Compositions Comprising Lipid Nanoparticles and Antifungal Polyenes - Patent application

Abstract

The present invention relates to a composition comprising an aqueous phase, a polyene antifungal agent and lipid nanoparticles dispersed in the aqueous phase, said lipid nanoparticles comprising an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, and the polyene antifungal agent dispersed in the aqueous phase and / or part of the lipid nanoparticle. The present invention further relates to lipid nanoparticles comprising an antifungal polyene, an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, and the polyene antifungal agent part of the lipid nanoparticle, and a method for preparing the same. The present invention also relates to a composition or lipid nanoparticle of the present invention for use in the prevention or treatment of infections caused by fungi, preferably fungi of the order Mucorales.

Description

[Technical field]

[0001] Introduction / Field of the Invention The technical field of the present invention is the treatment of fungal diseases, in particular fungal diseases caused by the order Mucorales. [Background technology]

[0002] Mucormycosis is a life-threatening invasive fungal disease caused by species belonging to the order Mucorales. It is a difficult infection to treat in immunocompromised patients and is resistant to most antifungal drugs. Practical treatment is based on surgery, basic disease control, and aggressive antifungal therapy whenever possible. Currently, there are only three active antifungal agents against Mucorales: amphotericin B (L-AmB) formulated as liposomes, which is recommended for first-line therapy, and posaconazole (PSC) and isavuconazole (ISC) as alternative options. Despite these treatments, mortality remains unacceptable and varies widely from 30 to 90%, depending on the disease type.

[0003] As a result, there is a need to provide new treatments for patients infected with Mucorales fungi.

[0004] Combinations of antifungal agents have been tested in vitro and in vivo to improve their efficacy against Mucorales. Unfortunately, no combination of antifungal agents has been shown to be more effective against mucormycosis in patients than antifungal agents alone. Therefore, several authors have investigated combinations of antifungal agents with non-antifungal molecules. Some of these combinations have increased antifungal efficacy in vitro or in vivo in animal models, but also presented some problems, e.g., improving virulence or toxicity of Mucorales.

[0005] Although AmB is a highly active antifungal drug, it has limited solubility and is a highly cytotoxic substance. The toxicity of AmB and its bioaccumulation in organs, such as the liver, lungs and kidneys, are undesirable properties that seriously affect treated patients. With the development of liposomal formulations, especially liposomal dispersion Ambisome®, the toxicity of AmB has been attenuated due to the small size of liposomes, which allows long-term circulation and distribution in organs. However, even with such liposomal formulations, the problem of dose-dependent nephrotoxicity still remains. In addition, such liposomal formulations are difficult and expensive to manufacture.

[0006] Cryptococcus neoformans is a yeast (fungus) responsible for a life-threatening invasive infection worldwide, named cryptococcosis. C. neoformans causes meningoencephalitis, primarily in immunocompromised patients, and is responsible for approximately 200,000 deaths annually. Thus, this yeast represents a major public health problem worldwide.

[0007] A combination of AmB and 5-fluorocytosine is currently recommended for first-line treatment. Two AmB formulations are mainly used: AmB deoxycholate (AmBd) and liposomal AmB (L-AmB). L-AmB is the less toxic form and is preferred in developed countries. However, it is a much more expensive form and, as a result, is not widely used in developing countries. AmBd represents an essential component in the management of cryptococcosis in developing countries. Unfortunately, the efficacy of treatments against Cryptococcus remains low, with mortality rates of 35-40% in both resource-rich and resource-poor environments. Against this background, there is an urgent need for new treatments for cryptococcosis that are more effective and at the same time less expensive. [Prior art documents] [Non-patent literature]

[0008]

Non-licensed literature 1

Non-licensed Document 4

[0009] Thus, there is a continuing need in the art for optimized formulations based on polyene antifungals, such as AmB.

[0010] The present inventors have developed formulations that enhance the antifungal efficacy of AmB against Mucorales, as well as other fungi such as Cryptococcus, Aspergillus, and Candida, while simultaneously reducing its toxicity. [Means for solving the problem]

[0011] In a first object, the present invention relates to a composition comprising an aqueous phase, a polyene antifungal agent and lipid nanoparticles dispersed in the aqueous phase, said lipid nanoparticles comprising an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, and the polyene antifungal agent is dispersed in the aqueous phase and / or is part of the lipid nanoparticles.

[0012] The hydroxy fatty acid is preferably a C8 to C24 hydroxy fatty acid, preferably a C12 to C20 hydroxy fatty acid, more preferably a C14 to C18 hydroxy fatty acid.

[0013] Furthermore, the polyglycol ester of a fatty acid preferably contains from 1 to 100 ethylene oxide groups, preferably from 5 to 50 ethylene oxide groups, more preferably from 15 to 25 ethylene oxide groups, and even more preferably from 10 to 20 ethylene oxide groups.

[0014] The polyglycol ester of a fatty acid preferably comprises a C14 to C18 hydroxy fatty acid, preferably a C18 hydroxy fatty acid group, such as 12-hydroxystearic acid, and from 10 to 20 ethylene oxide groups.

[0015] The polyglycol ester of a hydroxy fatty acid is polyethylene glycol-15-hydroxystearate.

[0016] Further, the polyene antifungal agent may preferably be selected from amphotericin B, nystatin, partricin, natamycin or mixtures thereof.

[0017] Compositions according to the invention may contain between 1 g / L and 5 g / L, preferably between 1 g / L and 2 g / L, of a polyene antifungal agent and between 20 g / L and 100 g / L, preferably between 20 and 80 g / L, more preferably between 40 and 60 g / L, of a polyglycol ester of a hydroxy fatty acid. Such compositions are preferably for parenteral administration.

[0018] Compositions according to the invention may contain between 30 mg / L and 120 mg / L, preferably between 50 mg / L and 100 mg / L, of a polyene antifungal agent and between 600 mg / L and 2400 mg / L, more preferably between 1000 and 2000 mg / L, of a polyglycol ester of a hydroxy fatty acid. Such compositions are preferably for respiratory administration.

[0019] Furthermore, the size of the lipid nanoparticles is preferably between 1 and 250 nm, preferably between 10 and 150 nm, preferably between 50 and 100 nm.

[0020] In certain embodiments, the lipid nanoparticles of the composition according to the present invention do not comprise an antifungal polyene.

[0021] In certain embodiments, the aqueous phase of the composition according to the present invention does not contain a polyene antifungal agent.

[0022] Additionally, the lipid nanoparticles preferably comprise a polyglycol ester of a hydroxy fatty acid and a polyene antifungal agent in a weight ratio ranging from 5:1 to 100:1 polyglycol ester of a hydroxy fatty acid:polyene antifungal agent.

[0023] In one embodiment, the composition according to the invention further comprises a triazole antifungal or an antifungal subject to fungal resistance, in particular efflux pump-mediated resistance, wherein the triazole antifungal or an antifungal subject to fungal resistance is dispersed in the aqueous phase and / or is part of the lipid nanoparticles.

[0024] In one embodiment, the composition according to the invention further comprises a triazole antifungal or an antifungal agent affected by fungal resistance, in particular efflux pump-mediated resistance, wherein the triazole antifungal or the antifungal agent affected by fungal resistance is part of the lipid nanoparticle, preferably dispersed in the oily core of the lipid nanoparticle.

[0025] In certain embodiments, the triazole antifungal agent or antifungal agent affected by fungal resistance is part of the lipid nanoparticle, preferably solubilized in the oily internal phase of the nanoparticle.

[0026] In one embodiment, the composition according to the invention further comprises a triazole antifungal or an antifungal agent affected by fungal resistance, in particular efflux pump-mediated resistance, which is solubilized in the oil phase of the nanoparticles, and a polyene antifungal agent is solubilized in the aqueous phase and / or is part of the lipid nanoparticles.

[0027] In certain embodiments, the triazole antifungal agent or antifungal agent affected by fungal resistance and the polyene antifungal agent are part of the lipid nanoparticle.

[0028] The triazole antifungal agent may be selected, for example, from isavuconazole, itraconazole, posaconazole, voriconazole, fluconazole or mixtures thereof, preferably isavuconazole.

[0029] The antifungal agent affected by fungal resistance, in particular affected by efflux pump-mediated resistance, is preferably a lipophilic antifungal agent, more preferably an azole antifungal agent, in particular an azole antifungal agent, which may be selected, for example, from triazole antifungal agents and imidazole antifungal agents.

[0030] In one embodiment, the composition comprises a triazole antifungal and a polyene antifungal in a weight ratio ranging from 10 to 200 triazole antifungal:polyene antifungal, preferably from 10 to 150, more preferably from 10 to 100.

[0031] In a second object, the present invention also relates to lipid nanoparticles comprising an antifungal polyene, an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, and the polyene antifungal drug being part of the lipid nanoparticle.

[0032] In certain embodiments, the lipid nanoparticles further comprise a triazole antifungal agent or an antifungal agent affected by fungal resistance, particularly efflux pump-mediated resistance, dispersed in the oily internal phase of the lipid nanoparticles.

[0033] In a third object, the present invention provides a method for preparing lipid nanoparticles according to the second object of the present invention, comprising: a) providing an oily phase comprising at least a fatty substance and an antifungal polyene as defined above, b) providing an aqueous phase comprising a polyglycol ester of a hydroxy fatty acid; c) mixing the oily phase of step a) with the aqueous phase of step b); d) cooling the mixture obtained in step c) to obtain lipid nanoparticles. The present invention relates to a method comprising the steps of:

[0034] In embodiments in which the lipid nanoparticles comprise a triazole antifungal drug or an antifungal drug affected by fungal resistance, the oily phase provided in step a) further comprises a triazole antifungal drug.

[0035] In a fourth object, the present invention relates to a composition according to the first object of the invention or lipid nanoparticles according to the second object of the invention for use in the prevention or treatment of an infection caused by a fungus, preferably a fungus of the order Mucorales. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] composition A first object of the present invention relates to a composition comprising an aqueous phase, a polyene antifungal drug and lipid nanoparticles dispersed in the aqueous phase, said lipid nanoparticles comprising an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, and the polyene antifungal drug being dispersed in the aqueous phase and / or being part of the lipid nanoparticles.

[0037] The inventors have surprisingly found that the combination of antifungal polyenes and polyglycol esters of hydroxy fatty acids in the form of lipid nanoparticles makes it possible to obtain compositions that are very low in toxicity and at the same time maintain or even enhance the effectiveness of antifungal polyenes, especially against Mucorales, regardless of whether the polyene antifungal agent is dispersed in the aqueous phase and / or is part of lipid nanoparticles.The inventors have further shown that this synergistic effect can be observed not only against Mucorales, but also against Aspergillus and Cryptococcus.Interestingly, the inventors have further shown that the combination of antifungal polyenes, triazole antifungal agents and polyglycol esters of hydroxy fatty acids in the form of lipid nanoparticles significantly enhances the effectiveness of antifungal polyenes.

[0038] Polyglycol esters of hydroxy fatty acids The term "polyglycol esters of hydroxy fatty acids" as used herein refers to a mixture containing at least esters of hydroxy fatty acids obtained by ethoxylation of hydroxy fatty acids. The terms "polyglycol" and "poly(ethylene glycol)", "polyethylene glycol" (PEG) or "polyethylene oxide (PEO)" or "macrogol" or "carbovax" or "polyoxyethylene" (POE) are interchangeable. The mixture may contain about 60 wt% to about 75 wt% of esters, preferably mono- and diesters of hydroxy fatty acids, and about 25 wt% to 40 wt% of free polyethylene glycol, based on the weight of the mixture.

[0039] The phrase "between" as used herein should be understood to include the end points of the recited range.

[0040] Advantageously, the polyglycol esters of hydroxy fatty acids have a hydrophilic-lipophilic balance (HLB) of between 14 and 16, as determined by the method of Griffin.

[0041] The term "hydroxylated fatty acid", as used herein, refers to a fatty acid having at least one hydroxyl group attached to the alkyl chain of the fatty acid. Preferably, the fatty acid has a unique hydroxyl group attached to the alkyl chain of the fatty acid. Advantageously, the hydroxy fatty acid is a C8-C24 hydroxy fatty acid, preferably a C12-C20 hydroxy fatty acid, more preferably a C14-C18 hydroxy fatty acid. More preferably, the hydroxy fatty acid is hydroxystearic acid. More advantageously, the hydroxy fatty acid is 12-hydroxystearic acid.

[0042] Advantageously, the polyglycol ester of a fatty acid contains from 1 to 100 ethylene oxide groups, preferably from 5 to 50 ethylene oxide groups, more preferably from 15 to 25 ethylene oxide groups, even more preferably from 10 to 20. More advantageously, the polyglycol ester of a fatty acid contains 15 ethylene oxide groups.

[0043] In one embodiment, the polyglycol ester of a fatty acid comprises a C14 to C18 hydroxy fatty acid, preferably a C18 hydroxy fatty acid group, such as 12-hydroxystearic acid, and from 10 to 20 ethylene oxide groups.

[0044] In one embodiment, the polyglycol ester of a hydroxy fatty acid is polyethylene glycol-15-hydroxystearate.

[0045] In the present invention, the term "polyethylene glycol-15-hydroxystearate" refers to a mixture comprising at least an ester of 12-hydroxystearic acid obtained by ethoxylation of 12-hydroxystearic acid. Preferably, the polyethylene glycol-15-hydroxystearate has at least the formula:

[0046] [ka]

[0047] (wherein n=15). Polyethylene glycol-15-hydroxystearate is also known as 12-hydroxyoctadecanoic acid polymer with α-hydro-ω-hydroxypoly(oxy-1,2-ethanediyl); 12-hydroxystearic acid polyethylene glycol copolymer; macrogol 15 hydroxystearate; and polyethylene glycol 660 12-hydroxystearate. In some embodiments, the polyethylene glycol-15-hydroxystearate is Solutol® HS 15 (CAS number 70142-34-6), also available under the trade name Kolliphor® HS 15 (BASF AG, Germany). In some embodiments, the polyethylene glycol-15-hydroxystearate is a mixture comprising about 60 wt% to about 75 wt% of a polyglycol ester of 12-hydroxystearic acid and about 60 wt% to about 75 wt% of free polyethylene glycol, based on the weight of the mixture.

[0048] Antifungal polyenes The term "antifungal polyenes", as used herein, refers to antifungal agents characterized by a nearly planar macrolactone ring, usually containing eight or more atoms, with a series of conjugated double bonds (commonly called polyene macrolides). Depending on the number of conjugated double bonds, such polyenes can be classified as trienes, tetraenes, pentaenes, hexaenes, heptaenes, etc. Antifungal polyenes usually contain a hydrophilic "head" bearing a mycosamine group. Such antifungal polyenes are also commonly called "antifungal macrolides".

[0049] Advantageously, the antifungal polyene may be selected from amphotericin B (AmB), nystatin, natamycin (also known as pimaricin), partricin, or mixtures thereof. Preferably, the antifungal agent is amphotericin B.

[0050] Compositions according to the invention may contain high concentrations of antifungal polyenes without cytotoxicity, making them particularly suitable for parenteral administration as well as respiratory administration, particularly intrapulmonary respiratory administration as described in more detail below.

[0051] Advantageously, the composition according to the invention comprises between 1 g / L and 5 g / L, preferably between 1 g / L and 2 g / L, of a polyene antifungal agent and between 20 g / L and 100 g / L, preferably between 20 and 80 g / L, more preferably between 40 and 60 g / L, of a polyglycol ester of a hydroxy fatty acid. Such a composition is preferably for parenteral administration, more preferably for intravenous administration as detailed below.

[0052] Advantageously, the composition according to the invention comprises between 30 mg / L and 120 mg / L, preferably between 50 mg / L and 100 mg / L, of a polyene antifungal agent and between 600 mg / L and 2400 mg / L, more preferably between 1000 and 2000 mg / L, of a polyglycol ester of a hydroxy fatty acid. Such a composition is preferably for respiratory administration, more preferably for respiratory pulmonary administration as detailed below.

[0053] Advantageously, the composition according to the invention comprises a polyglycol ester of a hydroxy fatty acid and a polyene antifungal in a weight ratio ranging from 5:1 to 100:1, preferably from 10:1 to 100:1, preferably from 10:1 to 90:1, preferably from 10:1 to 50:1, preferably from 10:1 to 40:1, preferably from 10:1 to 30:1, preferably from 10:1 to 25:1. Preferably, the weight ratio of polyglycol ester of a hydroxy fatty acid to a polyene antifungal is in the range from 15:1 to 25:1.

[0054] As described in more detail below, compositions according to the present invention may include an antifungal polyene dispersed in the aqueous phase and / or as part of a lipid nanoparticle.

[0055] Antifungal triazoles In certain embodiments, a composition according to the present invention further comprises a triazole antifungal agent.

[0056] The term "antifungal triazoles", as used herein, refers to antifungal agents characterized by containing a triazole fraction, i.e. a five-membered ring with two carbon atoms and three nitrogen atoms, and derivatives thereof.

[0057] The triazole antifungal may be selected, for example, from isavuconazole, itraconazole, posaconazole, voriconazole, fluconazole or mixtures thereof. Preferably, the triazole antifungal is isavuconazole.

[0058] In one embodiment, the composition comprises a triazole antifungal and a polyene antifungal in a weight ratio ranging from 10 to 200 triazole antifungal:polyene antifungal, preferably from 10 to 150, more preferably from 10 to 100.

[0059] In certain embodiments, the composition according to the present invention further comprises a triazole antifungal agent, wherein the triazole antifungal agent is dispersed in the aqueous phase and / or is part of the lipid nanoparticles.

[0060] In the present invention, "dispersed" may preferably mean "solubilized".

[0061] In certain embodiments, the triazole antifungal agent is part of the lipid nanoparticle, preferably dispersed in the oily internal phase of the nanoparticle.

[0062] In one embodiment, a composition according to the invention comprises a triazole antifungal agent dispersed in the oily internal phase of the nanoparticles and a polyene antifungal agent dispersed in the aqueous phase and / or which is part of the lipid nanoparticles.

[0063] In certain embodiments, the triazole antifungal agent and the polyene antifungal agent are dispersed in an aqueous phase.

[0064] In certain embodiments, the triazole antifungal and the polyene antifungal are part of a lipid nanoparticle, the triazole antifungal being preferably dispersed in the oily internal phase of the lipid nanoparticle, and the polyene antifungal being preferably dispersed in the oily core of the lipid nanoparticle and / or being a component of the envelope.

[0065] As described in the experimental section below, the present inventors have shown that isavuconazole greatly increases the activity of amphotericin B when combined with polyglycol esters of hydroxy fatty acids.

[0066] The present inventors have shown that the combination of a polyene antifungal drug, AmB, with a triazole antifungal drug, isavuconazole (ISA), in polyglycol ester of hydroxy fatty acids-based nanoparticles, PEG H15S-based nanoparticles, has significantly higher antifungal efficacy compared to a solution of AmB, a solution of AmB with ISA, and PEG H15S-nanoparticles containing AmB.

[0067] The inventors believe that, if incorporated into the nanoparticles of the present invention, triazole antifungal drugs that currently cannot be administered by nebulization or are very ineffective will be available by nebulization route.Without wishing to be bound by any particular theory, the inventors believe that triazoles are very lipophilic, so when administered intrapulmonary as aerosol, they are absorbed into the bloodstream very quickly, and therefore do not have time to act on fungi in the lungs.In contrast, the inventors believe that when triazoles are administered as aerosolized nanoparticles according to the present invention, the absorption of triazoles from the lungs should be slowed down, thereby increasing their residence time in the lungs, and therefore giving them more time to exert their antifungal effect in the lungs.

[0068] Therefore, according to the inventors, the interest of incorporating a combination of a polyene antifungal, such as AmB, and a triazole antifungal, such as isavuconazole, in the nanoparticles according to the invention would be twofold: to lower the MIC of said antifungal in a surprising manner, and at the same time to allow the administration of the triazole by nebulization, resulting in a very long lung residence time.

[0069] In one embodiment, the compositions of the invention comprise a polyene antifungal and a triazole antifungal, wherein the polyene antifungal is preferably AmB and the triazole antifungal is preferably isavuconazole.

[0070] Antifungal drugs affected by resistance The present inventors have further demonstrated that the combination of a polyene antifungal drug, AmB, in a polyglycol ester of hydroxy fatty acid-based nanoparticle, PEG H15S-based nanoparticle, penetrates Cryptococcus cells much more effectively than nanoparticles not loaded with Amb. Indeed, as shown in the experimental section of this application, lipid nanoparticles according to the present invention containing AmB as a polyene antifungal drug penetrated Cryptococcus cells 16 times more effectively than PEG H15S-based nanoparticles not containing a polyene antifungal drug.

[0071] Without being bound by theory, the inventors believe that these data suggest that lipid nanoparticles according to the present invention, when comprising a combination of a polyene antifungal drug and an antifungal drug affected by fungal resistance, such as isavuconazole, may help in enhancing the intracellular concentration of the antifungal drug affected by fungal resistance in fungal cells. Indeed, some antifungal drugs, such as azoles (including isavuconazole), are substrates for fungal membrane efflux pumps, which may reduce their intracellular concentration and reduce their effectiveness, resulting in antifungal drug resistance, such as azole resistance. The use of lipid nanoparticles according to the present invention may allow the effect of these pumps to be avoided and the intracellular concentration of the antifungal drug affected by fungal resistance to be increased. Thus, lipid nanoparticles according to the present invention may be of use in overcoming fungal resistance, particularly efflux pump-mediated fungal resistance.

[0072] In certain embodiments, the lipid nanoparticles according to the present invention comprise an antifungal agent that is affected by fungal resistance, in particular by efflux pump-mediated resistance.

[0073] The expression "antifungal drugs affected by efflux pump-mediated resistance" or "antifungal drugs that have obtained EPMR", as used in the present invention, refers to antifungal drugs that may be difficult or impossible to accumulate intracellularly in some fungal cells to toxic levels due to the activity of fungal membrane-associated transporters acting as efflux pumps, making antifungal drug treatment less effective or ineffective. In fact, it is known that some fungal membrane-associated transporters, such as primary active transporters related to the superfamily of ATP-binding cassette (ABC) proteins or secondary active transporters belonging to the major facilitator superfamily (MFS), can recognize some antifungal drugs as substrates and allow them to be effluxed from fungal cells. In some resistant fungal strains, fungal efflux pumps ensure that antifungal drugs do not accumulate to lethal levels.

[0074] In one embodiment, the lipid nanoparticles according to the invention comprise an antifungal polyene, preferably AmB, and an antifungal agent that is subject to resistance, in particular subject to EPMR, preferably an azole antifungal agent. The polyene antifungal agent is preferably dispersed in the oily core of the lipid nanoparticle and / or is a constituent of the envelope, preferably at least a portion of the polyene antifungal agent is part of the envelope. The antifungal agent that is subject to fungal resistance is preferably a lipophilic antifungal agent that is dispersed in the oily core of the lipid nanoparticle.

[0075] In certain embodiments, the antifungal agent affected by fungal resistance, particularly to which an EPMR has been obtained, is a lipophilic antifungal agent, preferably an azole antifungal agent.

[0076] The term "azole antifungal drugs", as used herein, refers to antifungal drugs characterized by containing a 5-membered nitrogen-containing heterocyclic ring system and its derivatives containing other non-carbon atoms such as sulfur or oxygen. The azole group of antifungal drugs is usually divided into two subgroups: triazole antifungals and imidazole antifungals as defined above. Examples of imidazole antifungals are clotrimazole, econazole, miconazole, ketoconazole.

[0077] In one embodiment, the lipid nanoparticles according to the present invention comprise an antifungal polyene, preferably AmB, and an azole antifungal drug, which may be a triazole antifungal drug, preferably isavuconazole, or an imidazole antifungal drug.

[0078] Lipid Nanoparticles (LNPs) The term "lipid nanoparticles", as used in the present invention, relates to nanometer-sized lipid particles of the vesicular type comprising an oily internal phase (or oily core) and an envelope (or shell) comprising polyglycol esters of hydroxy fatty acids.

[0079] The size of the lipid nanoparticles is preferably between 1 and 250 mm, preferably between 10 and 150 nm, preferably between 50 and 100 nm.

[0080] The polydispersity index (PDI) of the lipid nanoparticles according to the present invention is preferably less than 0.4, more preferably less than 0.2.

[0081] The sizes given above correspond to the Z-average, i.e. the mean value of the intensity-weighted size distribution of the lipid nanoparticles, determined by dynamic light scattering at 25° C. using a Zetasizer Nano ZS instrument from Malvern Instrument (Malvern Panalytical).

[0082] As used herein, PDI is a numerical value calculated from a two-parameter fit to correlation data (cumulant analysis). The index is dimensionless and is scaled so that values ​​below 0.05 are predominantly observed in highly monodisperse standards. PDI values ​​above 0.7 indicate that the sample has a very broad particle size distribution and is probably not suitable for analysis by dynamic light scattering (DLS) techniques. Different size distribution algorithms work with data that fall between the two extreme values ​​of PDI (i.e., 0.05-0.7). In the present invention, size and PDI parameters are determined as defined in ISO standard parameters 13321:1996E and ISO 22412:2008.

[0083] The oily core, which is liquid or semi-liquid at room temperature, preferably comprises a biocompatible, biodegradable lipophilic compound with low water miscibility selected from vegetable oils, such as soybean oil, corn oil, olive oil, safflower oil, triglycerides; tocopherols, such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol; fish oils: sterols; vitamin D, such as cholecalciferol and ergocalciferol; or mixtures thereof.

[0084] The term "biocompatible and biodegradable", as used herein, refers to compounds that may be absorbed and excreted by the body, preferably with low accumulation in tissues.

[0085] The triglyceride may comprise one or more triglycerides, in particular one or more triglycerides of at least one fatty acid, preferably a C6 to C12 fatty acid, more preferably a C6 to C10 fatty acid.

[0086] A commercially available triglyceride composition that may be used is Labrafac lipophile WL 1349 available from Gattefosse Corporation.

[0087] In addition to the non-ionic surfactants, the envelope may contain other excipients, in particular chosen from amphoteric surfactants, such as phospholipids, or anionic surfactants, such as fatty acids, to stabilize the shell.

[0088] Advantageously, the amphoteric surfactant is an amphiphilic lipid selected from the phospholipid family, in particular from phosphatidylcholine or lecithin. The amphiphilic lipids used in the present invention are preferably solid at room temperature, as they promote the formation of a semi-solid interface around a liquid or semi-liquid core.

[0089] The amphipathic lipid is preferably selected from the group of lecithins, more preferably lecithins exhibiting a transition temperature above 35° C., such as dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dibehenylphosphatidylcholine (DBPC), palmitoyl-stearoylphosphatidylcholine (PSPC), palmitoyl-behenylphosphatidylcholine (PSPC), stearoyl-behenylphosphatidylcholine (SBPC).

[0090] The lipid nanoparticles preferably contain hydrogenated lecithin. More specifically, the hydrogenated lecithin used according to the invention has a high percentage of saturated phosphatidylcholine. The term "high percentage" is understood to mean an amount of hydrogenated (or saturated) phosphatidylcholine greater than 85% relative to the total mass of the lecithin. A commercially available lecithin composition that may be used is Phospholipon® 90H, whose hydrogenated phosphatidylcholine content is greater than 90% and whose transition temperature is approximately 54°C.

[0091] Advantageously, the lipid nanoparticles comprise an amphiphilic lipid compound and a polyglycol ester of a hydroxy fatty acid in a mass ratio ranging between 10:2 and 25:1 amphiphilic lipid:polyglycol ester of a hydroxy fatty acid.

[0092] Advantageously, the envelope of the lipid nanoparticles, in particular of the lipid nanoparticles according to the invention, further comprises a sterol.

[0093] The sterol may be selected from an animal sterol, such as cholesterol, a plant sterol, such as campesterol, a sterol derivative, such as vitamin D, or mixtures thereof.

[0094] Advantageously, the lipid nanoparticles comprise sterol in an amount between 0% and 15% by weight, preferably between 5% and 10% by weight, relative to the total weight of the composition according to the invention.

[0095] In the compositions according to the invention, the polyene antifungal agent is dispersed in the aqueous phase and / or is part of the lipid nanoparticles.

[0096] The term "part of the lipid nanoparticle" referring to the antifungal polyene means that the antifungal polyene is dispersed in the oily core of the lipid nanoparticle and / or is a component of the envelope. In some embodiments, the antifungal polyene is embedded in the envelope and / or adsorbed on the outer surface of the envelope.

[0097] The expression "part of the lipid nanoparticle" referring to the antifungal triazole means that the antifungal polyene is dispersed in the oily core (i.e., the oily internal phase) of the lipid nanoparticle and / or is a constituent of the envelope. In a preferred embodiment, the antifungal triazole is dispersed in the oily internal phase of the lipid nanoparticle. In another embodiment, the antifungal triazole is embedded in the envelope and / or adsorbed on the outer surface of the envelope.

[0098] As stated above, the term "dispersed" preferably means "solubilized."

[0099] In certain embodiments, compositions according to the present invention comprise an antifungal polyene dispersed in the aqueous phase of the composition.

[0100] In this embodiment, the lipid nanoparticles preferably do not contain an antifungal polyene. The advantage of this embodiment is that it is cheaper, less complicated to prepare, and potentially more stable than compositions having lipid nanoparticles that contain an antifungal polyene.

[0101] The inventors surprisingly show that the toxicity of AmB dispersed in an aqueous phase was significantly reduced in the presence of PEG15HS incorporated into lipid nanoparticles compared to a composition containing both AmB and PEG15HS in solution.

[0102] In another embodiment, the antifungal polyene is part of the lipid nanoparticle. In this embodiment, the aqueous phase does not contain the antifungal polyene. Preferably, the antifungal polyene is AmB.

[0103] An advantage of this embodiment is that it ensures that the mass ratio of polyglycol ester of hydroxy fatty acid:polyene antifungal is maintained all the way to the site of action of the polyene antifungal. Without wishing to be bound by any particular theory, the inventors believe that if the antifungal polyene is present in the aqueous phase, there is a risk that the polyglycol ester of hydroxy fatty acid and the polyene antifungal will not reach the site of action in an optimal ratio.

[0104] In one embodiment, the lipid nanoparticles comprise a polyene antifungal and a triazole antifungal, the polyene antifungal is preferably AmB and the triazole antifungal is preferably isavuconazole.

[0105] The advantage of this embodiment is that, as shown in the experimental section below, the combination of a polyene antifungal, such as AmB, with a triazole antifungal, such as isavuconazole (ISA), in a polyglycol ester of hydroxy fatty acid-based nanoparticle, such as a PEG H15S-based nanoparticle, has a much higher antifungal efficacy than a solution of AmB, a solution of AmB with ISA, and a PEG H15S-nanoparticle containing AmB. Thus, the advantage of this embodiment is that the combination of a polyene antifungal and a triazole antifungal in the lipid nanoparticle according to the invention reduces the MIC of both the polyene antifungal and the triazole antifungal.

[0106] A further advantage of the nanoparticles of this embodiment is that by incorporating a triazole antifungal into the lipid nanoparticles according to the invention, the absorption of the triazole antifungal by the lungs will be delayed, thus increasing its residence time and therefore its in vivo antifungal efficacy, which would make it particularly interesting to administer such lipid nanoparticles by nebulization.

[0107] In one embodiment, the lipid nanoparticles comprise a polyene antifungal agent and an antifungal agent affected by fungal resistance, the polyene antifungal agent is preferably AmB, and the antifungal agent affected by fungal resistance is preferably an azole antifungal agent, e.g., a triazole antifungal agent or an imidazole antifungal agent.

[0108] An advantage of this embodiment is that such lipid nanoparticles may help overcome fungal EPMR, as discussed above.

[0109] In a second object, the present invention relates to a lipid nanoparticle as defined in the first object according to the invention, wherein the lipid nanoparticle comprises an antifungal drug as part of the lipid nanoparticle.

[0110] Advantageously, these lipid nanoparticles comprise a polyglycol ester of a hydroxy fatty acid and a polyene antifungal in a weight ratio ranging from 400:1 to 50:1, preferably from 300:1 to 50:1, more preferably from 200:1 to 50:1, even more preferably from 125:1 to 75:1.

[0111] Advantageously, these lipid nanoparticles comprise between 5% and 60% by weight, preferably between 5% and 50% by weight, more preferably between 30% and 40% by weight, of polyglycol esters of hydroxy fatty acids relative to the total weight of the lipid nanoparticle.

[0112] Advantageously, these lipid nanoparticles contain between 0.01% and 10% by weight, preferably between 0.1% and 5% by weight, more preferably between 0.5 and 2% by weight, of a polyene antifungal drug relative to the total weight of the lipid nanoparticle.

[0113] Method for preparing lipid nanoparticles according to the present invention In a third object, a method is provided for preparing lipid nanoparticles according to the second object of the present invention, comprising the steps of: a) providing an oily phase comprising at least a fatty substance and an antifungal polyene as defined above, b) providing an aqueous phase comprising a polyglycol ester of a hydroxy fatty acid; c) mixing the oily phase of step a) with the aqueous phase of step b); d) cooling the mixture obtained in step c) to obtain lipid nanoparticles.

[0114] When the lipid nanoparticles comprise a triazole antifungal agent, the oily phase provided in step a) further comprises a triazole antifungal agent.

[0115] Preferably, step c) is carried out by applying temperature cycles between 90° C. and 60° C. with steps of 4° C. / min. Preferably, 2, 3, 4 or 5 temperature cycles are carried out.

[0116] Preferably, step d) of cooling the mixture obtained in step c) is carried out by cooling the mixture to a temperature comprised below 60°C, preferably to a temperature above 55°C and below 60°C, even more preferably to a temperature of 58°C, to obtain lipid nanoparticles.

[0117] The mixing step c) may be carried out via techniques known to those skilled in the art, for example by high pressure homogenization, high speed stirring, sonication, phase inversion temperature (PIT), or solvent injection.

[0118] In certain embodiments, the lipid nanoparticles of the present invention are in the form of a suspension or in the form of a dry powder.

[0119] In the latter case, the lipid nanoparticles of the present invention in the form of dry powder can be obtained by spray-drying or freeze-drying an aqueous dispersion of lipid nanoparticles, said aqueous dispersion preferably containing a stabilizer.Such stabilizer is preferably selected from sugars, such as lactose, trehalose and raffinose; cyclodextrins; biopolymers, such as starch and dextrins; synthetic polymers with molecular weights of more than 5000 g / mol but less than 25000 g / mol, such as polyvinyl alcohol, polyvinylpyrrolidone (PVP) and linear or branched polyethylene glycol, or mixtures thereof.

[0120] How to treat In a fourth object, the present invention relates to a composition according to the invention of the first object or a lipid nanoparticle according to the invention of the second object for preventing or treating an infection caused by a fungus.

[0121] According to one embodiment, the infection is caused by at least one fungus of the order Mucorales, preferably including but not limited to the genus Rhizopus (e.g., R. arrhizus spp., R. microsporus spp.), Lichtheimia (e.g., L. ramosa spp., L. corymbifera spp.), Rhizomucor (e.g., R. pusillus spp.), Mucorales (e.g., R. cephalosporum spp.), Rhizomucor ... The infection is caused by at least one fungus of a genera selected from the group consisting of Mucor (e.g., the species M. circinelloides), Cunninghamella (e.g., C. bertholletiae), Saksenaea (e.g., S. vasiformis) and Apophysomyces (e.g., A. elegans).

[0122] According to another embodiment, the infection is caused by, but is not limited to, Aspergillus (e.g., A. clavatus, A. flavus, A. fumigatus, A. felis, A. nidulans, A. niger, A. terreus, A. lentulus, or A. versicolor), Fusarium (e.g., F. oxysporum, F. solani, F. verticillioides, F. erythroderma ... verticillioides, F. chlamydosporum, F. dimerum, F. fujikuroi, or F. incarnatum), Scedosporium (e.g., S. apiospermum, S. auraticum, S. boydii, or S. prolificans), Lomentospora (e.g., Lomentospora prolificans), prolificans), Blastomyces (e.g., B. capitatus, B. dermatitidis, and B. brasiliensis), Coccidioides (e.g., C. immitis, C. posadasii), Histoplasma (e.g., Histoplasma capsulatum), capsulatum), Candida spp. (e.g. C. albicans, C. parapsilosis, C. tropicalis, C. krusei, C. dubliniensis, C. famata, C. glabrata, C.glabrata, C. auris, and Cryptococcus (e.g., C. gattii, C. albidus, C. laurentii, or C. neoformans).

[0123] According to one embodiment, the infection is caused by a fungus of the Aspergillus genus, in particular Aspergillus fumigatus.

[0124] According to one embodiment, the infection is caused by a fungus of the genus Cryptococcus, in particular Cryptococcus gattii or Cryptococcus neoformans, preferably Cryptococcus neoformans.

[0125] According to certain embodiments, the infection is caused by a fungus exhibiting antifungal drug resistance, preferably by a fungus exhibiting efflux pump-mediated resistance, more preferably by a fungus exhibiting azole resistance, the fungus being in particular from the order Mucorales, or from the genus Rhizopus (e.g., R. alhydus spp., R. microsporus spp.), Lichtheimia (e.g., L. ramosa spp., L. corymbifera spp.), Rhizomucor (e.g., R. pusillus spp.), or from the genus Rhizomucor (e.g., R. pusillus spp.). species), Mucocele (e.g. M. circineroides species), Cunninghamella (e.g. C. batholetiae), Saxenaea (e.g. S. bassiformis) and Apophysomyces (e.g. A. elegans), Aspergillus (e.g. A. clavatus, A. flavus, A. fumigatus, A. felis, A. nidulans, A. niger, A. terreus, A. lentulus or A. versicolor), Fusarium (e.g. F. oxysporum , F. solani, F. verticillioides, F. chlamydosporum, F. dimerum, F. fujikuroi, or F. incarnatum), Scedosporium (e.g., S. apiospermum, S. auraticum, S. boydii, or S. prolificans), Lomentospora (e.g., Lomentospora prolificans), Blastomyces (e.g., B. capitatus, B. dermatitidis, and B. braziliensis), Coccidioides ( For example, it may be from a genus selected from the group including C. immitis, C. posadacii), Histoplasma (e.g., Histoplasma capsulatum), Candida (e.g., C. albicans, C. parapsilosis, C. tropicalis, C. kruzei, C. dubliniensis, C. famata, C. glabrata) and Cryptococcus (e.g., C. gattii, C. albidus, C. laurentii or C. neoformans).

[0126] In certain embodiments, the infection is caused by an antifungal drug-resistant fungus, preferably an efflux pump-mediated resistance fungus, more preferably an azole-resistant fungus, and the fungus is of the genus Aspergillus, Candida, or Cryptococcus.

[0127] According to yet another aspect, the present invention provides a method for preventing or treating diseases and disorders caused by fungal species, comprising administering said composition or lipid nanoparticles to a subject in need thereof.

[0128] According to some embodiments, the composition or lipid nanoparticles are administered at an advanced stage of a fungal infection, preferably an infection caused by a fungus of the Mucorales order.

[0129] The present invention also relates to the use of the composition or lipid nanoparticles described herein for the manufacture of a medicament for the prevention or treatment of diseases and disorders caused by fungal species.

[0130] Mucormycosis As used herein, the term "mucormycosis" refers to an infection caused by a fungus of the Mucorales order.

[0131] According to some embodiments, the composition or lipid nanoparticles are useful for the treatment of diseases caused by fungi of the order Mucorales, preferably selected from the group consisting of rhinocerebral (sinus and brain) mucormycosis, pulmonary (lung) mucormycosis, gastrointestinal mucormycosis, cutaneous (skin) mucormycosis and disseminated mucormycosis. According to currently preferred embodiments, the method of the invention is useful for the treatment of pulmonary mucormycosis and disseminated mucormycosis.

[0132] In certain embodiments, mucormycosis is caused by an antifungal drug-resistant Mucorales fungus, preferably by an efflux pump-mediated resistance Mucorales fungus, more preferably by an azole-resistant Mucorales fungus.

[0133] Aspergillosis As used herein, the term "aspergillosis" refers to infection caused by fungi of the genus Aspergillus.

[0134] According to certain embodiments, the composition or lipid nanoparticles are useful for the treatment of diseases caused by fungi of the genus Aspergillus, preferably selected from the group consisting of invasive aspergillosis, chronic pulmonary aspergillosis, allergic bronchopulmonary aspergillosis and Aspergillus bronchitis.

[0135] In certain embodiments, the aspergillosis is caused by an antifungal drug-resistant Aspergillus fungus, preferably an efflux pump-mediated resistance Aspergillus fungus, more preferably an azole-resistant Aspergillus fungus.

[0136] Cryptococcosis As used herein, the term "cryptococcosis" refers to infection caused by fungi of the genus Cryptococcus, most often caused by Cryptococcus neoformans.

[0137] According to some embodiments, the composition or lipid nanoparticles are useful for the treatment of diseases caused by fungi of the genus Cryptococcus, preferably selected from cryptococcal meningoencephalitis, pulmonary cryptococcosis, and cutaneous cryptococcosis. In some embodiments, the composition or lipid nanoparticles are useful for the treatment of Cryptococcus neoformans meningoencephalitis.

[0138] In certain embodiments, the cryptococcosis is caused by an antifungal drug-resistant fungus of the genus Cryptococcus, preferably by an efflux pump-mediated resistance Cryptococcus, more preferably by an azole-resistant Cryptococcus.

[0139] Mode of administration The compositions or lipid nanoparticles of the present invention may be administered by any convenient route of administration. Unless otherwise specified, the route of administration is defined as detailed in the standard FDAC-DRG-00301 dated November 14, 2017.

[0140] In certain embodiments, the compositions or lipid nanoparticles of the present invention are for enteral administration (i.e., administration directly into the intestine), oral administration (i.e., administration by or through the oral cavity), sublingual administration (i.e., administration under the tongue), buccal administration (i.e., administration generally in the oral cavity towards the cheek), or rectal administration (i.e., administration into the rectum).

[0141] In certain embodiments, the compositions or lipid nanoparticles of the present invention are for parenteral administration (i.e., administration by injection, infusion, or implantation). As used herein, parenteral administration includes intravenous administration, intramuscular administration, and subcutaneous administration.

[0142] The compositions or lipid nanoparticles of the invention for parenteral administration are preferably in the form of an injectable suspension, especially for intravenous administration.

[0143] The compositions or lipid nanoparticles of the invention for parenteral administration, preferably intravenous administration, are preferably administered in the form of an injectable suspension that allows the maximum concentration of antifungal polyene achieved in the plasma to be between 1 and 15 mg / L, preferably between 1 and 10 mg / L, preferably between 1 and 5 mg / L.

[0144] In certain embodiments, the compositions or lipid particles are for respiratory administration (i.e., administration into the airways, e.g., by oral inhalation, also referred to herein as inhalation administration), preferably for intrapulmonary respiratory administration.

[0145] As used herein, "pulmonary respiratory administration" refers to respiratory administration in which the composition or lipid nanoparticles are delivered to the lungs or their bronchi and concentrated in the alveolar epithelium. Pulmonary administration is complementary to parenteral (preferably intravenous) administration.

[0146] The compositions or lipid nanoparticles of the invention for respiratory administration, preferably respiratory pulmonary administration, may be administered as an aerosol of a powder, an aqueous solution or an aqueous suspension based on the compositions or lipid nanoparticles of the invention.

[0147] The compositions or lipid nanoparticles of the invention for respiratory administration, preferably respiratory pulmonary administration, are preferably administered in the form of an aerosol that allows the maximum concentration of antifungal polyene achieved in the pulmonary epithelial lining fluid, measured by bronchoalveolar lavage, to be between 15 mg / L and 60 mg / L, preferably between 15 and 60 mg / L, most preferably between 30 and 60 mg / L.

[0148] The term "aerosol" as used herein refers to a dispersion of solid particles or droplets in a gas suitable for targeting the lower respiratory tract, preferably the lungs. The compositions or lipid nanoparticles of the invention for respiratory administration preferably comprise a discontinuous phase of lipid nanoparticles of the invention or droplets of the compositions of the invention dispersed in a continuous gas phase.

[0149] Compositions or lipid nanoparticles of the invention for respiratory administration, preferably intrapulmonary administration, may be administered using a nebulizer or dry powder inhaler.

[0150] A nebulizer is defined as a device that can aerosolize liquid materials (solutions or dispersions) in the form of inhalable droplets. A nebulizer allows the administration of the composition using a mask or tip placed in the subject's mouth and / or nose. In a particular embodiment of the present invention, the aqueous suspension of lipid nanoparticles of the present invention is administered using a nebulizer, which may be, for example, a jet nebulizer or a mesh nebulizer.

[0151] The composition or lipid nanoparticles of the present invention are useful for treating fungal infections, particularly infections caused by fungi of the Mucorales, Aspergillus or Cryptococcus genera, and the active ingredient is included in an amount to achieve its intended purpose. In this range, the composition or nanoparticles should be administered in an effective amount.

[0152] The term "effective amount" refers to an amount of a compound sufficient to produce a desired therapeutic result. In particular, the lipid nanoparticle compositions of the present invention are administered in an amount sufficient to show the desired effect.

[0153] The optimal dosage of the composition or lipid nanoparticles of the present invention to be administered can be easily determined by those skilled in the art and will vary with the composition or lipid nanoparticles used, the strength of the preparation, the mode of administration, and the severity of the infection to be treated. Furthermore, it will be necessary to adjust the dosage and interval depending on factors related to the specific patient to be treated, including the patient's age, weight, diet, and administration time. The frequency and / or dosage associated with simultaneous or separate administration may be modified by those skilled in the art depending on the patient's function, medical condition, administration form, etc.

[0154] The invention will be more clearly understood with the aid of the following illustrative, non-limiting examples. [Brief description of the drawings]

[0155] [Figure 1] Figure 1 shows the MIC of Amphotericin B (AmB) against Lichtheimia ramosa measured in the presence of AmB in solution in RPMI and in the presence of PEG15HS in the form of blank lipid nanoparticles (BLNP) dispersed in RPMI. The curves correspond to duplicates of the same assay. [Diagram 2]FIG. 1 shows the hemolytic activity of AmB (1, 5, 15 mg / L) solubilized in PBS mixed with PEGHS15 (0, 10, 20, 50, 100, 200, 400, 800, 1600, 3200 mg / L) loaded onto BLNP against human red blood cells dispersed in PBS. [Diagram 3] Figure 1 shows the hemolytic activity of AmB (15, 30, 45, 60 mg / L) solubilized in RPMI mixed with PEGHS15 (0, 10, 20, 50, 100, 200, 400, 800, 1600, 3200, 6400 mg / L) loaded on BLNP against human erythrocytes dispersed in RPMI. A15: 15 mg / L AmB; A30: 30 mg / L AmB; A45: 45 mg / L AmB; A60: 60 mg / L AmB. [Figure 4] 1 shows the percentage of hemolysis as a function of formulation of LNPs containing PEG15HS and 15 mg / L AmB (LNP) or LNPs containing PEG15HS, cholesterol and 15 mg / L AmB (LNP+CH) in PBS. AMB: Control assay using 15 mg / L AmB dispersed in PBS. [Diagram 5] FIG. 1 shows a comparison of AmB concentrations in LNPs containing AmB and PEG15HS (LNP-009 and LNP-014), LNPs containing AmB and Brij® S20 (LNP-015), and LNPs containing AmB Brij® O20 (LNP-016). [Figure 6] Figure 1 shows a comparison of the percentage of hemolytic activity of LNPs containing AmB and PEG15HS (LNP-014), LNPs containing AmB and Brij® S20 or Brij® O20 (LNP-015 or LNP-016, respectively). AMB: hemolytic activity of 15 mg / L AmB in solution. Red blood cells and LNPs or AmB were present in PBS, pH=7.4, and incubated at 37°C for 1 hour. [Figure 7]FIG. 1 shows structures of compounds characteristic of A) Solutol® HS15 with n=15; B) Brij® O20 with n=20; C) Brij® S10 with n=10, or Brij® S20 when n=20; D) Brij® C10 with n=10. [Figure 8] Figure 1 shows the MIC of AmB (mg / L) versus PEG15HS concentration (mg / L) for 12 Mucorales strains. AmB and PEG15HS are dispersed in the aqueous phase. Circles represent the MIC of AmB determined during one checkerboard experiment, and solid lines represent individual AmB MICs predicted by the Emax model based on three checkerboard experiments. [Figure 9] Figure 1 shows the MICs of AmB against Lichtheimia ramosa measured for different concentrations of detergents as solutions. AmB and the different detergents are solubilized in the aqueous phase. The circles represent the MICs of AmB determined during one checkerboard experiment, and the solid lines represent the individual AmB MICs predicted by the Emax model based on three checkerboard experiments. [Figure 10] Figure 1 shows the hemolytic activity of AmB, surfactants and AMB-surfactant combinations. The hemolytic activity of Solutol® HS15, Brij® 020 and Brij® C10 (0, 10, 20, 50 and 100 mg / L), AmB (5 mg / L) and combinations of AmB (5 mg / L) with Solutol® HS15, Brij® 020 or Brij® C10 (0, 10, 20, 50 and 100 mg / L) was measured on human erythrocytes suspended in RPMI (a) or PBS (b) after 1 hour incubation at 37°C. Hemolysis was measured at 540 nm using a plate reader. Experiments were performed in duplicate. AmB5: AmB at 5 mg / L. [Figure 11]Figure 11 shows the hemolytic activity of AmB, Solutol® HS15 and AMB-Solutol® HS15 combinations. The hemolytic activity of AmB (0, 1, 5, 15, 30, 45, 60 mg / L), PEG15HS (0, 10, 20, 50, 100, 200 and 400 mg / L) and combinations of AmB (0, 1, 5, 15, 30, 45, 60 mg / L) with PEG15HS (0, 10, 20, 50, 100, 200 and 400 mg / L) in solution was measured on human red blood cells dispersed in RPMI (Figure 11a) and PBS (Figure 11b) after 1 hour incubation at 37°C. Hemolysis was measured at 540 nm using a plate reader. Experiments were performed in duplicate in two experiments. ns: not significant; *p<0.05. [Figure 12] FIG. 1 shows the fluorescence intensity ratio between the Cryptococcus cell marker (CellTracker Green CMFDA) and the lipid nanoparticle marker (DiL) after 18 hours of incubation. EXAMPLES

[0156] In the following examples, unless otherwise stated, the properties of the formulations were measured by the following methods:

[0157] Minimum inhibitory concentration (MIC) The MIC for each fungus was determined in the presence of different concentrations of AmB (solubilized in the LNP suspension or loaded into the LNP) as described by the EUCAST broth microdilution antifungal susceptibility testing methodology (EUCAST DEFINITIVE DOCUMENT E.Def 9.3.2 April 2020. EUCAST antifungal MIC method for mould).

[0158] hemolytic activity Human blood samples from different healthy volunteers were collected in EDTA tubes and centrifuged at 3000×g for 5 min. Plasma was removed and red blood cells were washed four times with 2 ml of 0.9% NaCl by centrifugation at 3000×g for 5 min.

[0159] Erythrocytes were suspended in PBS, pH 7.4, and the concentration was adjusted to 5.10^8 cells / mL. Erythrocytes were then incubated with AmB, either solubilized as a solution in LNP or loaded into LNP, for 1 h at 37°C under shaking, and then centrifuged at 3000×g for 5 min. 100 μL of the supernatant was then transferred into a flat-bottomed clear 96-well plate, and the absorbance was measured at 540 nm. Untreated erythrocytes dispersed in PBS were used as a negative control, and erythrocytes treated with 1% (v / v) TritonX-100 were used as a positive control. The percentage of hemolysis was calculated using the following formula: 100*(Asample--Acontrol) / (aTritonX-100-Anegative), where Asample is the absorbance measured for each experimental condition, and Anegative and aTritonX-100 are the absorbances of the negative and positive controls, respectively.

[0160] Preparation of lipid nanoparticles Lipid nanoparticles with (LNP) or without (BLNP) AmB were prepared according to the procedure described by Anton et al. (Anton, N.; Gayet, P.; Benoit, J.-P.; Saulnier, P. Nano-emulsions and nanocapsules by the PIT method: An investigation on the role of the temperature cycling on the emulsion phase inversion. Int. J. Pharm. 2007, 344, 44-52).

[0161] Briefly, the oil phase of lipid nanoparticles made from triglycerides (Labrafac®) was mixed with water under magnetic stirring in the presence of NaCl and two surfactants (Solutol HS15® and Phospholipon®). This oil-in-water emulsion was heated above the phase inversion temperature (PIT) (90°C) to obtain a water-in-oil emulsion. It was then cooled below the PIT (60°C) and returned to the formation of an oil-in-water emulsion. These temperature cycles were performed three times and then the system was quenched by adding 12-10 mL of chilled water (4°C).

[0162] Example 1 Materials and methods for compositions containing AmB and AmB-free PEG15HS-based lipid nanoparticles (BLNPs) dispersed in an aqueous phase Formulations containing AmB in the form of a solute and increasing concentrations of PEG15HS incorporated into lipid nanoparticles (LNPs) were prepared and assayed. The LNPs did not contain AmB and are hereafter referred to as "blank lipid nanoparticles" (BLNPs).

[0163] 1.1.1. Preparation of lipid nanoparticles containing PEG15HS BLNPs containing increasing concentrations of PEG15HS were prepared as described above.

[0164] The ingredients are detailed in Table 1.

[0165] [Table 1]

[0166] 1.1.2. Preparation of a formulation containing AmB and BLNP in solution. The BLNPs are then mixed with a solution containing 1, 5, 15, 30, 45, or 60 mg / mL AmB as a solute.

[0167] AmB-BLNP mixtures were prepared by mixing a volume of BLNP prepared in 1.1.1 with the same volume of a two-fold concentrated solution of AmB in RPMI or DPBS.

[0168] A liquid formulation is obtained that contains AmB in dissolved form and PEG15HS incorporated into LNPs dispersed in the liquid formulation.

[0169] 1.1.3.Stocks A clinical isolate of Lichtheimia ramosa was used. The isolate was plated on a 75 cm plate containing 15 mL of Sabouraud dextrose agar (Sigma-Aldrich, St. Quentin Fallavier, France). 3 The spores were subcultured from frozen stocks (-80°C) in flasks of 10 ml each at 37°C for 3 days. Spores were harvested by immersing the flasks in 10 mL of sterile water. The suspension was filtered through an 11 μm pore size nylon filter (Millipore, Carrigtwohill, Ireland) to remove hyphal elements. The concentration of the spore suspension was then adjusted in sterile water to 2 × 10 5 An inoculum of spores / mL was obtained.

[0170] Culture medium MIC and hemolytic activity measurements were performed using RPMI 1640 (with L-glutamine, pH indicator, bicarbonate free) (Sigma-Aldrich) supplemented with 2% (w / v) dextrose and buffered to pH 7 with 0.165 mol / L MOPS (Sigma-Aldrich). The medium was sterilized using a 0.22 μm pore size filter. Sterile Dulbecco's phosphate buffered saline (DDPBS, Dominique Dutscher, Bernolsheim, France), pH 7, was also used as medium to measure the hemolytic activity of AmB.

[0171] 1.2.Results 2.2.1. Effectiveness against Mucorales The MICs of the formulations for Lichtheimia ramosa were assayed.

[0172] The results are shown in Figure 1.

[0173] The results show that in the presence of 15-30 mg / L of PEG15HS incorporated into the LNP, the MIC of AmB decreases from 0.5 mg / L to 0.01 mg / L.

[0174] 2.2.1. Hemolytic activity The results are shown in Figure 3.

[0175] In PBS, 5 to 15 mg / L of AmB induces hemolysis. PEG15HS loaded into LNPs reduces this hemolytic effect of AmB at concentrations up to 100 mg / L.

[0176] In RPMI, high concentrations of AmB (15, 30 mg / L) induce hemolysis. PEGHS15 loaded into LNPs prevents this hemolytic effect of AmB at a concentration of 800 mg / L.

[0177] Example 2 A composition comprising lipid nanoparticles comprising AmB and PEG15HS (LNP). 2.1. Materials and Methods Formulations containing AmB and PEG15HS incorporated into lipid nanoparticles (LNPs) were prepared and assayed.

[0178] LNPs containing increasing concentrations of PEG15HS were prepared as described above.

[0179] The ingredients are detailed in Table 2.

[0180] [Table 2]

[0181] 2.2.Results 2.2.1.AmB content in LNP To determine the AmB content in the LNPs, 50 μL of the LNP suspension was solubilized in 950 μL of DMSO. The absorbance of these solutions was measured at a wavelength of 407 nm using an Infinite M200 Pro microplate reader (Tecan®, France). The concentration of AmB in these solutions was estimated in the concentration range (10 to 0.25 mg / L) according to a standard curve of free AmB in DMSO. The assay was validated using quality control solutions of 8, 2 and 0.4 mg / L. The AmB concentrations (μg / ml or mg / L) of the solutions obtained by mixing 50 μL of freshly prepared LNP suspension with 950 μL of DMSO are reported in Table 3 below.

[0182] 2.2.2. Size distribution and PDI The size distribution of LNPs was determined by dynamic light scattering measurements using a Malvern NanoZS (Malvern, Orsay, France). LNP suspensions were diluted (1:60 v / v) with MilliQ water and analyzed in triplicate at 25 °C. LNPs were characterized by the median of the intensity-weighted size distribution and by the polydispersity index (PDI), a measure of the broadness of the size distribution. The PDI is a number calculated from a two-parameter fit (cumulant analysis) to the correlation data. This index is dimensionless and is scaled such that values ​​below 0.05 are predominantly observed in highly monodisperse standards. PDI values ​​above 0.7 indicate that the sample has a very broad particle size distribution and is probably not suitable for analysis by dynamic light scattering (DLS) techniques. Different size distribution algorithms work with data that fall between the two extreme values ​​of the PDI (i.e., 0.05 to 0.7). The calculations used to determine size and PDI parameters are defined in ISO standards 13321:1996E and ISO 22412:2008.

[0183] The results are shown in Table 3 below.

[0184] [Table 3]

[0185] 2.2.2. Hemolytic activity Human blood samples from different healthy volunteers were collected in EDTA tubes and centrifuged at 3000×g for 5 min. Plasma was removed and red blood cells were washed four times with 2 ml of 0.9% NaCl by centrifugation at 3000×g for 5 min.

[0186] Red blood cells were suspended in PBS, pH 7.4, and the concentration was adjusted to 5.10 8 The concentration of AmB was adjusted to 1000 cells / mL. The erythrocytes were then incubated with 15 mg / L AmB solubilized as a solution in LNPs made from PEG15HS (LNP-008, LNP-009, LNP014) or other surfactants (LNP-015 and LNP-016) or loaded into LNPs for 1 hour at 37°C under shaking, then centrifuged at 3000×g for 5 minutes. 100 μL of the supernatant was then transferred into a flat-bottomed clear 96-well plate (SARSTEDT, France) and the absorbance was measured at 540 nm using a plate reader (Infinite M200 PRO, Tecan®, France). Untreated erythrocytes dispersed in PBS were used as a negative control, and erythrocytes treated with 1% (v / v) TritonX-100 were used as a positive control. The percentage of hemolysis was calculated using the following formula: 100*(Asample--Acontrol) / (aTritonX-100-Anegative), where Asample is the absorbance measured for each experimental condition, and Anegative and aTritonX-100 are the absorbances of the negative and positive controls, respectively.

[0187] The results are shown in Figure 4.

[0188] Comparative Example 3: Composition containing lipid nanoparticles containing AmB and a surfactant other than PEG15HS 3.1. Materials and Methods LNPs corresponding to LNP-009 according to Example 2, in which PEG15HS is replaced with Brij® S20 and Brij® O20, are prepared using the components detailed in Table 5 below.

[0189] [Table 4]

[0190] 3.2.Results The results shown in FIG. 5 indicate that the AmB concentration in LNPs containing AmB and PEG15HS (LNP-009 and LNP-014) is higher than or equal to that in LNPs containing AmB and Brij® S20 or Brij® O20 (LNP-015 or LNP-016, respectively).

[0191] The results shown in FIG. 6 indicate that the hemolytic activity of LNPs containing AmB and PEG15HS (LNP-014) is higher than or equivalent to that of LNPs containing AmB and Brij® S20 or Brij® O20 (LNP-015 or LNP-016, respectively).

[0192] Comparative Example 4: Formulations containing AmB dispersed in an aqueous phase and various surfactants (not according to the invention) 4.1. Materials and Methods 4.1.1. Stocks Twelve clinical isolates of the order Mucorales identified by ARNr sequencing were used: Rhizopus arrhizus (5), Rhizopus microsporus (1), Lichtheimia corymbifera (3), Lichtheimia ramosa (1), Mucor circinelloides (1), and Rhizomucor pusillus (1). Isolates were cultured on 75 cm plates containing 15 mL of Sabouraud dextrose agar (Sigma-Aldrich, St. Quentin Fallavier, France). 3 The spores were subcultured from frozen stocks (-80°C) in flasks of 10 ml each at 37°C for 3 days. Spores were harvested by immersing the flasks in 10 mL of sterile water. The suspension was filtered through an 11 μm pore size nylon filter (Millipore, Carrigtwohill, Ireland) to remove hyphal elements. The concentration of the spore suspension was then adjusted in sterile water to 2 × 10 5 An inoculum of spores / mL was obtained.

[0193] Culture medium RPMI 1640 (containing L-glutamine and a pH indicator but no bicarbonate) (Sigma-Aldrich) was supplemented with dextrose to a final concentration of 2% (w / v) and buffered to pH 7 with 0.165 mol / L MOPS (Sigma-Aldrich). The medium was sterilized using a 0.22 μm pore size filter.

[0194] Phosphate buffered saline (PBS) at 0.05 mol / L with pH 7.4 was also used as the medium to measure the hemolytic activity of AmB and UV spectra were recorded.

[0195] 4.1.3. Preparation of solutions AmB powder (Sigma-Aldrich) was dissolved in dimethyl sulfoxide to obtain a stock solution of 8192 mg / L and stored at -80°C for 6 months. On the day of use, AmB and freshly made surfactant solutions (in RPMI or PBS) were diluted with the same medium (RPMI or PBS) to obtain the desired concentrations. The surfactants used (polyethylene glycol (15)-hydroxystearate (also called Solutol® HS15 or Kolliphor® HS15, or macrogol 15 hydroxystearate); polyoxyethylene (10) stearyl ether (Brij® S10); polyoxyethylene (20) stearyl ether (Brij® S20); polyoxyethylene (20) oleyl ether (Brij® 020); and polyoxyethylene (10) cetyl ether (Brij® C10)) have the chemical structures shown in Figure 7 and were purchased from Sigma-Aldrich.

[0196] Checkerboard assay The AmB minimum inhibitory concentration (MIC) of each strain of Mucorales was determined in RPMI in the presence of different concentrations of surfactant as described by the EUCAST broth microdilution antifungal susceptibility testing methodology with modifications to the checkerboard procedure. Checkerboards were performed by adding 100 μL of two-fold dilutions of AmB (0.008 to 4 mg / L) and surfactant solutions (0.04 to 2752 mg / L) in 96-well plates. One hundred microliters of 10 5 Spore suspensions at a concentration of spores / mL were added to the wells and the plates were incubated for 24 h at 37° C. The MIC was visually read after 24 h as the lowest concentration of AmB that completely inhibited the growth of the fungal strain. Triplicate experiments were performed for each strain.

[0197] The inhibitor E was developed by Chauzy et al. (Chauzy, A.; Buyck, J.; de Jonge, BLM; Marchand, S.; Gregoire, N.; Couet, W. Pharmacodynamic Modelling of β-Lactam / β-Lactamase Inhibitor Checkerboard Data: Illustration with Aztreonam-Avibactam. Clin. Microbiol. Infect. 2019, 25, 515.e1-515.e4, doi:10.1016 / j.cmi.2018.11.025) to evaluate the enhancing effect of non-antibiotic molecules on the efficacy of antibiotics against bacteria. max The model (Equation 1) is used in the present invention to determine the surfactant concentration (C surf MIC values ​​of AmB against various Mucorales strains in relation to

[0198]

number

[0199] The mean reduction in was reported.

[0200]

number

[0201] In Equation 1, MIC0 refers to the MIC of AmB measured without surfactant, and MIC ∞ is the lowest AmB MIC measured in the presence of surfactant. The maximum ratio of reduction in AmB MIC corresponds to the maximum efficacy (E max ), where MIC0 / MIC ∞ EC was calculated as the ratio of 50 E maxThe concentration of surfactant that produces 50% of the original concentration characterizes the efficacy of the surfactant. These parameters were evaluated using WinNonlin software (version 6.2, Certara, NJ, USA) as described by Chauzy et al.

[0202] 4.1.5. Hemolytic activity The hemolytic activity of AmB in the presence of detergent was measured according to the protocol described in Serrano et al. (Serrano, DR; Hernandez, L.; Fleire, L.; Gonzalez-Alvarez, I.; Montoya, A.; Ballesteros, MP; Dea-Ayuela, MA; Miro, G.; Bolas-Fernandez, F.; Torrado, JJ Hemolytic and Pharmacokinetic Studies of Liposomal and Particulate Amphotericin B Formulations. Int J Pharm 2013, 447, 38-46, doi:10.1016 / j.ijpharm.2013.02.038).

[0203] Human blood samples from different healthy volunteers were collected in EDTA tubes and centrifuged at 3000×g for 5 min. Plasma was removed and red blood cells were washed four times with 2 ml of 0.9% NaCl by centrifugation at 3000×g for 5 min.

[0204] Red blood cells were suspended in RPMI or PBS to a concentration of 5.10. 8The red blood cells were then incubated with different combinations of AmB (0.5 mg / L) and surfactants (0, 5, 10, 20, 50, 100 mg / L) for 1 h at 37° C. under shaking, and then centrifuged at 3000×g for 5 min. 100 μL of the supernatant was then transferred into a flat-bottomed clear 96-well plate (SARSTEDT, France) and the absorbance was measured at 540 nm using a plate reader (Infinite M200 PRO, Tecan®, France). Untreated red blood cells dispersed in RPMI or PBS were used as negative controls, and red blood cells treated with 1% (v / v) TritonX-100 were used as positive controls. The percentage of hemolysis was calculated using the following formula: 100*(A sample --A control ) / (A TritonX-100 -A negative )(wherein, A sample is the absorbance measured for each experimental condition, and A negative and A TritonX-100 are the absorbances of the negative and positive controls, respectively). Experiments were performed in duplicate. After these preliminary experiments, the experiments were repeated with combinations of AmB (0, 1, 5, 15 mg / L) and Solutol® HS15 (0, 5, 10, 20, 50, 100, 200, 400 mg / L) in PBS, and with combinations of AmB (0, 15, 30, 45 and 60 mg / L) and Solutol® HS15 (0, 5, 10, 20, 50, 100, 200, 400 mg / L) in RPMI. Experiments were performed in duplicate in four experiments for RPMI and in duplicate in two experiments for PBS.

[0205] 4.2.Results 4.2.1. Effectiveness of PEG15HS and other surfactants to improve the efficacy of AmB against Mucorales in solution The efficacy of PEG15HS to improve the efficacy of AmB against several genera and strains of Mucorales involved in mucormycosis was assayed.

[0206] The MICs of all isolates for AmB were below 1 mg / L, while MIC values ​​of over 1024 mg / L were obtained for Solutol® HS15 alone. Under these conditions, it may be difficult to use the fractional inhibitory concentration (FIC) index analysis, which is traditionally used to evaluate the efficacy of antimicrobial combinations, to evaluate the effect of Solutol® HS15 on the efficacy against AmB.

[0207] Furthermore, the decrease in the MIC of AmB as a function of Solutol® HS15 concentration showed an exponential decay profile (Figure 8).

[0208] The curves in FIG. 8 were further analyzed using an inhibition Emax model to determine the maximum efficacy (E max ) and effectiveness (EC 50 ) was used as a comparison variable. The results are shown in Table 4.

[0209] [Table 5]

[0210] E of Solutol® HS15 to improve the antifungal activity of AmB max varied from 2.5 to 63.8 depending on the isolate (Table 1). The highest values ​​were obtained for Lichtheimia ramosa species. max The values ​​were also relatively high against the Lichtheimia corymbifera species, except for one strain (L. corymbifera 2), but the efficacy of AmB alone against this strain was already high, as indicated by its low MIC value of 0.06 mg / L. Therefore, it seems difficult to further lower the already low MIC of AmB against this strain. Against the tested strains of Rhizomucor pusillus and Mucor circineloides, the AmB-Solutol® HS15 combination also showed E values ​​of 20 and 16.5, respectively. maxHowever, for two of the Rhizopus species tested (one Microsporus and five strains from the genus Alhyzus (formerly Oryzae)), the combination appeared to be less effective, with E max Excluding one strain of Alhyzus with a value of 6.4, E max The value was below 4.

[0211] If the Emax value is greater than 4, Solutol® HS15 is generally very potent and has an EC 50 The values ​​were below 0.5 mg / L, indicating that Solutol® HS15 improved the efficacy of AmB at low concentrations. For most Rhizopus strains, the EC 50 Values ​​were mostly above 1 mg / L and occasionally rose to as high as 53.45 mg / L.

[0212] The results obtained with Solutol® HS15 showed that this surfactant was able to improve the effectiveness of AmB by more than four-fold against several strains of the Mucorales order at low concentrations.

[0213] To assess whether this effect was specific to Solutol® HS15 or could be obtained with other non-ionic polyethoxylated surfactants, the antifungal efficacy of AmB in association with various Brij® was also tested against L. ramosa, the Mucorales strain that responded most to the AmB-Solutol® combination. The Brij® surfactants tested differed in chain length for the hydrophobic (acyl group) and hydrophilic (polyethylene oxide) moieties (Figure 7).

[0214] The results shown in Figure 9 indicate that Brij S10, Brij S20, Brij 020 and Brij C10 were also able to enhance the efficacy of AmB against L. ramosa. The four nonionic surfactants tested showed high potency and EC 50values ​​were above 0.1 mg / L (Table 1). However, Brij® 020 and Brij® C10 showed potencies close to that of Solutol® HS15 (23.27 and 20.4, respectively), whereas Brij® S10 and Brij® S20 were less effective, with potencies of 4.1 and 9.2, respectively (Table 4).

[0215] 4.2.2. Hemolytic activity The results presented above show that certain non-ionic polyethoxylated surfactants, mainly Solutol® HS15, Brij® 020 and Brij® C10, can enhance the antifungal activity of AmB against various isolates of the Mucorales order. However, these results are only interesting if the combination has a good benefit / risk ratio, as well as if the potential human cytotoxicity of AmB is increased. Indeed, the permeabilization of mammalian cell membranes by AmB is a potential deleterious effect that may also be enhanced by these non-ionic surfactants.

[0216] To assess this potential adverse effect, the hemolytic activity of 5 mg / L AmB alone or in the presence of various concentrations of Solutol® HS15, Brij® 020 and Brij® C10 (0, 10, 20, 50 and 100 mg / L) was measured on human red blood cells dispersed in RPMI (Figure 10a) or PBS (Figure 10b). The results are shown in Figure 10.

[0217] Interestingly, Solutol® HS15 alone did not induce hemolysis in the range of concentrations tested in both media, while Brij® 020 and Brij® C10 alone both induced high hemolysis (>90% hemolysis) in both media, even at the lowest tested concentration (10 mg / L).

[0218] At 5 mg / L, AmB alone did not cause hemolysis in RPMI (FIG. 10a), whereas 7.7±0.7% of red blood cells were hemolyzed when dispersed in PBS (FIG. 10b).

[0219] In RPMI and PBS, Solutol® HS15 at concentrations up to 100 mg / L did not enhance the hemolytic activity of AmB (FIG. 4).

[0220] Further hemolytic experiments were carried out with different concentrations of AmB (1, 5, 15, 30, 45 and 60 mg / L) and Solutol® HS15 (0, 10, 20, 50, 100, 200 and 400 mg / L) in solution.

[0221] The results are shown in Figure 11. Figure 11a shows the results in RPMI, and Figure 11b shows the results in PBS.

[0222] For concentrations up to 100 mg / L, Solutol® HS15 did not enhance the hemolytic activity of AmB, regardless of the medium and AmB concentration. However, for Solutol® HS15 at concentrations of 200 and 400 mg / L, the hemolytic activity of AmB increased in both media. This increase in hemolytic activity was more pronounced in RPMI than in PBS. For example, for 15 mg / L AmB, hemolysis was 0.6±0.6% in RPMI for Solutol® HS15 at 100 mg / L, and increased to 20.5±4.7% for Solutol® HS15 at 400 mg / L. In PBS, hemolysis was 52.0±6.7% in RPMI for Solutol® HS15 at 100 mg / L, and increased to 66.4±6.3% for Solutol® HS15 at 400 mg / L. Again, the hemolytic activity of AmB in PBS was much higher than that in RPMI: for example, for 15 mg / L pure AmB, 49.0 ± 2.1% hemolysis was observed in PBS, while less than 0.1% was obtained in RPMI at this AmB concentration.

[0223] Example 5 A composition comprising a lipid nanoparticle (LNP) comprising AmB PEG15HS and isavuconazole (ISA). 5.1. Materials and Methods Prior to LNP formulation, the combination of AmB, ISA and PEG15HS in solution was evaluated.

[0224] The minimum inhibitory concentration (MIC) of AmB for each strain of Mucorales was determined in RPMI in the presence of different concentrations of ISA or PEG15HS as described by the EUCAST broth microdilution antifungal susceptibility testing methodology with a modification of the checkerboard procedure. Checkerboards were performed by adding 100 μL of two-fold dilutions of AmB (0.008 to 4 mg / L) and ISA (0.008 to 4 mg / L) or PEG15HS (0.04 to 2752 mg / L) in 96-well plates. One hundred microliters of 10 5 Spore suspensions at a concentration of spores / mL were added to the wells and the plates were incubated for 24 h at 37° C. The MIC was visually read after 24 h as the lowest concentration of AmB that completely inhibited the growth of the fungal strain. Triplicate experiments were performed for each strain.

[0225] The MIC of AmB for each strain of the Mucorales order was determined in RPMI in the presence of different concentrations of ISA and a constant concentration of PEG15HS (100-fold higher than AmB) as described by the EUCAST broth microdilution antifungal susceptibility testing methodology using a modified checkerboard procedure. Checkerboards were performed by adding 100 μL of two-fold dilutions of AmB (0.008 to 4 mg / L) + PEG15HS (0.8 to 400 mg / L) and ISA (0.008 to 4 mg / L) in a 96-well plate. 5 Spore suspensions at a concentration of spores / mL were added to the wells and the plates were incubated for 24 h at 37° C. The MIC was visually read after 24 h as the lowest concentration of AmB that completely inhibited the growth of the fungal strain. Triplicate experiments were performed for each strain.

[0226] Preparation of LNPs Formulations containing AmB+PEG15HS loaded lipid nanoparticles (LNPs) and AmB+PEG15HS+ISA loaded LNPs were prepared as follows:

[0227] Briefly, a mass (80-120 mg) of oil phase consisting of triglycerides (Labrafac® or different mass ratios of tributyrin, i.e. 100% Labrafac® to 100% tributyrin) was added with AmB (0.025-0.2 mg) and ISA (0.25-5 mg) and then mixed with 80-120 mg of PEG15HS (Solutol HS15®) and 500-800 μl of water to form an oil-in-water emulsion. This emulsion was then heated to 85° C. to obtain a water-in-oil emulsion and then cooled to 40° C. Such a temperature cycle was performed three times and then the system was rapidly cooled to 4° C. The formulated nanoparticles were then characterized by size distribution and average zeta potential measurements. Examples of AmB and AmB-ISA loaded LNP formulations are detailed in Table 5:

[0228] [Table 6]

[0229] Strains used The strains used were Rhizopus alhizus 1, Lichheimia corymbifera and Lichheimia ramosa, which correspond to the most frequently encountered species in human mucormycosis. These three strains were isolated from patients with pulmonary mucormycosis.

[0230] Two reference strains of Aspergillus fumigatus (ATCC16424 and ATCC204305) and a clinical azole-resistant strain (AfR2) were used.

[0231] 5.2.Results 5.2.1 Efficacy of LNP against Mucorales and Aspergillus fumigatus The combination of AMB and ISA in solution was compared to the combination of AMB ISA and PEG15HS in solution, and the results are shown in Table 6 below:

[0232] [Table 7]

[0233] The results show that the combination of ISA and AMB in solution reduced the MIC of AmB by 4-8 fold. The combination of AmB and PEG15HS in solution reduced the MIC of AmB by 16-32 fold. The combination of AmB, ISA and PEG15HS in solution reduced the MIC of AmB by 125 fold. The combination of AmB, ISA and PEG15HS in solution reduced the MIC of ISA by 8-32 fold.

[0234] 5.2.2. Size distribution and PDI The size distribution of LNPs was determined by dynamic light scattering measurements using a Malvern NanoZS (Malvern, Orsay, France). LNP suspensions were diluted (1:60 v / v) with MilliQ water and analyzed in triplicate at 25 °C. LNPs were characterized by the median of the intensity-weighted size distribution and by the polydispersity index (PDI), a measure of the broadness of the size distribution. The PDI is a number calculated from a two-parameter fit to correlation data (cumulant analysis). This index is dimensionless and is scaled such that values ​​below 0.05 are predominantly observed in highly monodisperse standards. PDI values ​​above 0.7 indicate that the sample has a very broad particle size distribution and is probably not suitable for analysis by dynamic light scattering (DLS) techniques. Different size distribution algorithms work with data that fall between the two extreme values ​​of the PDI (i.e., 0.05 to 0.7). The calculations used to determine the size and PDI parameters are defined in ISO standard documents 13321:1996E and ISO 22412:2008. The results are shown in Table 7 below:

[0235] [Table 8]

[0236] 5.2.3 Efficacy of LNP against Mucorales and Aspergillus fumigatus The MICs of AmB in solution, ISA in solution, AmB-loaded LNP, and AmB-ISA-loaded LNP were evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Briefly, MICs were determined by adding 100 μL of two-fold dilutions (0.008 to 4 mg / L) of AmB in solution, ISA in solution, AmB-LNP, or AmB-ISA-LNP in a 96-well plate. 100 microliters of 10 5 Spore suspensions at a concentration of spores / mL were added to the wells and the plates were incubated at 37° C. for 24 hours (Mucorales) or 48 hours (A. fumigatus). The MIC was read visually after 24 hours as the lowest concentration of AmB that completely inhibited the growth of the fungal strain. Three experiments were performed for each strain. The results are shown in Tables 8 and 9:

[0237] [Table 9]

[0238] [Table 10]

[0239] The results show that formulating AmB in nanoparticles with SOLUTOL H15S® improves the efficacy of AmB against the major strains of Mucorales causing pulmonary infections by 33-fold compared to that observed in solution (Table 8). These results are also observed against Aspergillus fumigatus strains that are sensitive and resistant to azole antifungals causing pulmonary infections (Table 9). In Tables 8 and 9 above, "Ratio (AmB)" refers to the ratio of the MIC of AmB alone to the MIC of AmB in nanoparticles, and "Ratio (ISA)" refers to the ratio of the MIC of ISA alone to the MIC of ISA in nanoparticles.

[0240] The presence of ISA potently enhances this advantage in efficacy by 125-500 fold compared to that of AmB in solution.

[0241] Example 6 Efficacy of a composition comprising lipid nanoparticles (LNPs) containing AmB PEG15HS against C. neoformans. Cryptococcus neoformans is a yeast (fungus) responsible for a life-threatening invasive infection worldwide, named cryptococcosis. C. neoformans causes meningoencephalitis, primarily in immunocompromised patients, and is responsible for approximately 200,000 deaths annually. Thus, this yeast represents a major public health problem worldwide.

[0242] A combination of AmB and 5-fluorocytosine is currently recommended for first-line treatment. Two AmB formulations are mainly used: AmB deoxycholate (AmBd) and liposomal AmB (L-AmB). L-AmB is the less toxic form and is preferred in developed countries. However, it is a much more expensive form and is therefore not widely used in developing countries. AmBd represents an essential component in the management of cryptococcosis in developing countries. Unfortunately, the efficacy of treatments against Cryptococcus remains low, with mortality rates of 35-40% in both resource-rich and resource-poor settings. Against this background, novel treatments are urgently needed.

[0243] Repurposing existing drugs and improving the efficacy of existing antifungal agents are priority avenues of investigation. As demonstrated in the above examples, the non-ionic surfactant polyethylene glycol (15)-hydroxystearate (PEG15HS) can improve the efficacy of AmB against Mucorales and Aspergillus fumigatus. This combination reduces the MIC of AmB up to 60-fold against Mucorales. As PEG15HS is an FDA-approved surfactant for parenteral use, the combination of AmB and PEG15HS can be used against invasive fungal infections.

[0244] Lipid nanoparticles (LNPs) are water-dispersible lipid systems that allow the combination of multiple molecules. Formulating AmB and PEG15HS in LNPs can allow homogenous distribution of both compounds and potential intracellular action. Since AmB is the first-line treatment for cryptococcosis, the combination of AmB and PEG15HS may be useful for cryptococcosis. Furthermore, without wishing to be bound by theory, the inventors believe that preparing AmB and PEG15HS in LNPs on an industrial scale would be much simpler than preparing L-AmB.

[0245] 6.1. Materials and Methods Eight clinical strains of Cryptococcus neoformans were used, as shown in Table 10 below:

[0246] [Table 11]

[0247] Isolates were subcultured from frozen stocks (−80°C) on Sabouraud dextrose agar (Sigma-Aldrich, St. Quentin Fallavier, France) for 48 h at 37°C before use.

[0248] Minimum inhibitory concentration (MIC) determinations were performed using RPMI 1640 (containing L-glutamine, pH indicator, and without bicarbonate) (Sigma-Aldrich, Saint-Quentin-Fallavier, France) supplemented with 2% (w / v) dextrose and buffered to pH 7 with 0.165 mol / L MOPS (Sigma-Aldrich). The medium was sterilized using a 0.22 μm pore size filter.

[0249] AmB powder (Sigma-Aldrich) was dissolved in dimethyl sulfoxide to give a stock solution of 1000 mg / L and stored at -80°C for 6 months.

[0250] On the day of use, AmB and freshly made AmB-loaded LNPs were diluted with RPMI medium to obtain the desired concentrations. AmB-loaded LNPs were prepared as described in Example 5.

[0251] The MICs of AmB alone and AmB formulated in LNP were evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Briefly, MICs were determined by adding 100 μL of two-fold dilutions of AmB (0.008 to 4 mg / L) and LNP-AmB (0.04 to 1024 mg / L) in 96-well plates. 5 One hundred microliters of fungal suspension at CFU / mL was added to the wells and the plates were incubated for 48 hours at 30° C. The MIC was defined as the lowest concentration of AmB that inhibited 90% of the growth of the fungal strain and was read after 24 hours of incubation by measuring the absorbance at 530 nm using an Infinite M200 Pro microplate reader (Tecan®, France). Triplicate experiments were performed for each strain.

[0252] 6.2.Results / Discussion MICs of all isolates for AmB ranged from 0.25 to 1 mg / L, while MIC values ​​above 1024 mg / L were obtained for blank-LNPs. MICs of AmB-loaded LNPs varied from 0.03 to 0.125 mg / L. The reduction in MICs obtained with AmB-LNPs ranged from 8 to 32 depending on the isolates (Table 11). Furthermore, for concentrations below their MICs, AmB-loaded LNPs slowed the growth rates of the tested cryptococcal strains, while AmB alone at concentrations below its MIC did not affect the growth profile of the tested cryptococcal strains.

[0253] [Table 12]

[0254] Example 7 Lipid nanoparticles loaded with Amb penetrate cryptococcal cells more effectively than nanoparticles not loaded with Amb. 7.1. Materials and Methods Cryptococcus neoformans staining Prior to nanoparticle treatment, C. neoformans were stained for 45 min with 5 μg / ml CellTracker Green CMFDA, a stable, non-toxic fluorescent probe for viable cells.

[0255] The fungi were then seeded into 8-well chamber slides suitable for high-resolution fluorescence observation using an Olympus FluoView FV-3000 confocal laser scanning microscope (ibidi, France).

[0256] Nanoparticle Treatment The fungi were then incubated with DIL-labeled empty LNPs (AmB-blank LNPs) or DIL-labeled AmB-loaded LNPs (AmB-loaded LNPs) for 18 h. Untreated fungi were used as a reference.

[0257] Imaging of Cryptococcus neoformans by confocal laser scanning microscopy Planktonic Cryptococcus neoformans were observed using a 100x oil immersion objective and visualized with CellTracker Green (excitation 488 nm - emission range: 500-540 nm) and DiL (excitation 555 nm - emission range: 570-670 nm) dyes with sequential excitation at 488 and 555 nm. Horizontal plane images of 20 to 30 stacks (1024 × 1024 pixels, corresponding to 127 × 127 μm) with a z-step of 0.5 μm were acquired for each treatment condition.

[0258] 3D projections of C. neoformans were constructed using the Easy 3D feature of the IMARIS software (Bitplane, Inc.). While creating the surfaces, the same segmentation parameters (grain size, absolute intensity and lack of post-segmentation filter) were applied to all 3D constructs, regardless of treatment.

[0259] Images captured by confocal laser scanning microscope images after 18 hours of incubation were analyzed to determine the fluorescence intensity ratio between the Cryptococcus cell marker (CellTracker Green CMFDA) and the lipid nanoparticle marker (DiL).

[0260] 7.2.Results The results are shown in Figure 12.

[0261] Analysis of confocal laser scanning microscopy images showed that, quite unexpectedly, AmB-loaded LNPs penetrated Cryptococcus cells 16-fold more efficiently than AmB-blank LNPs (FIG. 12).

[0262] These data suggest that AmB-loaded LNPs may enhance the entry of other antifungal drugs (especially lipophilic ones, especially azoles, such as isavuconazole) into fungal cells. Indeed, antifungal drugs, such as azoles (including isavuconazole), are substrates for membrane efflux pumps, which reduces their intracellular concentration and therefore their efficacy. The use of AmB-loaded LNPs may circumvent the effects of these pumps and allow higher intracellular concentrations of isavuconazole. Efflux proteins are known to play a role in azole excretion and resistance, i.e., fungal resistance to azoles. Thus, AmB-loaded LNPs may be useful in overcoming the resistance of antifungal azoles by improving their delivery to fungal cells, thereby improving the efficacy of such antifungal drugs.

Claims

1. A composition comprising an aqueous phase, a polyene antifungal agent, and lipid nanoparticles dispersed in the aqueous phase, said lipid nanoparticles comprising an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, and the polyene antifungal agent is dispersed in the aqueous phase and / or is part of the lipid nanoparticles.

2. 2. The composition of claim 1, wherein the hydroxy fatty acid is a C8 to C24 hydroxy fatty acid, preferably a C12 to C20 hydroxy fatty acid, more preferably a C14 to C18 hydroxy fatty acid.

3. 3. A composition according to claim 1 or 2, wherein the polyglycol ester of a fatty acid contains from 1 to 100 ethylene oxide groups, preferably from 5 to 50 ethylene oxide groups, more preferably from 15 to 25 ethylene oxide groups, even more preferably from 10 to 20 ethylene oxide groups.

4. 4. The composition according to any one of claims 1 to 3, wherein the polyglycol ester of a fatty acid comprises a C14 to C18 hydroxy fatty acid, preferably a C18 hydroxy fatty acid group, such as 12-hydroxystearic acid, and from 10 to 20 ethylene oxide groups.

5. 5. The composition according to claim 1, wherein the polyglycol ester of a hydroxy fatty acid is polyethylene glycol-15-hydroxystearate.

6. 6. The composition of any one of claims 1 to 5, wherein the polyene antifungal agent is selected from amphotericin B, nystatin, partricin, natamycin or a mixture thereof.

7. 7. A composition according to any one of claims 1 to 6, comprising between 1 g / L and 5 g / L, preferably between 1 g / L and 2 g / L, of a polyene antifungal agent and between 20 g / L and 100 g / L, preferably between 20 and 80 g / L, more preferably between 40 and 60 g / L, of a polyglycol ester of a hydroxy fatty acid.

8. 7. A composition according to any one of claims 1 to 6, comprising between 30 mg / L and 120 mg / L, preferably between 50 mg / L and 100 mg / L of a polyene antifungal agent and between 600 mg / L and 2400 mg / L, more preferably between 1000 and 2000 mg / L of a polyglycol ester of a hydroxy fatty acid.

9. 9. The composition according to any one of claims 1 to 8, wherein the lipid nanoparticles have a size preferably between 1 and 250 mm, preferably between 10 and 150 nm, preferably between 50 and 100 nm.

10. 10. The composition of claim 1, wherein the lipid nanoparticles do not contain an antifungal polyene.

11. 10. The composition of claim 1, wherein the aqueous phase does not contain a polyene antifungal agent.

12. 12. The composition of any one of claims 1 to 11, wherein the lipid nanoparticles comprise a polyglycol ester of a hydroxy fatty acid and a polyene antifungal agent in a mass ratio of polyglycol ester of a hydroxy fatty acid:polyene antifungal agent in the range of 5:1 to 100:

1.

13. 13. A composition according to any one of claims 1 to 12, further comprising a triazole antifungal or an antifungal agent affected by fungal resistance, in particular efflux pump-mediated resistance, wherein the triazole antifungal or the antifungal agent affected by fungal resistance is dispersed in the aqueous phase and / or is part of the lipid nanoparticles.

14. 14. The composition of claim 13, wherein the triazole antifungal agent is selected from isavuconazole, itraconazole, posaconazole, voriconazole, fluconazole, or a mixture thereof.

15. A lipid nanoparticle comprising an antifungal polyene, an oily internal phase and an envelope, the envelope comprising a polyglycol ester of a hydroxy fatty acid, the polyene antifungal drug being part of the lipid nanoparticle.

16. 16. The lipid nanoparticle of claim 15, further comprising a triazole antifungal or an antifungal agent affected by fungal resistance, in particular efflux pump-mediated resistance, wherein the triazole antifungal or the antifungal agent affected by fungal resistance is part of the lipid nanoparticle, preferably dispersed in the oily internal phase of the nanoparticle.

17. A method for preparing lipid nanoparticles according to claim 15 or 16, comprising: a) providing an oily phase comprising at least a fatty substance and an antifungal polyene as defined above, b) providing an aqueous phase comprising a polyglycol ester of a hydroxy fatty acid; c) mixing the oily phase of step a) with the aqueous phase of step b); d) cooling the mixture obtained in step c) to obtain lipid nanoparticles; The method includes:

18. The method of claim 17, wherein the oily phase provided in step a) further comprises a triazole antifungal drug or an antifungal drug affected by fungal resistance, provided that the lipid nanoparticles comprise a triazole antifungal drug or an antifungal drug affected by fungal resistance.

19. A composition according to any one of claims 1 to 14 or lipid nanoparticles according to claim 15 or 16 for use in the prevention or treatment of an infection caused by a fungus.

20. 20. The composition for use according to claim 19, wherein the fungus is preferably of the order Mucorales, of the genus Aspergillus or of the genus Cryptococcus, preferably Cryptococcus neoformans.

Images