[0228] The poly(lactide-co-glycolide) (lactide-glycolide copolymer) used in this example had a lactide-glycolide ratio of 50:50, was ester-terminated, and had a weight-average molecular weight of 38,000 to 54,000 Da.

[0229] PACA particles Several parameters for the production of PACA particles were changed as described below.

[0230] Monomers: Different types of PACA monomers were used to encapsulate compound 1A. The objective was to solubilize compound 1A in the oil phase without initiating a polymerization reaction. Poly(ethylhexyl cyanoacrylate) (PEHCA) acted with compound 1A. Poly(butyl cyanoacrylate) (PEBCA) was also tested. During these tests, it was found that compound 1A initiated polymerization of PEBCA, resulting in unsatisfactory particles.

[0231] pH: Polymerization of particles at low pH (e.g., pH=1) produced particles with superior properties (size, size distribution, and colloidal stability). A higher pH of pH4 was used for the oxidation of compound 1A at low pH. Retention of compound 1A in the oil phase during manufacturing was found to reduce its sensitivity to pH. Lower pH levels were tested to determine whether the particles had higher loading and loading efficiency. LC-DAD-QTOF analysis of these formulations demonstrated that no degradation occurred, and therefore pH is not a critical parameter for the stability of compound 1A.

[0232] Surfactants: A mixture of Brij L35 and Kolliphor HS 15 was typically used through the tests described herein. Kolliphor was found to initiate the polymerization process.

[0233] Stabilizer: Ascorbic acid was tested for its effect on the stability of compound 1A in solution. Ascorbic acid was added to the aqueous phase during polymerization. Compound 1A was found to be chemically dehydrated with high concentrations of ascorbic acid. Ascorbic acid was found to decrease the stability of compound 1A at high concentrations.

[0234] Vanillin was tested for its effect on the stability of compound 1A. A series of experiments were conducted with and without vanillin. In these experiments, vanillin was found to aid in the dissolution of compound 1A. It was also observed that the addition of vanillin decreased the stability of the compound 1A / acrylate suspension. Since dissolved compound 1A was observed to react more readily with the acrylate monomer compared to undissolved compound 1A, one possible explanation for this effect may be improved solubility of compound 1A in the oil phase.

[0235] Generally, PACA particles contain the following components: • Aqueous phase: Brij L35, Kolliphor HS 15, and HCl to adjust pH. Oil phase: Acrylate monomers (ethylhexyl cyanoacrylate, ethyl butyl cyanoacrylate, 1-heptyl cyanoacrylate, and 2-phenylethyl cyanoacrylate / butyl cyanoacrylate), mygliol, vanillin, compound 1A. 1-heptyl cyanoacrylate and 2-phenylethyl cyanoacrylate / butyl cyanoacrylate could not dissolve compound 1A. Important findings: PACA particles can stabilize compound 1A (2-week test) in suspension. Vanillin has a solubilizing effect on compound 1A. PACA particles can be manufactured at low pH without causing unacceptable levels of degradation of compound 1A. • The water-soluble antioxidants ascorbic acid and 6-O-palmityl-L-ascorbic acid failed to improve the stability of compound 1A at the test concentration of ascorbic acid.

[0236] Figure 5 shows the LC-UV trace for one of the best-performing PACA formulations of compound 1A.

[0237] PLGA particles PLGA nanoparticles were investigated as a vehicle for compound 1A. PLGA possesses certain advantageous properties over PACA. For example, PLGA polymers are pre-formed. This means that polymer excipients do not involve reactivity-related problems. Furthermore, PLGA allows the use of organic solvents, thereby enabling the complete dissolution of compound 1A before encapsulation.

[0238] The method used for preparing the PLGA particles described herein was a nanoprecipitation method. This procedure involves slowly adding an organic phase (containing PLGA, compound 1A, and other optional hydrophobic components, such as vanillin) to an aqueous solution containing a surfactant. Alternatively, a microfluidic can be used, for example, by mixing the organic phase and aqueous phase in a fixed ratio in a continuous flow through a microfluidic channel.

[0239] During the initial testing of PLGA particles, the organic phase was added to the aqueous solution using magnetically driven stirring. The inventors tested four different surfactants at 1 to 3 different concentrations. Furthermore, the inventors tested numerous organic solvents and mixtures of organic solvents. The conditions tested are summarized in Table 30.

[0240] Table 30 provides an overview of the conditions tested during PLGA manufacturing. The percentage for compound 1A represents the theoretical maximum dry weight loading. [Table 30]

[0241] The current maximum loading achieved for compound 1A is 2.4%, but the inventors hope to increase this to >5%.

[0242] Generally, PLGA particles include: • Aqueous phase: Poly(vinyl alcohol) (PVA), Pluronic F68 (F68), Pluronic F127 (F127), Tween 80. Concentrations are given as (w / v) percentages. • Organic phase: Organic solvent, compound 1A, and potentially other hydrophobic excipients (e.g., vanillin) Important findings: NMP was the best performing solvent for the preparation of PLGA particles. Pluronic FI27 and Tween 80 were among the best-performing surfactants for the preparation of PLGA particles described herein. Vanillin did not improve drug loading of PLGA particles. Compound 1A remains stable during particle manufacturing. • The concentration of compound 1A is low in the final suspension due to dilution during preparation. The concentration of compound 1A can be increased by tangential flow filtration.

[0243] Figure 6 shows the LC-UV trace for one of the best-performing PLGA formulations of compound 1A.

[0244] lipid particles Lipid-based particles were investigated as vehicles for compound 1A. The solubility of compound 1A in different lipids and oils was investigated as shown in the list below. Stearic acid Myristic acid Palmitic acid Isopropyl myristate Isopropyl palmitate 1-nonanol Linalool • Eugenol • trans-cinnamaldehyde • Linalyl acetate p-Anisaldehyde Tetraglycol None of the tested oils or lipids dissolved compound 1A well. Compound 1A was only partially soluble in trans-cinnamaldehyde.

[0245] Example 20 Using a desolvation method, an organic solvent such as ethanol, acetone, or N-methylpyrrolidone is slowly added to an aqueous solution of albumin. This causes the albumin to precipitate. The precipitated protein can be crosslinked using glutaraldehyde or transglutaminase. The protein particles are purified by centrifugation and resuspension, followed by dialysis.

[0246] Example 21 Albumin nanoparticles are produced by adding urea to an albumin solution. This destabilizes the tertiary structure of the protein, exposing its hydrophobic domains to the aqueous phase and leading to particle formation. The precipitated protein can be crosslinked using glutaraldehyde or transglutaminase. The protein particles are purified by centrifugation and resuspension, followed by dialysis.

[0247] Example 22 The protein is destabilized as described in Example 21, but this is done at a high temperature instead. This also induced a certain amount of crosslinking of the protein.

[0248] Example 23 The same procedures as those described in Examples 20-22 were used in this example, except that the protein was fibroin.

[0249] Example 24 The same procedures as those described in Examples 20-22 were used in this example, except that the protein was gelatin.

[0250] Example 25 For any of Examples 20-24, compound 1A is swelled into nanoparticles from a solution in N-methylpyrrolidone or DMSO.

[0251] Other embodiments Those skilled in the art will see that various modifications and variations of the described invention will be apparent without departing from the scope and spirit of the invention. While the invention has been described in relation to specific embodiments, it should be understood that the claimed invention is not limited to such specific embodiments. Finally, preferred embodiments of the present invention are described in separate sections. [Embodiment 1] The following structure: [ka] A pharmaceutical composition comprising a plurality of nanoparticles containing a pharmaceutical active ingredient which is a compound or a pharmaceutically acceptable salt thereof. [Embodiment 2] The aforementioned active pharmaceutical ingredient is [ka] The pharmaceutical composition according to Embodiment 1, or a pharmaceutically acceptable salt thereof. [Embodiment 3] A pharmaceutical composition according to embodiment 1 or 2, further comprising a pharmaceutically acceptable polymer excipient. [Embodiment 4] The pharmaceutical composition according to Embodiment 3, wherein the plurality of nanoparticles include a pharmaceutically acceptable polymer excipient. [Embodiment 5] The pharmaceutical composition according to Embodiment 4, wherein the pharmaceutical active ingredient is encapsulated in nanocapsules. [Embodiment 6] The pharmaceutical composition according to any one of embodiments 3 to 5, wherein the pharmaceutically acceptable polymer excipient is poly(alkylcyanoacrylate) or polyphosphazene. [Embodiment 7] The pharmaceutical composition according to Embodiment 6, wherein the pharmaceutically acceptable polymer excipient is poly(alkylcyanoacrylate). [Embodiment 8] The pharmaceutical composition according to Embodiment 7, wherein the pharmaceutically acceptable polymer excipient is poly(ethylhexyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(n-hexyl cyanoacrylate), poly(4-methylpentyl cyanoacrylate), poly(ethylbutyl cyanoacrylate), poly(butyl cyanoacrylate), or poly(octyl cyanoacrylate). [Embodiment 9] The pharmaceutical composition according to Embodiment 8, wherein the pharmaceutically acceptable polymer excipient is poly(ethylhexyl cyanoacrylate). [Embodiment 10] The pharmaceutical composition according to any one of Embodiments 3 to 5, wherein the pharmaceutically acceptable polymer excipient is a lactic acid-glycolic acid copolymer. [Embodiment 11] The pharmaceutical composition according to any one of Embodiments 3 to 5, wherein the pharmaceutically acceptable polymer excipient is a protein. [Embodiment 12] The pharmaceutical composition according to Embodiment 11, wherein the pharmaceutically acceptable polymer excipient is a protein, such as casein, albumin, fibroin, gelatin, or a combination thereof. [Embodiment 13] Poly(ethylhexyl cyanoacrylate) and the following structure:

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Claims

1. The following structure: 【Chemistry 1】 A pharmaceutical composition comprising a plurality of nanoparticles containing a pharmaceutical active ingredient which is a compound or a pharmaceutically acceptable salt thereof, further comprising a pharmaceutically acceptable polymer excipient, wherein the pharmaceutically acceptable polymer excipient is selected from poly(alkylcyanoacrylate), polyphosphazene, lactic acid / glycolic acid copolymer, and protein.

2. The aforementioned active pharmaceutical ingredient is 【Chemistry 2】 The pharmaceutical composition according to claim 1, or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable polymer excipient is a poly(alkylcyanoacrylate) selected from poly(ethylhexylcyanoacrylate), poly(ethylcyanoacrylate), poly(n-hexylcyanoacrylate), poly(4-methylpentylcyanoacrylate), poly(ethylbutylcyanoacrylate), poly(butylcyanoacrylate), and poly(octylcyanoacrylate).

4. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable polymer excipient is a protein such as casein, albumin, fibroin, gelatin, or a combination thereof.

5. A lyophilized pharmacopoeia according to any one of claims 1 to 4.

6. A pharmaceutical composition according to any one of claims 1 to 4, further comprising a plurality of microbubbles.

7. The pharmaceutical composition according to claim 6, wherein the microbubbles include perfluorocarbons, hydrocarbons, sulfur fluoride gas, air, components of air, or mixtures thereof.

8. The microbubbles contain nitrogen (N ２ ), oxygen (O ２ ), argon (Ar), carbon dioxide (CO ２ ), helium (He), neon (Ne), methane (CH4) ４ The pharmaceutical composition according to claim 7, comprising ), or a mixture thereof.

9. A pharmaceutical composition according to any one of claims 1 to 8, further comprising a pharmaceutically acceptable surfactant.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutically acceptable surfactant is a nonionic surfactant.

11. The pharmaceutical composition according to claim 9, wherein the pharmaceutically acceptable surfactant is polyoxyethylene ether, polyoxyethylene fatty acid ester, sorbitan ester, polysorbate, polyethoxylated castor oil, polyoxyethylene / polyoxypropylene block copolymer, or a combination thereof.

12. The pharmaceutical composition according to claim 11, wherein the polyoxyethylene fatty acid ester is polyoxyethylated 12-hydroxystearic acid, and / or the polyoxyethylene ether is polyoxyethylene lauryl ether.

13. A pharmaceutical composition according to any one of claims 1 to 12, further comprising a pharmaceutically acceptable stabilizer.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable stabilizer is vanillin, butylated hydroxytoluene, butylated hydroxyanisole, or vitamin E.

15. The pharmaceutical composition according to claim 13 or 14, comprising 0.1 to 10% (w / w) of a pharmaceutically acceptable stabilizer relative to the particle mass.

16. A pharmaceutical composition according to any one of claims 1 to 15, further comprising a pharmaceutically acceptable oil.

17. The pharmaceutical composition according to claim 16, wherein the pharmaceutically acceptable oil is selected from the group consisting of medium-chain triglycerides, long-chain triglycerides, and combinations thereof.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutically acceptable oil is one or more medium-chain triglycerides selected from the group consisting of Miglyol, Captex, and Kollisolv.

19. A pharmaceutical composition according to any one of claims 16 to 18, comprising 0.5 to 5% (w / w) of pharmaceutically acceptable oil relative to the particle mass.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the plurality of nanoparticles have a number-average diameter of 20 to 200 nm when measured by dynamic light scattering.

21. The pharmaceutical composition according to any one of claims 1 to 19, wherein the plurality of nanoparticles have a number average diameter of 30 to 150 nm when measured by nanoparticle tracking analysis.

22. A pharmaceutical composition according to any one of claims 1 to 21, which is an aqueous composition.

23. The pharmaceutical composition according to claim 22, wherein the pH is 4.0 to 8.

0.

24. The pharmaceutical composition according to any one of claims 1 to 23, further comprising a cosolvent that is a polar organic solvent.

25. The pharmaceutical composition according to claim 24, wherein the polar organic solvent is dimethyl sulfoxide, N-methyl-2-pyrrolidone, N,N-dimethylformamide, or a combination thereof.

26. A pharmaceutical composition according to any one of claims 1 to 25, comprising 1 to 15% dry (w / w) of a pharmaceutical active ingredient as measured by liquid chromatography.

27. A pharmaceutical composition for treating a subject suffering from a fungal infection, wherein the subject is a species of Candida, Cryptococcus, Aspergillus, Colletotrichum, Geotrichum, Hormonema, Lecythophora, Pecilomyces, Penicillium, or Rhodotorula. A pharmaceutical composition according to any one of claims 1 to 26, for a person suffering from a fungal infection caused by a fungal species of the following genera: orula, Fusarium, Saccharomyces, Trichoderma, Trichophyton, Scopularilopsis, Histoplasma, Blastomyces, or Coccidioides.

28. The pharmaceutical composition according to claim 27, wherein the subject is suffering from a fungal infection caused by a fungal species of the genus Candida, Aspergillus, or Cryptococcus.

29. The pharmaceutical composition according to claim 28, wherein the subject is suffering from a fungal infection caused by an azole-resistant Aspergillus species.

30. A pharmaceutical composition according to any one of claims 27 to 29, which is administered intravenously, by inhalation, intranasally, orally, sublingually, buccally, percutaneously, intradermally, intramuscularly, vaginally, parenterally, intra-arterially, intracranially, intrathecally, subcutaneously, intraorbitally, intraventricularly, intraspinally, intraperitoneally, or topically.

31. pharmaceutically acceptable polymer excipients and the following structures: 【Transformation 3】 A method for producing a plurality of nanoparticles comprising a compound or a pharmaceutically acceptable salt thereof, the method comprising the step of polymerizing a monomer precursor of the pharmaceutically acceptable polymer excipient in a liquid containing the monomer precursor and the compound or a pharmaceutically acceptable salt thereof, wherein the polymerization step produces a plurality of nanoparticles. The method, wherein the monomer precursor is an alkylcyanoacrylate, and the pharmaceutically acceptable polymer excipient is a poly(alkylcyanoacrylate).

32. The compound has the following structure: 【Chemistry 4】 The method according to claim 31, wherein the compound is or a pharmaceutically acceptable salt thereof.

33. The method according to claim 31 or 32, wherein the liquid further comprises a pharmaceutically acceptable surfactant, a pharmaceutically acceptable stabilizer, and / or a pharmaceutically acceptable oil.

34. The method according to any one of claims 31 to 33, wherein the plurality of nanoparticles have a number average diameter of 20 to 200 nm when measured by dynamic light scattering.

35. The method according to any one of claims 31 to 33, wherein the plurality of nanoparticles have a number average diameter of 30 to 150 nm when measured by nanoparticle tracking analysis.

36. The method according to any one of claims 31 to 35, wherein the liquid is an aqueous composition.

37. The method according to claim 36, wherein the pH of the liquid is 0.5 to 8.

0.

38. The method according to any one of claims 31 to 37, further comprising adding a plurality of microbubbles.

39. The method according to any one of claims 31 to 38, further comprising freeze-drying the plurality of nanoparticles.

40. The method according to any one of claims 31 to 39, further comprising dialysis of the plurality of nanoparticles with deionized water.

41. The method according to any one of claims 31 to 40, further comprising adjusting the pH of the liquid to a range of 4.0 to 8.

0.

42. The method according to claim 41, wherein the step of adjusting the pH is performed during the polymerization step.

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